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Jul 2, 2009 - As part of these continuing studies, we report here on the radiolabeling with technetium-99m (99mTc) and stability of a series of SAAC ...
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Bioconjugate Chem. 2009, 20, 1625–1633

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Comprehensive Radiolabeling, Stability, and Tissue Distribution Studies of Technetium-99m Single Amino Acid Chelates (SAAC) Kevin P. Maresca,† Shawn M. Hillier,† Frank J. Femia,† Craig N. Zimmerman,† Murali K. Levadala,‡ Sangeeta R. Banerjee,‡ Justin Hicks,§ Chitra Sundararajan,§ John Valliant,§ Jon Zubieta,‡ William C. Eckelman,† John L. Joyal,† and John W. Babich*,† Molecular Insight Pharmaceuticals, Inc., 160 Second Street, Cambridge, Massachusetts 02142, Department of Chemistry, Syracuse University, Syracuse, New York 13244, and Department of Chemistry, McMaster University, Hamilton, Canada ON L8S4MI. Received April 24, 2009; Revised Manuscript Received May 27, 2009

Technetium tricarbonyl chemistry has been a subject of interest in radiopharmaceutical development over the past decade. Despite the extensive work done on developing chelates for Tc(I), a rigorous investigation of the impact of changing donor groups and labeling conditions on radiochemical yields and/or distribution has been lacking. This information is crucially important if these platforms are going to be used to develop molecular imaging probes. Previous studies on the coordination chemistry of the {M(CO)3}+ core have established alkylamine, aromatic nitrogen heterocycles, and carboxylate donors as effective chelating ligands. These observations led to the design of tridentate ligands derived from the amino acid lysine. Such amino acid analogues provide a tridentate donor set for chelation to the metal and an amino acid functionality for conjugation to biomolecules. We recently developed a family of single amino acid chelates (SAAC) that serve this function and can be readily incorporated into peptides via solid-phase synthesis techniques. As part of these continuing studies, we report here on the radiolabeling with technetium-99m (99mTc) and stability of a series of SAAC analogues of lysine. The complexes studied include cationic, neutral, and anionic complexes. The results of tissue distribution studies with these novel complexes in normal rats demonstrate a range of distribution in kidney, liver, and intestines.

INTRODUCTION Advances in molecular imaging and targeted radiotherapy of cancer are intimately dependent on technological advancements in radiochemistry. Recent reports show significant clinical benefit of targeted radiotherapeutics for the treatment of cancer; however, the arsenal of available compounds is limited despite the significant unmet clinical need (1-4). In addition, the desire to produce high specific activity radiopharmaceuticals for both imaging and therapy that bind to low-density, molecular targets has been clearly demonstrated (5). The ability to incorporate readily available radionuclides with optimal decay characteristics into targeting molecules has been the foremost consideration in developing diagnostic radiopharmaceuticals. In this respect, 99mTc has become the mainstay of diagnostic nuclear medicine and in some chemical form is used in more than 85% of the diagnostic scans performed each year in hospitals (6). The preferential use of 99mTc radiopharmaceuticals reflects the ideal nuclear properties of the isotope, as well as its convenient availability from commercial generator columns. 99mTc emits a 140 keV γ-ray with 89% abundance which is close to optimal for imaging with commercial gamma cameras. The minimal tissue-damaging linear energy transfer radiation allows the injection of activities of more than 1 GBq with low radiation exposure to the patient. The six-hour halflife is sufficiently long for pharmaceutical preparation and in ViVo accumulation in the target, yet short enough to minimize radiation dose to the patient or environmental repercussions. Furthermore, the combination of the medically useful radionuclides, 99mTc and rhenium-186/188 (186/188Re), is attractive for * Corresponding author. E-mail [email protected]. † Molecular Insight Pharmaceuticals, Inc. ‡ Syracuse University. § McMaster University.

developing molecular imaging and molecular radiotherapeutics due to the similarities in their coordination chemistry and their excellent physical decay characteristics, which enable imaging and therapy, respectively. The coordination chemistries of 99mTc and 186/188Re are remarkably similar in regard to the {M(CO)3L3}+ core, where the coordination complexes of 99mTc and 186/188Re are isostructural (7-9). Radiopharmaceuticals may be classified into two broad categories: (i) those with biological distribution determined strictly by blood flow, or perfusion, which target high capacity systems such as glomerular filtration, phagocytosis, hepatocyte clearance and bone absorption, and (ii) those with biological distribution determined by specific enzymatic or receptor mediated binding interactions, i.e., low-density sites. The latter approach, which necessitates high specific activity complexes, is achieved using two methods to chelate 99mTc in a receptor specific molecule, the conjugate/pendent approach and the integrated approach. The conjugate or pendant approach, Scheme 1, involves tethering a 99mTc chelate moiety to a species known to possess high affinity binding to a receptor (10-13). This strategy has an inherent disadvantage in that incorporation of the metal chelate complex often decreases the affinity of the “native” biomolecule, requiring optimization of both the chelator and the physical-chemical properties of the tether to regain affinity, often at the expense of a favorable pharmacokinetic profile. The very nature of the bifunctional chelate is constructed from three independent components adding complexity to the optimization process: a side arm for linkage to the bioactive molecule, the tether between the side arm linker and the donor groups, and a set of appropriate ligand donor groups for coordination to the metal site, in this case, 99mTc. The “bifunctional” ligand serves two purposes. First, it complexes the metal (M) radionuclide securely, holding it to the radiopharmaceutical without leakage

10.1021/bc900192b CCC: $40.75  2009 American Chemical Society Published on Web 07/02/2009

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Maresca et al.

Scheme 1. Representation of the Pendant Approach for the Design of Radiopharmaceutical and Radioimaging Agents

in ViVo, and second, it provides a side arm for antibody, peptide, or small molecule attachment while maintaining maximal integrity of the bioactive molecule (BAM). The bifunctional chelators must be capable of conjugation under rapid, mild, preferably aqueous conditions reasonably close to physiological pH, with minimal need for purification. In addition, the moiety that chelates the 99mTc must demonstrate significant kinetic stability under biological conditions. Conversely, the integrated approach replaces a portion of the high affinity receptor ligand with a 99mTc chelate, so as to preserve the binding affinity (14-16). The SAAC system is an example of the integrated approach which simplifies the process of identifying the appropriate combination of linker and chelator. By combining the tether and chelator into one molecule, SAAC systems transform this from a two variable to a one variable challenge. In the case of the lysine SAAC systems (Figure 1), the components consist of constant functionalities (B,C,D) for conjugation to the biomolecules of interest and a set of functional donor groups (A). One advantage of this approach is that the SAAC systems can be tailored to produce desired pharmacokinetic profiles in a more straightforward manner, via modification of the chelator (A) since B, C, and D are held constant in lysine. Furthermore, SAAC ligands are compatible with solid-phase peptide synthesis and to this end have been successfully exploited to created vast libraries of SAACcontaining peptides to enable high-throughput screening. The fluorescent SAAC analogue, rhenium tricarbonyl 2-amino-6(bis(quinolin-2-ylmethyl)amino)hexanoic acid (Re-DqK), has been successfully integrated into the chemotactic peptide sequence fMLFK which has been used for imaging bacterial infection (17), and into the trans-activating transcriptional activator (TAT) peptide for imaging neuronal stem cells (18). The most stable and readily accessible oxidation states are often characterized by chemically robust core structures which may be exploited as platforms for the development of radiopharmaceutical reagents. The most extensively developed core structure is {Tc(V)O}3+, particularly with tetradentate ligands of the NxS(4-x) type. Past research has focused primarily on attachment of the bifunctional chelators for complexation of the {Tc(V)O}+3 core into small tripeptides and tetrapeptides (19-22). This has been accomplished predominantly using N3S and N4 ligands. Other structural subunits which have attracted attention include the {TcN}2+, {TcO2}+, and technetium 6-hydrazinoni-

Figure 1. Components of the lysine SAAC analogues binding the group (VII) metals (M ) Re or Tc).

cotinic acid {Tc(HYNIC)x}3+ (where x ) 1 or 2) cores. More recently, the Tc(I) core, technetium tricarbonyl {Tc(CO)3}+ has been shown to provide an ideal geometry for the labeling of chelators of the {Tc(CO)3}+ core with high specific activity. The Tc(I)-tricarbonyl core offers a number of advantages for the design of novel radiopharmaceuticals: (i) a facile route exists to the important intermediate [M(H2O)3(CO)3]+ through the work of Alberto and co-workers (23, 24); (ii) [M(H2O)3(CO)3]+ is water-soluble and the water molecules readily undergo ligand exchange with appropriate donor ligands; (iii) the complexes containing the {Tc(CO)3}+ core are chemically robust and maintain their integrity under the most forcing conditions. Complex formation is facilitated by the facile substitution chemistry at the aqua sites of the {M(CO)3(H2O)3}+ (M ) Tc, Re) and {Re(CO)3X (H2O)2} (where X ) Cl, Br) intermediate species, and the coordination preferences of Tc(I) and Re(I) for nitrogen and oxygen donors. Research in our laboratories focused on the derivatization of a single amino acid (L-lysine) for incorporation of the {Tc(CO)3}+ core. The advantages of this strategy include (i) decreased size and complexity of the chelator due to the ability to modify a single amino acid terminus, a feature which should minimize perturbations of the peptide receptor targeting small molecule or peptide by reducing the molecular weight and steric constraints of the bifunctional unit; (ii) the ease of derivatization of the amino acid or amino acid analogue; (iii) the use of a robust Tc-core, {Tc(CO)3}+, which is readily available from the [Tc(CO)3(H2O)]+ precursor and undergoes substitution reactions at the aqua ligand sites with donor ligands (25-28). By reducing the size of the bifunctional unit, the lipophilicity of the complexes may be minimized, leading to diminished liver and gastrointestinal uptake and accumulation. These novel chelators should lead to 99mTc-radiopharmaceuticals with improved whole body clearance and thus enhanced image quality especially in the abdominal regions. The use of {99mTc(CO)3(H2O)3}+ as the starting material leads to readily formed complexes of the fac-[99mTc(CO)3(L)3]+ core. Recent work has demonstrated the high affinity of the {Tc(CO)3}+ core for this chelator system (29-32). The thermodynamic stability of the {Tc(I)(CO)3}+ core also adds in ViVo stability, limiting complicating presence of metabolites and side products that may negatively effect the imaging properties of the final complexes and negate the true tracer characteristics. We recently synthesized and characterized a series of bifunctional chelators derivatized from the ε-amine of L-lysine as stable, low molecular weight chelates for 99mTc and rhenium, including bis(2-pyridine), bis(dithiazole), bis(o-phenols), and 2-pyridine monoacetic acid (33) with the goal of investigating the impact of donor groups and labeling conditions on radiochemical yields and/or distribution that could be readily inserted into a solid-phase peptide synthesizer. Modification of the chelating moieties presents a facile way to vary the structural and physiochemical properties of our SAAC systems. The chelators prepared led to cationic, neutral and anionic complexes. Herein, are described the {99mTc(I)(CO)3+} SAAC systems: [99mTc(CO)3(η3-{2-amino-6-(bis(pyridin-2-ylmethyl)amino)hexanoic acid}] (7), [99mTc(CO)3(η3-{2-amino-6-(bis(thiazol-2-

Comprehensive Studies of

99m

Tc SAAC

ylmethyl)amino)hexanoic acid}] (8), [99mTc(CO)3(η3-{2-amino6-((carboxymethyl)(pyridin-2-ylmethyl)amino)hexanoic acid}] (9), and [99mTc(CO)3(η3-{6-[bis(2-hydroxy-benzyl)amino]-2(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid}] (10). All the ligands were evaluated for radiolabeling optimization, stability, and in addition, ligands 7-9 were evaluated for tissue distribution studies in normal rats providing crucial data relating characteristics of the chelates to pharmacokinetic profiles.

EXPERIMENTAL SECTION General Methods. All reactions were carried out in dry glassware under an atmosphere of argon unless otherwise noted. Reactions were purified by column chromatography, under medium pressure using a Biotage SP4 or by preparative highpressure liquid chromatography using a Varian Prostar 210 preparative HPLC system equipped with a semipreparative Vydac C18 reverse-phase column (250 mm × 10 mm × 5 µm) connected to a Varian Prostar model 320 UV-visible detector and monitored at a wavelength of 254 nm. The final product purifications were achieved using a binary solvent gradient of 5-95% B over 21 min (A ) triethyl ammonium phosphate (TEAP) pH 3, B ) methanol). Analytical HPLC of the radioiodinated compounds was performed using the same method with an analytical Vydac C18 reverse-phase column (250 mm × 4.6 mm × 5 µm). Microwave reactions were performed in a Biotage Initiator 60 microwave using 10 mL reactor vials. 1H NMR spectra were obtained on a Bruker 400 MHz instrument. Spectra are reported as ppm and are referenced to the solvent resonances in CDCl3, DMSO-d6, or methanol-d4. Elemental analysis was performed by Prevalere Life Sciences, Inc. Highresolution mass spectra were determined by M-Scan Inc. using positive ion electrospray with a Q-Tof API US hybrid quadrupole/time-of-flight mass spectrometer. 99mTc was used as a Na99mTcO4 solution in saline, as a commercial 99Mo/99mTc generator eluant (Cardinal Health). The 99mTc-containing solutions were always kept behind sufficient lead shielding. The use of [99mTc(CO)3(H2O)3]+ was prepared from commercially available Isolink kits (Mallinckrodt). The synthesis of the [ε-{N,N-di(pyridyl-2-methyl)}-R-(Fmoc)lysine], [ε-{N,N-di(thiazole-2-methyl)}-R-(Fmoc)lysine], and [ε-{N-(pyridyl-2-methyl)N-(acetic acid}-R-(Fmoc)lysine] were reported previously by our group (33). All solvents were purchased from Sigma Aldrich. Reagents were purchased from Sigma Aldrich (St. Louis, MO), Bachem (Switzerland), Akaal (Long Beach, CA), or Anaspec (San Jose, CA). The following abbreviations are used: Fmoc ) Fluorenylmethyloxycarbonyl, DMAP ) N, N-dimethylaminopyridine, DMF ) N,N -dimethylformamide, DCM ) dichloromethane, NaOH ) sodium hydroxide, ID/g ) injected dose per gram, PBS ) phosphate buffered saline, RCP ) radiochemical purity, RCY ) radiochemical yield.

SYNTHESIS All ligands were prepared using standard reductive amination techniques and have been described previously by Zubieta et al. (33) 2-Amino-6-(bis(pyridin-2-ylmethyl)amino)hexanoic Acid (DpK) (1). In a 100 mL round-bottom flask was placed 2-(9Hfluoren-9-ylmethoxycarbonylamino)-6-(bis(pyridin-2-ylmethyl)amino)hexanoic acid (0.20 g, 0.36 mmol) and an equal molar amount of DMAP. The solids were dissolved in 5 mL of DMF and 1 mL of methanol. The reaction mixture was allowed to stir at room temperature for 12 h. The crude reaction was concentrated and purified by column chromatography (SiO2) (20% methanol/methylene chloride as eluent) to afford the final product as an off-white solid (100 mg, 0.30 mmol, 83% yield). 1 H NMR (CDCl3) δ 8.64 (d, 2H). 8.24 (t, 2H), 7.80 (d, 2H), 7.72 (t, 2H), 4.31 (s, 2H), 3.20 (m, 2H), 3.14 (s, 9H), 3.04 (m,

Bioconjugate Chem., Vol. 20, No. 8, 2009 1627

2H), 2.25 (m, 2H),1.30 (m, 2H). ESMS m/z: 328 (M+H)+. Anal. (C18H24N4O2) C, H, N. 2-Amino-6-(bis(thiazol-2-ylmethyl)amino)hexanoic Acid (DtK) (2). In a 100 mL round-bottom flask was placed 2-(9Hfluoren-9-ylmethoxycarbonylamino)-6-(bis(thiazol-2-ylmethyl)amino)hexanoic acid (0.050 g, 0.089 mmol) and an equal molar amount of DMAP. The solids were dissolved in 5 mL of DMF and 1 mL of methanol. The reaction mixture was allowed to stir at room temperature for 12 h. The crude reaction was concentrated and purified by column chromatography (SiO2) (0-40% methanol/methylene chloride as eluent) to afford the final product as an off-white solid (18 mg, 0.053 mmol, 60% yield). 1H NMR (D2O) δ 7.73 (d, 2H). 7.57 (d, 2H), 4.09 (s, 4H), 3.69 (t, H), 2.63 (t, 2H), 1.81 (m, 2H), 1.61 (m, 2H), 1.35 (m, 2H). ESMS m/z: 340 (M+H)+. Anal. (C14H20N4O2) C, H, N. 2-Amino-6-((carboxymethyl)(pyridin-2-ylmethyl)amino)hexanoate (PAMA-K) (3). In a 100 mL round-bottom flask was placed t-butyl 2-(9H-fluoren-9-ylmethoxycarbonylamino)6-pyridin-2-ylmethyl)amino)hexanoate (0.070 g, 0.122 mmol) and an equal molar amount of DMAP. The solids were dissolved in 5 mL of DMF and 1 mL of methanol. The reaction mixture was allowed to stir at room temperature for 12 h. The crude reaction was concentrated and purified by column chromatography (SiO2) (0-40% methanol/ methylene chloride as eluent). Subsequently, the t-butyl-6-pyridin-2-ylmethyl)amino)hexanoate was dissolved in 4 mL dichloromethane and 4 mL of trifluoroacetic acid. The reaction mixture was allowed to stir at room temperature for 1.5 h. The solution was blown dry under a stream of nitrogen. The crude reaction was purified by column chromatography (SiO2) (0-40% methanol/ methylene chloride as eluent) to afford the final product as an off-white solid. (21 mg, 0.068 mmol, 55% yield). 1H NMR (CDCl3) 1H NMR (d6DMSO) δ 8.62 (d, H), 8.15 (s, 2H), 7.92 (m, H), 7.56 (d, H), 7.47 (m, H), 4.40 (d, 2H), 3.87 (m, 2H), 3.01 (m, H), 2.50 (m, 2H), 1.75 (m, 2H), 1.62 (m, 2H), 1.39 (m, 2H). ESMS m/z: 295 (M+H)+. Anal. (C14H21N3O4) C, H, N. 2-Amino-6-(bis-(2-hydroxybenzyl)amino)hexanoic Acid (Diphenol-K) (4). In a 100 mL round-bottom flask was placed 2-(9H-fluoren-9-ylmethoxycarbonylamino)-6-[bis-(2-hydroxybenzyl)amino]-hexanoic acid (0.050 g, 0.086 mmol), and an equimolar amount of DMAP and 5 mL of DMF and 1 mL of methanol was added. The reaction mixture was allowed to stir at room temperature for 12 h. The crude reaction was purified by column chromatography (SiO2) (0-40% methanol/methylene chloride as eluent) to afford the final product as an off-white solid (16 mg, 0.047 mmol, 52% yield). 1H NMR (CDCl3) δ 7.10 (m, 4H), 6.90 (m, 4H), 4.20 (d, 2H), 3.92 (m, 2H), 3.0 (m, H), 2.50 (m, 2H), 1.78 (m, 2H), 1.55 (m, 2H), 1.42 (m, 2H). ESMS m/z: 358 (M+H)+. Anal. (C20H26N2O4) C, H, N. 2,2′-(pyridin-2-ylmethylazanediyl)diacetic Acid (PADA) (5). In a 100 mL round-bottom flask was placed pyridin-2ylmethanamine (1.0 g, 9.26 mmol) dissolved in 20 mL of toluene. The solution was placed in an ice bath for dropwise addition of ethyl bromo acetate. The reaction was allowed to stir at 0 °C for 30 min. The crude reaction was concentrated and purified by column chromatography (SiO2) (10% methanol/ methylene chloride as eluent) to afford the diethyl 2,2′-(pyridin2-ylmethylazanediyl)diacetate (0.40 g, 1.42 mmol). The diethyl 2,2′-(pyridin-2-ylmethylazanediyl)diacetate (0.40 g, 0.142 mmol) was dissolved in 5 mL of methanol and 5 mL of 2 N NaOH. The reaction was refluxed for 18 h. The crude reaction was purified by column chromatography (SiO2) (0-40% methanol/ methylene chloride) to afford the final product as an off-white solid. (310 mg, 0.138 mmol, 96% yield). 1H NMR (d6-DMSO) δ 8.10 (s, H), 7.84 (dd, H), 7.60 (d, H), 7.47 (m, H), 4.30 (d,

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Figure 2. The SAAC ligands (1-4) and their respective and lipophilicity.

Maresca et al.

99m

Tc-SAAC complexes (7-10) prepared and evaluated for radiolabeling yield, stability,

2H), 3.45 (m, 4H). LCMS m/z: 224 (M+H)+. Anal. (C10H12N2O4) C, H, N. General Preparation of 99mTc-Complexes. The {99mTc(I)(CO)3}+ labeling was accomplished in two steps using commercially available IsoLink kits (Covidien) to form the [99mTc(CO)3(H2O)3]+ intermediate, which was heated at 75 °C for 30 min with the appropriate SAAC (Figure 2) (10-4 to 10-6 M, pH 2-9, 75-100 °C) to form complexes [99mTc(CO)3(η3-{2amino-6-(bis(pyridin-2-ylmethyl)amino)hexanoic acid}] ) [99mTc(CO)3(DpK)]+ (7), [99mTc(CO)3(η3-{2-amino-6-(bis(thiazol2-ylmethyl)amino)hexanoic acid}] ) [99mTc(CO)3(DtK)]+ (8), [99mTc(CO)3(η3-{2-amino-6-((carboxymethyl)(pyridin-2-ylmethyl)amino)hexanoic}] ) [99mTc(CO)3(PAMA-K)] (9), and [99mTc(CO)3(η3-{6-[bis(2-hydroxy-benzyl)amino]-2-(9H-fluoren9-ylmethoxycarbonylamino)hexanoicacid}])[99mTc(CO)3(diphenolK)]-(10). Likewise, the control ligands PADA and L-histidine (Figure 3) were labeled in a similar manner (10-6 M, pH 7, 75 °C) to form their 99mTc-complexes. Upon cooling, the reactions were purified by preparatory reverse-phase (RP) HPLC and the purity assessed by analytical RP-HPLC.

Microwave Preparation of [99mTc(CO)3(DpK)]+ (7). The { Tc(I)(CO)3}+ labeling was accomplished in two steps using the [99mTc(CO)3(H2O)3]+ intermediate, which was heated at 100 °C for 5 min with 2-amino-6-(bis(pyridin-2-ylmethyl)amino)hexanoic acid (DpK) (1) (10-4 M, PBS pH 7.4) in 10 mL reactor tubes within the Biotage Initiator 60 microwave to form the complex [99mTc(CO)3(η3-{2-amino-6-(bis(pyridin-2-ylmethyl)amino)hexanoic acid}] ) [99mTc(CO)3(DpK)]+ (7). Determination of the Optimal pH for the Radiolabeling. The effect of pH on the radiochemical yield (RCY) of the radiolabeling reactions was investigated at a constant temperature, concentration, and reaction time. Solutions of the ligands at a concentration of 10-6 M were radiolabeled at a temperature of 75 °C and analyzed for RCY at pH ) 2, 5, 6, 7, 8, and 9 using sodium acetate and phosphate buffers. The optimal pH for all of the individual chelators was determined from the RCY at 30 min. Determination of Complex Stability. The stability of the HPLC purified final complexes was assessed by simultaneous incubation with cysteine and histidine (final ligand concentration 99m

Comprehensive Studies of

99m

Tc SAAC

Bioconjugate Chem., Vol. 20, No. 8, 2009 1629

Figure 3. Structure of control ligands PADA (5) and histidine (6).

10-5 M) at pH ) 7.4 and 37 °C at 1, 4, and 18 h. The complexes were prepared, purified by RP-HPLC to achieve carrier free complexes, and then incubated with an excess of cysteine and histidine. A solution of the 99mTc-SAAC complex was incubated under identical reaction conditions in the absence of cysteine and histidine as a control. Determination of Log P Values. The Log P values of the 99m Tc(I)-complexes were determined as follows. The 99mTcSAAC complexes were prepared and purified by RP-HPLC. The desired peak was collected, and the sample was evaporated under a stream of nitrogen. The residue was dissolved in 25 µL of saline and placed in an equal volume of n-octanol (3 mL) and 25 mM pH ) 7.4 phosphate buffer (3 mL). The samples were mixed under vortex for 20 min and centrifuged at 8000 rpm for 5 min, and three 100 µL aliquots were removed from both the aqueous and the organic layers for analysis on a gamma counter (Wallac 1282). Subsequently, 1 mL of the phosphate buffer-Tc-complex solution was removed, and the process was repeated with fresh n-octanol, for a total number of six extractions to ensure full extraction of all the organic components. The partition coefficients were calculated using the equation: P ) (activity concentration in n-octanol)/(activity concentration in aqueous layer). The Log P values reported were calculated from the average of the different measurements. Rat Tissue Distribution Studies. The distribution and pharmacokinetics of selected 99mTc-SAAC complexes (7-10) were evaluated in normal male Sprague-Dawley rats (180-200 g) administered via the tail vein as a bolus injection (approximately 10 µCi/rat) in a constant volume of 0.1 mL. The animals (n ) 5 per time point) were euthanized by asphyxiation with carbon dioxide at 5, 30, 60, and 120 min post injection. Tissues (blood, heart, lungs, liver, spleen, kidneys, adrenals, stomach, intestines (with contents), testes, skeletal muscle, bone, and brain) were dissected, excised, weighed wet, and counted in an automated γ-counter (LKB Model 1282, Wallac Oy, Finland). Tissue time-radioactivity levels expressed as percent injected dose per gram of tissue (%ID/g) were determined.

RESULTS AND DISCUSSION The novel tridentate ligands possess a unique primary amine which allows for the facile synthesis of large numbers of structural variants with diverse physiochemical properties. The synthetic ease of preparation of N-substituted derivatives allowed for the preparation of SAAC ligands with the potential to form neutral, cationic, and anionic 99mTc complexes. The chemistry utilized to prepare the bifunctional SAAC chelators was based on the use of the reductive alkylation reaction (34, 35). The compounds were purified by column chromatography using silica gel to afford the pure prototype SAAC ligands, 2-amino6-(bis(pyridin-2-ylmethyl)amino)hexanoic acid (1), 2-amino-6(bis(thiazol-2-ylmethyl)amino)hexanoic acid (2), 2-amino-6((carboxymethyl)(pyridin-2-ylmethyl)amino)hexanote (3), and 2-amino-6-(bis-(2-hydroxybenzyl)amino)hexanoic acid (4) in fair to moderate yields. Radiolabeling of 99mTc-SAAC. Radiolabeling of SAAC systems was accomplished to form 7-10 on either the free

Figure 4. RCY as a function of pH for the formation of 99mTc-SAAC complexes at 75 °C for 30 min at a concentration of 10-6 M.

R-amino acids or as the appropriately N-protected amino acid derivative utilizing similar methodology, demonstrating the ease of preparation and the flexibility in the design of the SAAC systems. The 99mTc(I)(CO)3+ radiolabeling was accomplished in two steps using the commercially available IsoLink kits (Covidien) to form the [99mTc(CO)3(H2O)3]+ intermediate, which was reacted with the appropriate SAAC ligand (10-6 to 10-4 M) in an equal volume mixture of 1:1 acetonitrile and phosphate buffer. The sealed vial was heated at 100 °C for 30 min. Upon cooling, the reaction was analyzed for purity via RP-HPLC. The radiochemical purity (RCP) after HPLC purification, resulting in “carrier-free” products, was determined via HPLC and shown to be consistently g95%. Although initial results demonstrated radiolabeling at concentrations as low as 10-6 M, the RCY was e80%. To achieve a RCY of >95% at 75 °C, the reaction concentration needed to be increased to 10-4 M. Microwave Radiolabeling of [99mTc(CO)3(DpK)]+ (7). Heating with a Biotage Initiator 60 microwave was explored as an alternative to conventional heating, with the goal of reducing the time necessary to produce the 99mTc-SAAC complex in the highest yields. The conditions to produce 7 in >95% RCY were achieved (>97% RCY; n ) 5) by employing microwave heating at 100 °C for 5 min at a concentration of 10-4 M in PBS, pH 7.4 (data not shown). The RCY decreased to 8) or lower (pH < 8) pH values where the RCY decreased significantly; however, for ligands 2 and 4, slightly acidic and slightly basic reaction conditions were favored, respectively. Further evaluation demonstrated that the starting material [99mTc(CO)(H2O)]+ is extremely stable at pH > 6 but undergoes significant (∼20%) degradation upon heating at temperatures required for effective radiolabeling (75-100 °C at epH 5. When the radiolabeling was conducted at pH 50% RCY (at optimal pH) when the amount of SAAC ligand used was 10-6 M. The identities of the 99mTc-complexes were readily confirmed by RP-HPLC analysis after coinjection with the nonradioactive rhenium analogues as shown in Figure 5. 99m Tc Challenge Experiments. The 99mTc-SAAC complexes were analyzed by RP-HPLC for stability against ligand challenge with excess histidine (100 mM) and cysteine (100 mM) in PBS, pH ) 7.4 at 37 °C for 18 h. The 99mTc-SAAC complexes (carrier-free) demonstrated no degradation by HPLC analysis (see Figure 6) for 7. In contrast, the negatively charged 99mTcDiphenol-K complex proved to be much less stable, with significant degradation encountered upon purification by RPHPLC, and therefore it was excluded from the tissue distribution study. Determination of Lipophilicity (Log P Values). Log P values of the complexes were determined using the HPLCpurified 99mTc-SAAC complexes in n-octanol and 25 mM phosphate buffer (pH ) 7.4). The Log P values (Table 1) demonstrated similar partition coefficients for all three of the stable complexes evaluated. The Log P values ranged from -1.80 to -2.20 regardless of differences in charge on the metal center or the heterocycle employed.

Table 1. Comparisons of the Partition Coefficients (Log P) and HPLC Retention Times for the 99mTc-SAAC Complexes 99m

Tc-complex

HPLC Rt [min]

Log P

7 8 9 10 Tc-Histidine Tc-PADA

13.5 13 14 17.2 10.2 16.6

-1.9 -2.2 -1.8 n.d.a n.d.a n.d.a

99m 99m a

n.d. ) not determined.

Rat Tissue Distribution Studies. Animal tissue distribution studies were performed on three of the SAAC complexes, [99mTc(CO)3{η3-(DpK)}] (7), [99mTc(CO)3{η3-(DtK)}] (8), and [99mTc(CO)3{η3-(PAMAK)}] (9). A summary of the results for all three complexes in selected organs is shown in Table 2. Each SAAC complex demonstrated a unique hepatobiliary or renal clearance profile. Tissue distribution studies with complexes 7, Figure 7, and 9, Figure 9, demonstrated rapid clearance from the blood and primary excretion through the kidneys and liver. The activity decreased in all tissues as a function of time, except in the gastrointestinal (GI) tract. There was no prolonged retention of radioactivity in any of the other organs analyzed. Qualitatively, [99mTc(CO)3{η3-(PAMAK)}] (9) cleared more rapidly from the kidney and liver, while [99mTc(CO)3{η3-(DpK)}] (7) cleared more rapidly from the blood. In contrast, the blood clearance of [99mTc(CO)3{η3-(DtK)}] (8) (2.33 ( 0.26%ID/g at 30 min) was significantly slower than both 7, 0.07 ( 0.01%ID/g at 30 min (p < 0.001), and 9, 0.18 ( 0.02%ID/g at 30 min (p < 0.001). In contrast to complexes 7 and 9, which were efficiently cleared, for [99mTc(CO)3{η3-(DtK)}] (8), Figure 8, the radioactivity was retained in both the liver and kidneys throughout the time course of the experiment. In addition, [99mTc(CO)3{η3-(DtK)}] (8) was retained longer in most nontarget organs as shown in Figure 8. Overall, [99mTc(CO)3{η3-(PAMAK)}] (9) demonstrated lower hepatobiliary uptake at 30 min, 1.45 ( 0.21%ID/g, compared to 2.75 ( 0.11%ID/g for 7 (p < 0.001) and 1.93 ( 0.15%ID/g for 8 (p < 0.015). The cationic complexes, [99mTc(CO)3{η3-(DpK)}] (7) and 99m [ Tc(CO)3{η3-(DtK)}] (8), demonstrated increased clearance through the liver and GI compared to the neutral complex, [Tc(CO)3{η3-(PAMAK)}] (9). For example at 120 min, [99mTc(CO)3{η3-(DpK)}] (7) and [99mTc(CO)3{η3-(DtK)}] (8) both exhibited approximately 10-fold more radioactivity in the liver 2.12 ( 0.06% ID/g (p < 0.001) and 1.74 ( 0.12% ID/g (p < 0.001), respectively, compared to the uncharged complex [99mTc(CO)3{η3-(PAMAK)}] (9) 0.20 ( 0.03% ID/g, which suggests increased lipophilic character of the cationic 7 and 8.

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Bioconjugate Chem., Vol. 20, No. 8, 2009 1631

Table 2. Comparison of the Rat Tissue Distribution Results of %ID/g ( SEM

99m

Tc-SAAC Complexes in Blood and Excretory Tissues, Expressed as Average time (min)

compound

tissue

5

30

60

120

7

Blood Liver Kidney GI Blood Liver Kidney GI Blood Liver Kidney GI

0.58 ( 0.05 3.36 ( 0.44 6.05 ( 1.03 0.49 ( 0.08 3.32 ( 0.82 1.7 ( 0.42 3.26 ( 1.04 0.3 ( 0.15 0.63 ( 0.12 1.26 ( 0.27 6.4 ( 1.4 0.18 ( 0.04

0.07 ( 0.01 2.75 ( 0.11 4.95 ( 0.11 0.89 ( 0.07 2.33 ( 0.26 1.93 ( 0.15 4.71 ( 0.41 0.59 ( 0.13 0.18 ( 0.02 1.45 ( 0.21 3.25 ( 0.47 1.14 ( 0.23

0.025 ( 0.005 2.59 ( 0.08 4.93 ( 0.43 1.46 ( 0.09 1.4 ( 0.07 2.05 ( 0.41 4.78 ( 0.14 0.73 ( 0.29 0.071 ( 0.003 0.56 ( 0.1 1.14 ( 0.27 1.82 ( 0.35

0.013 ( 0.001 2.12 ( 0.06 3.89 ( 0.42 2.73 ( 0.57 0.76 ( 0.12 2.61 ( 0.31 4.83 ( 0.41 0.87 ( 0.2 0.034 ( 0.012 0.2 ( 0.06 0.47 ( 0.09 2.44 ( 1.18

8

9

It is unclear whether the increased hepatobiliary clearance is the result of the second heterocyclic ring (pyridine or thiazole) or the positively charged technetium metal center. Although the clearance of 7 is primarily via the hepatobiliary route, its clearance from the blood is the most rapid of the three SAAC complexes studied 0.013 ( 0.001% ID/g in the blood at 2 h compared to 1.09 ( 0.04% ID/g for 8 (p < 0.001) and 0.03 ( 0.01% ID/g for 9 (p < 0.01).

Figure 7. Tissue distribution results of [99mTc(CO)3{η3-(DpK)}](7) expressed as average %ID/g.

One goal of the SAAC labeling technology is to design complexes that preferentially clear the body via renal excretion. It was anticipated that the lipophilicity of the 99mTc-SAAC complexes would influence the route of clearance. Therefore, the Log P values of the 99mTc-SAAC complexes were measured as an independent assessment of the lipophilic nature of the complexes to gain insight into the impact of overall lipophilicity of the metal complex on tissue pharmacokinetics and how it may effect future bioconjugates. The experimentally determined Log P values suggested that the complexes were very hydrophilic, 7 (Log P ) -1.9), 8 (Log P ) -2.2), and 9 (Log P ) -1.8), were surprisingly very similar, and did not predict the differences in clearance described above. This lack of correlation between the Log P values and the clearance pathway suggests that the partition coefficient alone may be insufficient to assess the behavior of the 99mTc-SAAC complexes in ViVo. Furthermore, this implies that differences in tissue distribution, pharmacokinetics, and clearance may be governed by parameters other than lipophilicity, such as the overall charge of the complex, complex sterics, the basicity of the nitrogen donor and/or the other properties of the heterocyclic rings themselves. These studies suggest the physicochemical properties of the chelator portion of the SAAC complex affects the overall pharmacokinetics of the SAAC systems. While the impact of the chelator on the tissue pharmacokinetics of the biomolecule complex is anticipated to have a greater influence on small molecules and small peptides compared to larger peptides and proteins, it may be exploited in the design of chelators that, when incorporated into bioconjugates, offer preferred tissue distribution, pharmacokinetics, and clearance profiles. Impor-

Figure 8. Tissue distribution results of [99mTc(CO)3{η3-(DtK)}] (8), expressed as average %ID/g.

Figure 9. Tissue distribution results of [Tc(CO)3{η3-(PAMAK)}] (9), expressed as average %ID/g.

1632 Bioconjugate Chem., Vol. 20, No. 8, 2009

tantly, the ability to modify the pharmacokinetic properties of a SAAC-conjugate through chelator selection should provide an advantage in the design and development of novel radiopharmaceuticals. In summary, an examination of a set of SAAC ligands where the final technetium complex was neutral, cationic, or anionic at the metal center was accomplished. The SAAC systems could be efficiently radiolabeled in high yields with the {99mTc(CO)3}+ core to produce robust neutral or cationic 99mTc-SAAC complexes. The stability and optimal pH radiolabeling conditions were explored. The complexes were easily purified and are compatible with standard solid-phase peptide synthesis chemistries. The three stable SAAC complexes, cationic [99mTc(CO)3{η3-(DpK)}] (7) and [99mTc(CO)3{η3-(DtK)}] (8), neutral [99mTc(CO)3{η3-(PAMAK)}] (9), and their role in the uptake, retention, and clearance in normal rats was evaluated; however, the anionic complex, [99mTc(CO)3{η3-(Di-phenolK)}] (10), was unstable over time and was therefore eliminated from the rat tissue distribution studies. The pharmacokinetics of the complexes varied for each of the three stable complexes evaluated, with 9 demonstrating the most promise as a chelator for future technetium radiopharmaceuticals based on the efficient radiolabeling, high RCY, and favorable pharmacokinetic properties. The advantageous pharmacokinetic profiles included rapid blood clearance for both 7 and 9 with decreased radioactivity in all background tissues (excluding GI tract) as a function of time. These experiments suggest that the SAAC systems, particularly those that clear rapidly, like [99mTc(CO)3{η3(PAMAK)}] (9) should serve well as a novel radiolabeling strategy for peptide library synthesis and radiolabeling of other important biomolecules.

ACKNOWLEDGMENT This work was supported in part by grants (J.W.B.) from the Department Of Energy, grant no D2-FG02-99ER62791 and the National Institutes of Health, grant no 1R41A1054080-01. Supporting Information Available: Elemental analysis data for compounds 1-4. This material is available free of charge via the Internet at http://pubs.acs.org.

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