OtherBASIC SCIENCE INVESTIGATIONS

First Evaluation of a 99mTc-Tricarbonyl Complex, 99mTc(CO)3(LAN), as a New Renal Radiopharmaceutical in Humans

Malgorzata Lipowska, Haiyang He, Eugene Malveaux, Xiaolong Xu, Luigi G. Marzilli and Andrew Taylor
Journal of Nuclear Medicine June 2006, 47 (6) 1032-1040;

Abstract

99mTc-Mercaptoacetyltriglycine (99mTc-MAG3), 99mTc-dd- and ll-ethylene-dicysteine (99mTc-EC), and 99mTc-mercaptoacetamide-ethylene-cysteine (99mTc-MAEC) contain N3S or N2S2 ligands designed to accommodate the 4 ligating sites of the (99mTcO)3+ core; they are all excellent renal imaging agents but have renal clearances lower than that of 131I-orthoiodohippurate (131I-OIH). To explore the potential of the newly accessible but less polar [99mTc(CO)3]+ core with 3 ligating sites, we decided to build on the success of 99mTc-EC, with its N2S2 ligand and 2 dangling carboxylate groups; we chose an N2S ligand that also has 2 dangling carboxylate groups, lanthionine, to form 99mTc(CO)3(LAN), a new renal radiopharmaceutical. Methods: Biodistribution studies were performed on Sprague–Dawley rats with 99mTc(CO)3(LAN) isomers, meso-LAN and dd,ll-LAN (an enantiomeric mixture), coinjected with 131I-OIH. Human studies also were performed by coinjecting each 99mTc-labeled product (∼74 MBq [∼2 mCi]) and 131I-OIH (∼7.4 MBq [∼0.2 mCi]) into 3 healthy volunteers and then performing dual-isotope imaging by use of a camera system fitted with a high-energy collimator. Blood samples were obtained from 3 to 90 min after injection, and urine samples were obtained at 30, 90, and 180 min. Results: Biodistribution studies in rats revealed rapid blood clearance as well as rapid renal extraction for both preparations, with the dose in urine at 60 min averaging 88% that of 131I-OIH. In humans, both agents provided excellent renal images, with the plasma clearance averaging 228 mL/min for 99mTc(CO)3(meso-LAN) and 176 mL/min for 99mTc(CO)3(dd,ll-LAN). At 3 h, both 99mTc(CO)3(meso-LAN) and 99mTc(CO)3(dd,ll-LAN) showed good renal excretion, averaging 85% and 77% that of 131I-OIH, respectively. Plasma protein binding was minimal (10% and 2%, respectively), and erythrocyte uptake was similar (24% and 21%, respectively) for 99mTc(CO)3(meso-LAN) and 99mTc(CO)3(dd,ll-LAN). Conclusion: Although the plasma clearance and the rate of renal excretion of the 99mTc(CO)3(LAN) complexes were still lower than those of 131I-OIH, the results of this first application of a 99mTc-tricarbonyl complex as a renal radiopharmaceutical in humans demonstrate that 99mTc(CO)3(LAN) complexes are excellent renal imaging agents and support continued renal radiopharmaceutical development based on the 99mTc-tricarbonyl core.

The development of technetium radiopharmaceuticals has relied heavily on the (TcO)3+ core, with technetium in its +5 oxidation state, which is readily accessible by pertechnetate reduction in the presence of chelating ligands. Recently, the numerous synthetic advantages of the 99mTc-labeled water-stable organometallic precursor, [99mTc(CO)3(H2O)3]+ (with 99mTc in its low +1 oxidation state), have shifted the focus of 99mTc-labeled radiopharmaceutical development to agents with a fac-[99mTc(CO)3]+ core (1–11). Both cores are compact, form kinetically inert agents with suitable ligands, and are versatile for labeling many types of bioactive molecules. However, the fac-[99mTc(CO)3]+ moiety is nonpolar, has an almost spheric shape, and offers only 3 sites on an octahedral face for ligand attachment. N2S2 and N3S ligands designed to accommodate ligand attachment for the 4 coplanar sites of the polar (TcO)3+ core are generally unsuitable for the fac-[99mTc(CO)3]+ core; consequently, new ligands are needed.

To date, renal radiopharmaceuticals designed around the (TcO)3+ core have not achieved renal clearance in humans comparable to that of orthoiodohippurate (OIH). Thus, it seemed justified to redirect some of our effort to applying the tricarbonyl core approach to the goal of improving the performance of (TcO)3+ renal imaging agents. Our studies are focusing on relatively small ligands containing at least 3 N, O, or S ligating atoms (12,13). An important focus of our work was to determine whether the effects of the less polar tricarbonyl core on the biodistribution and pharmacokinetics of a radiopharmaceutical designed around this core would preclude developing tracers with high renal clearance. One of the first tridentately coordinating ligands that we selected to exploit for the fac-[99mTc(CO)3]+ core in the design of a novel renal radiopharmaceutical was lanthionine (3,3′-thiodialanine; LANH2) (Fig. 1). We selected this design because it mirrors that of one of the best N2S2 renal imaging agents, 99mTc-ethylene-dicysteine (99mTc-EC), in that it contains an O2C-CH2-NH-Tc-NH-CH2-CO2 sequence as well as 2 dangling carboxylate groups (Fig. 1). This similarity to 99mTc-EC and the promising initial results in rats led us to select this agent among those under study in our laboratories for our first assessment of a 99mTc-tricarbonyl core agent in humans. This report describing the biodistribution, excretion, and imaging characteristics of the new renal imaging agent, 99mTc(CO)3(LAN), in fact presents one of the first human studies with any type of radiopharmaceutical containing the 99mTc-tricarbonyl core.

FIGURE 1. 
FIGURE 1. 

Comparison of 99mTc-EC and 99mTc(CO)3(LAN), 2 agents with 2-carboxylate structural features.

MATERIALS AND METHODS

All chemicals and solvents were of reagent grade and were used without further purification. LANH2, a mixture of dd-, ll-, and meso-(dl)-LAN isomers, was purchased from TCI America. 99mTc-Pertechnetate (Math) was eluted from a 99Mo/99mTc generator (Amersham Health) with 0.9% saline. High-performance liquid chromatography (HPLC) analyses were performed by use of a Beckman System Gold Nouveau apparatus (for rat studies) and a Beckman System Gold Bioessential apparatus (for human studies) equipped with a model 170 radiometric detector, a model 166 ultraviolet light–visible light detector, and 32 Karat chromatography software; a Beckman C18 RP Ultrasphere octyldecyl silane 5-μm column (4.6 × 250 mm), a flow rate of 1 mL/min, and a mobile phase of ethanol (12%) and tetraethylammonium phosphate buffer (0.05 mol/L; pH 2.5) were used. The [99mTc(CO)3(H2O)3]+ precursor was prepared directly from Math in saline solution under 1 atm of CO as described previously (14).

99mTc Radiolabeling

LANH2 (1 mg) was dissolved in 1N HCl (0.1 mL), and the pH of the solution was adjusted to ∼9 with 1N NaOH. A sample (0.1 mL) of this solution was added to 1 mL of the freshly prepared [99mTc(CO)3(H2O)3]+ solution; the mixture was heated at 70°C for 30 min, cooled to room temperature, and analyzed by HPLC to show 3 resolved HPLC peaks with the following retention times: 6, 8, and 10 min (a minor peak). All 3 complexes were isolated by HPLC, and their radiochemical purities were found to be greater than 98%. The first eluting peak was assigned as 99mTc(CO)3(meso-LAN), and the second peak was assigned as 99mTc(CO)3(dd,ll-LAN) (an enantiomeric mixture of dd- and ll-LAN isomers). Those 2 complexes were buffered to pH 7.4 and tested by HPLC for stability up to 6 h; no measurable decomposition was observed, and they were tested in rats and humans. The third peak represented the less stable isomer containing the meso-LAN ligand and was not used in our studies. We assigned those configurations to the 99mTc(CO)3(LAN) isomers because similar results were obtained for Re(CO)3(LAN) complexes, which we have fully characterized by analytic and spectroscopic methods (15).

Rat Studies

Biodistribution Studies.

The animal experiments followed the principles of laboratory animal care and were approved by the Institutional Animal Care and Use Committee of Emory University. 99mTc(CO)3(meso-LAN) and 99mTc(CO)3(dd,ll-LAN) complexes were each evaluated in 5 Sprague–Dawley rats at 10 and 60 min. A solution of each 99mTc-labeled complex (3.7 MBq/mL [100 μCi/mL]) and 131I-OIH (925 kBq/mL [25 μCi/mL]) was prepared, and six 0.2-mL samples were drawn into insulin syringes. Five samples were used for doses; the sixth sample was diluted to 100 mL, and three 1-mL portions of the resulting solution were used as standards. Each rat was anesthetized with ketamine–xylazine (2 mg/kg of body weight) injected intramuscularly, with additional supplemental anesthetic as needed. The bladder was catheterized by use of heat-flared PE-50 tubing (Becton, Dickinson and Co.) for urine collection.

The radiopharmaceutical solution was injected intravenously via a tail vein; 5 animals were sacrificed at 10 min after injection, and 5 animals were sacrificed at 60 min after injection. A blood sample was obtained, and the heart, lungs, spleen, liver, intestines, stomach, and kidneys were removed. The whole liver was weighed, and random sections were obtained for counting. Blood, whole organs, and tissue samples were placed in tubes, and each sample was weighed. The radioactivity of the sample and standards was measured by use of a dual-channel well counter with 20% windows centered on the photo peaks of 99mTc (140 keV) and 131I (360 keV). Counts were corrected for background radiation, physical decay, and spillover of 131I counts into the 99mTc window. The percentage of the dose in each tissue or organ was calculated by dividing the counts in each tissue or organ by the total injected counts. The value given for the bowel represents combined stomach and intestine activities. The percentage injected dose in whole blood was estimated by assuming a blood volume of 6.5% of total body weight.

Metabolism Studies.

Rats were prepared according to the procedure described above for the biodistribution studies. A bolus injection of each 99mTc(CO)3(LAN) complex (∼7.4 MBq [0.2 mCi]) was intravenously administered to 2 rats; urine was collected for 30 min and analyzed by HPLC alone and with a purified complex added to determine whether the complex was metabolized or excreted unchanged in the urine.

Healthy Volunteer Studies

All studies were performed with the approval of the Radioactive Drug Research Committee and the Emory University Institutional Review Board; signed consent was obtained from each volunteer. Six healthy volunteers (4 men and 2 women; mean ± SD age, 30.3 ± 5.5 y; range, 25–38 y) participated in this study. Inclusion criteria were the absence of any history of kidney and bladder diseases and a normal review of systems. Pregnancy was excluded in women by means of a urine pregnancy test. Measurements of blood pressure, heart rate, and temperature were taken before and after injection for each volunteer; in addition, a complete blood count, standard chemistry panel, and urinalysis were obtained before and 24 h after injection. Volunteers were requested to drink approximately 500 mL of water before the study. 99mTc(CO)3(meso-LAN) and 99mTc(CO)3(dd,ll-LAN) complexes were each evaluated in 3 healthy volunteers. HPLC-purified complexes and phosphate-buffered saline (pH 7.4) were passed through a Sep-Pak Plus C18 cartridge (Waters Co.) (primed with 4 mL of ethanol) and a sterile Millex-GS 22-μm filter (Millipore Corp.) (primed with 4 mL of saline) into a sterile, pyrogen-free empty vial. The final concentration was 37 MBq/mL (1 mCi/mL), and the final pH was 7.4. Test samples of each complex were analyzed and determined to be sterile and pyrogen free.

Approximately 74 MBq (∼2 mCi) of each 99mTc(CO)3(LAN) complex were coinjected with 7.4–11.1 MBq (200–300 μCi) of 131I-OIH, and imaging was performed by use of a General Electric Infinia camera with an ∼1-cm (0.375-in.) crystal fitted with a high-energy collimator; a 20% window was centered over the 365-keV photopeak of 131I, and a second 20% window was centered over the 140-keV photopeak of 99mTc. Data were acquired in a 128 × 128 matrix with a 3-phase dynamic acquisition and processed on a General Electric Xeleris computer with QuantEM renal software. Blood samples were obtained at 3, 5, 10, 20, 30, 45, 60, and 90 min after injection, and plasma clearances for 131I-OIH and each 99mTc(CO)3(LAN) complex were determined by use of the single-injection, 2-compartment model of Sapirstein et al. (16). The volunteers voided at 30, 90, and 180 min after injection to determine the percentage of the dose in urine at each time period. Plasma protein binding (PPB) was determined by ultrafiltration (Centrifree micropartition system; Amicon Inc.) of 1 mL of plasma: PPB = (1.0 − [ultrafiltrate concentration/plasma concentration]) × 100. A Beckman γ-counter system was used to determine the concentrations of radioactivity in plasma, in erythrocytes, and in urine samples, with correction for 131I scatter into the 99mTc window. To determine whether the complex was metabolized or excreted unchanged in the urine, a 1-mL urine sample from the 30-min urine collection was obtained from each volunteer and analyzed by HPLC alone and with a purified complex added.

RESULTS

99mTc Radiolabeling

Lanthionine was effectively radiolabeled with 99mTc under mild conditions (30 min at 70°C, pH ∼9) to form well-defined complexes with the 99mTc-tricarbonyl core at a high yield. In all complexes, the LANH2 ligand coordinated tridentately and facially to yield a 99mTc(CO)3(N2S) coordination sphere, leaving both carboxyl groups uncoordinated. 99mTc(CO)3(meso-LAN) is a stable product of the meso-LAN ligand (there is also a less stable isomer containing the meso-LAN ligand that converts to a more stable product), and 99mTc(CO)3(dd,ll-LAN) is an enantiomeric mixture of dd- and ll-LAN isomers.

Rat Biodistribution Studies

Both 99mTc(CO)3(meso-LAN) and 99mTc(CO)3(dd,ll-LAN) showed rapid blood clearance in rats, with less than 6% of the injected dose remaining in the blood at 10 min after injection (Table 1). Both complexes also demonstrated rapid renal extraction and high specificity for renal excretion; the mean ± SD doses in urine at 60 min (as a percentage of 131I-OIH) were 89% ± 6% for 99mTc(CO)3(meso-LAN) and 87% ± 4% for 99mTc(CO)3(dd,ll-LAN). Less than 1% of the total activity was present in the spleen, heart, and lungs; moreover, there was minimal gastrointestinal activity: 4.6% for 99mTc(CO)3(dd,ll-LAN) and 1.7% for 99mTc(CO)3(meso-LAN).

View this table:
TABLE 1

Percentage Injected Dose in Rats of 99mTc(CO)3(meso-LAN) and 99mTc(CO)3(dd,ll-LAN) Compared with 131I-OIH in Blood, Urine, and Selected Organs at 10 and 60 Minutes (n = 5)

Healthy Volunteer Studies

There was no evidence of any toxicity, as determined by measurements of blood pressure, heart rate, or temperature, complete blood count, standard chemistry panel, or urinalysis, for any of the volunteers. The clearance of 99mTc(CO)3(meso-LAN) averaged 228 mL/min, and that of 99mTc(CO)3(dd,ll-LAN) was 176 mL/min (Table 2); both clearances were substantially lower than the clearance of 538 mL/min for 131I-OIH. PPB was minimal for both 99mTc(CO)3(LAN) isomers and averaged 10% for meso-LAN and 2% for dd,ll-LAN. Erythrocyte uptake levels were similar for the 2 isomers: 24% for meso-LAN and 21% for dd,ll-LAN. Both complexes had relatively rapid renal excretion, with the difference being that the dd,ll-LAN isomer was excreted more slowly than the meso-LAN isomer; the activities in urine [as a percentage of 131I-OIH, i.e., 99mTc(CO)3(LAN)/131I-OIH] at 30 and 180 min were 57% ± 6% and 85% ± 6%, respectively, for meso-LAN and 45% ± 3% and 77% ± 6%, respectively, for dd,ll-LAN (Table 2). Image quality was excellent with both agents (Fig. 2). The time to peak appeared to be slightly more prolonged with 99mTc(CO)3)(LAN) complexes than with 131I-OIH, and ratios of counts in kidneys at 20 min after injection to maximum counts for whole-kidney and cortical regions of interest appeared to be higher (Table 3). Representative 99mTc(CO)3)(LAN) images and renogram curves, as well as simultaneous 131I-OIH images and curves, are shown in Figure 2.

FIGURE 2. 
FIGURE 2. 
FIGURE 2. 

(A) 99mTc(CO)3(LAN) images and curves obtained from 27-y-old female volunteer who received an intravenous injection containing 62.9 MBq (1.7 mCi) of 99mTc(CO)3(meso-LAN) and 7.03 MBq (0.19 mCi) of 131I-OIH before 24 min of data acquisition. Demographics and renogram data—relative uptake (% uptake), time to maximum counts [Tmax (min)], time to half-maximum counts [T1/2 (min)], and ratio of counts at 20 min to maximum counts (20/max) for whole-kidney region of interest—are displayed in upper left panel. Upper middle panel shows flow images at 2 s per frame. Image containing injection site in volunteer's arm (upper right panel) showed no infiltration. Center panel shows good uptake by kidneys bilaterally and prompt excretion into bladder. Whole-kidney renogram curves are shown in lower left panel, and cortical renogram curves are shown in lower right panel. (B) 131I-OIH images and renogram curves obtained from volunteer described in A by use of identical regions of interest over whole kidney and cortex. 131I-OIH renogram curves are much noisier than 99mTc(CO)3(LAN) renogram curves because of lower dose of 131I-OIH and because camera was not optimized to image high-energy photons of 131I.

View this table:
TABLE 2

Clearance, Protein Binding, Erythrocyte Binding, and Urine Excretion of 99mTc(CO)3(LAN) Complexes Compared with 131I-OIH in Humans (n = 3)

View this table:
TABLE 3

Renogram Parameters for Whole-Kidney Regions of Interest with 99mTc(CO)3(meso-LAN) and 99mTc(CO)3(dd,ll-LAN) Compared with 131I-OIH in Humans (n = 3)

Metabolism Studies

Urine was analyzed by HPLC to determine whether the complexes were excreted intact. Greater than 95% of the activity recovered in urine from both rats and humans coeluted with the respective HPLC-purified 99mTc(CO)3(meso-LAN) and 99mTc(CO)3(dd,ll-LAN) tracers, proving that each complex was excreted unchanged (Fig. 3).

FIGURE 3. 
FIGURE 3. 

Urine samples from human volunteers injected with 99mTc(CO)3(meso-LAN) (A) and 99mTc(CO)3(dd,ll-LAN) (B) were subjected to γ-radioactive reversed-phase HPLC analysis. Corresponding reference HPLC traces show that both complexes were excreted unchanged in urine.

DISCUSSION

A major focus of our research has been to develop radiopharmaceuticals possessing high renal clearance (13,17–22). To obtain an agent with high renal clearance, 99mTc-labeled peptides and ligands are designed to target the organic anion tubular transporter of the proximal tubule (17,22,23). Small peptides are easy to synthesize and modify, are less likely than typical ligands to be immunogenic, and are more likely to exhibit rapid blood clearance. In most cases, the primary sites of interactions of the peptides are specific receptors on the outer surface of the cell membrane (extracellular). Thus, 99mTc-mercaptoacetyltriglycine (99mTc-MAG3), 99mTc-EC, and 99mTc-mercaptoacetamide-ethylene-cysteine (99mTc-MAEC) (22) are excreted primarily by tubular secretion, whereas the nonpeptide 99mTc-diethylenetriaminepentaacetic acid (99mTc-DTPA) is excreted by glomerular filtration and has a relatively low clearance compared with the other 99mTc-labeled renal agents.

All of these factors make small peptides excellent candidates for the development of target-specific radiopharmaceuticals. However, as mentioned earlier, agents based on the newer peptide ligands, although having clearances higher than that of 99mTc-MAG3, still have clearances lower than those of 131I-OIH and p-aminohippurate.

In an effort to define new cores for exploring ligands that could produce a superior 99mTc-labeled tubular agent, we decided to investigate the potential of the [99mTc(CO)3]+ core. This core recently attracted growing interest, particularly after Alberto et al. reported an aqueous preparation of the [99mTc(CO)3(H2O)3]+ precursor (14,24) and the introduction of the IsoLink boranocarbonate kit (Mallinckrodt).

As a relatively soft receptor, the [99mTc(CO)3]+ core prefers ligands with soft sp2 aromatic nitrogen and thioether donors (25–28). A bifunctional approach that incorporates ligating groups, such as pyridyl or imidazole groups, into amino acids or peptides has proved successful in labeling of the [99mTc(CO)3]+ core (2,29). However, we avoided incorporating pyridine rings into ligands to enhance labeling because pyridine rings tend to raise the overall lipophilicity of a complex; the latter situation usually leads to labeled agents with high levels of hepatobiliary uptake, an undesirable property in a renal radiopharmaceutical (30).

Lanthionine (Fig. 1) is a small peptide (dipeptide) containing 2 free carboxyl groups that would be recognized by the anionic renal tubular transport system. Moreover, it is a simple N2S ligand that efficiently produces uniform products when labeled with the 99mTc-tricarbonyl core. In humans, only 10% of 99mTc(CO)3(meso-LAN) and 2% of 99mTc(CO)3(dd,ll-LAN) are protein bound. These protein-binding levels are much lower than those for 99mTc-MAG3 (PPB, ∼80%), 99mTc-dd-EC (PPB, ∼28%), or syn-99mTc-d-MAEC (PPB, ∼87%). Reduced protein binding is a desirable property in a renal radiopharmaceutical because it facilitates clearance by glomerular filtration as well as tubular extraction (31). The clearance of both 99mTc-tricarbonyl agents exceeds the glomerular filtration rate; this fact indicates that these complexes must be transported by the renal tubules and, as anionic tracers, they likely share the same tubular transport process as 131I-OIH, 99mTc-MAG3, 99mTc-EC, and 99mTc-MAEC.

All 3 renal tubular agents with the (99mTcO)3+ core and high renal clearance in humans, 99mTc-MAG3, 99mTc-dd-EC, and syn-99mTc-d-MAEC, contain an oxo-technetium-glycyl sequence with a Math group syn to the oxo ligand (syn-Math); structure–distribution relationships suggest that the combination of the oxo and syn-Math groups is responsible for receptor recognition (32). Results in rodents generally show a similar dependence, with syn isomers showing higher clearance than anti isomers for agents with 1 carboxyl group. In rodents, however, the results for 99mTc-EC isomers do not show this dependence.

Labeling of a mixture of dd-, ll-, and dl-EC ligands resulted in a mixture of products that were resolved by HPLC into 3 peaks, one for the complexes with the chiral ligands (99mTc-dd-EC and 99mTc-ll-EC) and one for each of the 2 meso forms (syn- and anti-99mTc-dl-EC). In mice, biodistribution studies showed no significant differences in renal excretion, hepatobiliary excretion, or blood clearance for any of the 3 peaks (33–35). In rats, clearance, extraction efficiency, and biodistribution results were almost identical for all 4 separated 99mTc-EC isomers (17,36). In humans, however, our results showed that 99mTc-dd-EC and 99mTc-ll-EC had similar clearances (99mTc-EC/131I-OIH: 82% and 70%, respectively), which were significantly higher than the 40% clearance for syn-99mTc-dl-EC (17). Similarly, the percentage injected doses (99mTc-EC/131I-OIH) in urine at 0–30 min were 90% and 92% for 99mTc-dd-EC and 99mTc-ll-EC, respectively; that for syn-99mTc-dl-EC was 57%.

Our new 99mTc-tricarbonyl agents are based on a completely different core with different physical properties and do not contain the oxo-technetium-glycyl sequence, but they still exhibit a high specificity for renal excretion. In rats, there was no difference in the excretion of the 99mTc(CO)3(LAN) isomers at 60 min despite the absolute configurations of the asymmetric carbons; however, in humans, the meso-LAN isomer appeared to be superior to the dd,ll-LAN isomer (Table 2). It should be noted that 99mTc(CO)3(dd,ll-LAN) should have been a superior tracer relative to 99mTc(CO)3(meso-LAN) on the basis of a superficial analogy to 99mTc-EC biodistribution, because both agents contain 2 dangling carboxylate groups. This similar lack of dependence on stereochemistry in rodent biodistribution, combined with a different dependence on chiral versus meso stereochemistry, led us to analyze more thoroughly all of these structures to understand better the relationship between a particular structure and its renal clearance.

The 2 CO2 groups project in opposite directions in the isomer with the higher clearance and higher rate of excretion in urine, 99mTc(CO)3(meso-LAN), and in the same direction in the isomer with the lower clearance and lower rate of excretion in urine, 99mTc(CO)3(dd,ll-LAN) (Fig. 4). In this regard, our new results parallel those obtained with 99mTc-EC agents. For both dd- and ll-EC isomers, the 2 CO2 groups are on opposite sides of the structures. The lower extraction efficiency for the dl-EC isomer and 99mTc(CO)3(dd,ll-LAN) may be attributable to the steric properties of 2 bulky carboxylate groups (CO2) on the same side of the molecule or to electrostatic effects because the 2 CO2 groups are ionized and in close proximity to each other (Fig. 4). This feature appears to affect biodistribution in humans but not in rodents for agents with 2 carboxyl groups. This difference in the way in which the carboxyl groups project between the more classical 99mTc-EC agents and our new 99mTc(CO)3(LAN) agents is a direct consequence of the stereochemistry imposed by the cores; the Tc-tricarbonyl core imposes a triangular facial ligand coordination in an agent with a pseudooctahedral geometry, and the Tc-oxo core imposes a planar square-like ligand coordination in an agent with a pseudosquare-pyramidal geometry. It is interesting that 1 carboxyl group in the meso compound is situated very close to a carbonyl group, yet this agent has very high clearance. These findings offer hope that the effects of the relatively nonpolar carbonyl groups may not have an adverse effect on the recognition of the tracer by the proximal tubular receptor.

FIGURE 4. 
FIGURE 4. 

Schematic drawing of spatial relationships of carboxylate groups (CO2) to each other and to plane defined by donor atoms in 99mTc-EC and 99mTc(CO)3(LAN) complexes.

Another approach toward understanding the effects of changes at the Tc center on the biologic properties of 99mTc-labeled radiopharmaceuticals involves a comparison of complexes containing the same chelating ligand but different Tc cores. In recent biodistribution experiments with mice, Rattat et al. (37) studied the characteristics of 3 different DTPA complexes: 99mTc-DTPA (with a Tc-oxo core), 99Tc(CO)3(DTPA) (with a Tc-tricarbonyl core), and 99mTc(CO)2(NO)(DTPA) (with a Tc-dicarbonyl-nitrosyl core). 99mTc-DTPA, a renal imaging radiopharmaceutical with the “classic” core, was excreted rapidly by the kidneys and had a low overall uptake in all other organs. Labeling of DTPA with the 99mTc-tricarbonyl core led to an agent with a decreased excretion rate, a slightly higher liver uptake, and a longer retention in blood. Introduction of the 99mTc-dicarbonyl-nitrosyl core resulted in a significant increase in liver uptake, whereas excretion by the kidneys dropped to a negligible level, compared with the results for 99mTc-DTPA. These 3 different DTPA agents showed different physical and biological characteristics, and these differences can be attributed to the consequences of the modifications at the Tc center. However, because the exact chemical speciation of 99mTc(CO)3(DTPA) and 99mTc(CO)2(NO)(DTPA) has not been defined (38), the extent to which the spatial relationships of the carboxyl groups to each other and to the different cores influence biodistribution is unclear, and further studies are needed.

CONCLUSION

Results in rats showed that both 99mTc(CO)3(LAN) isomers are rapidly excreted in the urine and have a high specificity for renal excretion. Moreover, we described the first application of a 99mTc-tricarbonyl renal radiopharmaceutical in humans, and our results offer promise that a complex based on the [Tc(CO)3]+ core could be an excellent renal imaging agent with a high plasma clearance. Although the plasma clearance and the rate of renal excretion were still lower than those for 131I-OIH, these data provide support for the continued development of renal and other radiopharmaceuticals based on the 99mTc-tricarbonyl core. Additional ligand design and testing will be required to develop a 99mTc-labeled renal tracer that will provide a direct measurement of effective renal plasma flow.

Acknowledgments

This research was supported by National Institutes of Health grant DK38842. We thank Dr. Patricia A. Marzilli for her invaluable comments during the preparation of the article.

References

  1. 1.↵
    Hoepping A, Reisgys M, Brust P, et al. TROTEC-1: a new high-affinity ligand for labeling of the dopamine transporter. J Med Chem. 1998;441:4429–4432.
    OpenUrl
  2. 2.↵
    Egli A, Alberto R, Tannahill L, et al. Organometallic 99mTc-aquaion labels peptide to an unprecedented high specific activity. J Nucl Med. 1999;40:1913–1917.
    OpenUrlAbstract/FREE Full Text
  3. 3.
    Schibli R, Katti KV, Higginbotham C, Volkert WA, Alberto R. In vitro and in vivo evaluation of bidentate, water-soluble phosphine ligands as anchor groups for the organometallic fac-[99mTc(CO)3]+-core. Nucl Med Biol. 1999;26:711–716.
    OpenUrlPubMed
  4. 4.
    Schibli R, La Bella R, Alberto R, et al. Influence of the denticity of ligand systems on the in vitro and in vivo behavior of 99mTc(I)-tricarbonyl complexes: a hint for the future functionalization of biomolecules. Bioconjug Chem. 2000;11:345–351.
    OpenUrlCrossRefPubMed
  5. 5.
    Schibli R, Schubiger PA. Current use and future potential of organometallic radiopharmaceuticals. Eur J Nucl Med. 2002;29:1529–1542.
    OpenUrl
  6. 6.
    Dyszlewski M, Blake HM, Dahlheimer JL, Pica CM, Piwnica-Worms D. Characterization of a novel 99mTc-carbonyl complex as a functional probe of MDR1 P-glycoprotein transport activity. Mol Imaging. 2002;1:24–35.
    OpenUrlCrossRefPubMed
  7. 7.
    Saidi A, Seifert S, Kretzschmar M, Bergmann R, Pietzsch H-J. Cyclopentadienyl tricarbonyl complexes of 99mTc for the in vivo imaging of the serotonin 5-HT1A receptor in the brain. J Organomet Chem. 2004;689:4739–4744.
    OpenUrl
  8. 8.
    Mundwiler S, Candreia L, Hafliger P, Ortner K, Alberto R. Preparation of no-carrier-added technetium-99m complexes via metal-assisted cleavage from a solid phase. Bioconjug Chem. 2004;15:195–202.
    OpenUrlPubMed
  9. 9.
    Alberto R, Pak JK, van Staveren D, Mundwiler S, Benny P. Mono-, bi-, or tridentate ligands? The labeling of peptides with 99mTc-carbonyls. Biopolymers. 2004;76:324–333.
    OpenUrlPubMed
  10. 10.
    Kothari KK, Satpati D, Joshi S, Venkatesh M, Ramamoorthy N, Pillai MR. 99mTc carbonyl t-butyl isonitrile: a potential new agent for myocardial perfusion imaging. Nucl Med Commun. 2005;26:155–161.
    OpenUrlPubMed
  11. 11.↵
    Buchegger F, Bonvin F, Kosinski M, et al. Radiolabeled neurotensin analog, 99mTc-NT-XI, evaluated in ductal pancreatic adenocarcinoma patients. J Nucl Med. 2003;44:1649–1654.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    Lipowska M, Cini R, Tamasi G, Xu X, Taylor AT, Marzilli LG. Complexes having the fac-[M(CO)3]+ core (M=Tc, Re) useful in radiopharmaceuticals: x-ray and NMR structural characterization and density functional calculations of species containing two sp3 N donors and one sp3 O donor. Inorg Chem. 2004;43:7774–7783.
    OpenUrlCrossRefPubMed
  13. 13.↵
    He H, Lipowska M, Xu X, Taylor A, Carlone M, Marzilli LG. Re(CO)3 complexes synthesized via an improved preparation of aqueous fac-[Re(CO)3(H2O)3]+ as an aid in assessing 99mTc imaging agents: structural characterization and solution behavior of complexes with thioether-bearing amino acids as tridentate ligands. Inorg Chem. 2005;44:5437–5446.
    OpenUrlCrossRefPubMed
  14. 14.↵
    Alberto R, Schibli R, Egli A, Schubiger AP, Abram U, Kaden TA. A novel organometallic aqua complex of technetium for the labeling of biomolecules: synthesis of [99mTc(OH2)3(CO)3]+ from [99mTcO4]− in aqueous solution and its reaction with a bifunctional ligand. J Am Chem Soc. 1998;120:7987–7988.
    OpenUrl
  15. 15.↵
    Lipowska M, He H, Malveaux E, Xu X, Marzilli LG, Taylor A. New 99mTc-tricarbonyl complexes with dipeptide (lanthionine): synthesis, characterization, and evaluation as potential renal imaging agents [abstract]. J Nucl Med. 2005;46(suppl):363P.
    OpenUrl
  16. 16.↵
    Sapirstein L, Vidt DG, Mandel MJ, Hanusek G. Volumes of distribution and clearances of intravenously injected creatinine in the dog. Am J Physiol. 1955;181:330–336.
    OpenUrlFREE Full Text
  17. 17.↵
    Taylor A, Hansen L, Eshima D, et al. Comparison of technetium-99m-ll-EC isomers in rats and humans. J Nucl Med. 1997;38:821–826.
    OpenUrlAbstract/FREE Full Text
  18. 18.
    Hansen L, Hirota S, Xu X, Taylor AT, Marzilli LG. Nature of cysteine-based Re(V)=O(N2S2) radiopharmaceuticals at physiological pH ascertained by investigation of a new complex with a meso N2S2 ligand having carboxyl groups anti to the oxo group. Inorg Chem. 2000;39:5731–5740.
    OpenUrlPubMed
  19. 19.
    Lipowska M, Hansen L, Marzilli LG, Taylor A. The new renal imaging agent 99mTc(CO)3(ENDAC) and the chemistry of the Re(CO)3(ENDAC) [abstract]. J Nucl Med. 2001;42(suppl):259P.
    OpenUrl
  20. 20.
    Lipowska M, Hansen L, Cini R, et al. Synthesis of new N2S2 ligands and Re(V)O(N2S2) analogues of 99mTc renal imaging agents: characterization by NMR spectroscopy, molecular mechanics calculations, and x-ray crystallography. Inorg Chim Acta. 2002;339:327–340.
    OpenUrl
  21. 21.
    Lipowska M, Malveaux E, He H, Marzilli LG, Taylor A. Synthesis, characterization, and evaluation of novel 99mTc-tricarbonyl complex as potential renal imaging agent [abstract]. J Nucl Med. 2003;44(suppl):316P.
    OpenUrl
  22. 22.↵
    Taylor AT, Lipowska M, Hansen L, Malveaux E, Marzilli LG. 99mTc-MAEC complexes: new renal radiopharmaceuticals combining characteristics of 99mTc-MAG3 and 99mTc-EC. J Nucl Med. 2004;45:885–891.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    Shikano N, Kanai Y, Kawai K, Ishikawa N, Endou H. Transport of 99mTc-MAG3 via rat renal organic anion transporter 1. J Nucl Med. 2004;45:80–85.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    Alberto R, Ortner K, Wheatley N, Schibli R, Schubiger AP. Synthesis and properties of boranocarbonate: a convenient in situ CO source for the aqueous preparation of [99mTc(OH2)3(CO)3]+. J Am Chem Soc. 2001;123:3135–3136.
    OpenUrlCrossRefPubMed
  25. 25.↵
    Wust F, Skaddan MB, Leibnitz P, Spies H, Katzenellenbogen J, Johannsen B. Synthesis of novel progestin-rhenium conjugates as potential ligands for the progesterone receptor. Bioorg Med Chem. 1999;7:1827–1835.
    OpenUrlPubMed
  26. 26.
    Pietzsch H-J, Gupta A, Reisgys M, et al. Chemical and biological characterization of technetium(I) and rhenium(I) tricarbonyl complexes with dithioether ligands serving as linkers for coupling the Tc(CO)3 and Re(CO)3 moieties to biologically active molecules. Bioconjug Chem. 2000;11:414–424.
    OpenUrlPubMed
  27. 27.
    Alves S, Paulo A, Correia JDG, Domingos A, Santos I. Coordination capabilities of pyrazolyl containing ligands towards the fac-[Re(CO)3]+ moiety. J Chem Soc Dalton Trans. 2002;4714–4719.
  28. 28.↵
    Santos IG, Abram U, Alberto R, Lopez EV, Sanchez A. Tricarbonylrhenium(I) complexes with thiosemicarbazone derivatives of 2-acetylpyridine and 2-pyridine formamide showing two unusual coordination modes of tridentate thiosemicarbazone ligands. Inorg Chem. 2004;43:1834–1836.
    OpenUrlPubMed
  29. 29.↵
    Banerjee SR, Levadala MK, Lazarova N, et al. Bifunctional single amino acid chelates for labeling of biomolecules with the {Tc(CO)3}+ and {Re(CO)3}+ cores: crystal and molecular structures of [ReBr(CO)3(H2NCH2C5H4N)], [Re(CO)3{(C5H4NCH2)2NH}]Br, [Re(CO)3{(C5H4NCH2)2NCH2CO2H}]Br, [Re(CO)3{X(Y)NCH2CO2CH2CH3}]Br (X = Y = 2-pyridylmethyl; X = 2-pyridylmethyl, Y = 2-(1-methylimidazolyl)methyl; X = Y = 2-(1-methylimidazolyl)methyl), [ReBr(CO)3{(C5H4NCH2)NH(CH2C4H3S)}], and [Re(CO)3{(C5H4NCH2)N(CH2C4H3S)(CH2CO2)}]. Inorg Chem. 2002;41:6417–6425.
    OpenUrlPubMed
  30. 30.↵
    Vanbilloen HP, Bormans GM, De Roo MJ, Verbruggen AM. Complexes of technetium-99m with tetrapeptides, a new class of 99mTc-labelled agents. Nucl Med Biol. 1995;22:325–338.
    OpenUrlCrossRefPubMed
  31. 31.↵
    Eshima D, Eshima L, Hansen L, Lipowska M, Marzilli LG, Taylor A. Effect of protein binding on renal extraction of 131I-OIH and 99mTc-labeled tubular agents. J Nucl Med. 2000;41:2077–2082.
    OpenUrlAbstract/FREE Full Text
  32. 32.↵
    Fritzberg AR, Kuni CC, Klingensmith WC III, Stevens J, Whitney WP. Synthesis and biological evaluation of 99mTc-N,N′-bis(mercaptoacetyl)-2,3-diaminopropanoate: a potential replacement for 131I-o-iodohippurate. J Nucl Med. 1982;23:592–598.
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    Verbruggen AM, Nosco DL, Van Nerom CG, Bormans GM, Adriaens PJ, De Roo MJ. Technetium-99m-l,l-ethylenedicysteine: a renal imaging agent. I. Labeling and evaluation in animals. J Nucl Med. 1992;33:551–557.
    OpenUrlAbstract/FREE Full Text
  34. 34.
    Van Nerom C, Bormans G, Cleynhens B, Nosco D, De Roo M, Verbruggen A. Comparison of renal excretion characteristics of isomers l,l and d,d of 99mTc-ethylenedicysteine [abstract]. J Nucl Med. 1990;31(suppl):806P.
    OpenUrl
  35. 35.↵
    Verbruggen A, Bormans G, Van Nerom C, Cleynhens B, Osiadacz D, De Roo M. Is the syn and anti orientation of the oxotechnetium and carboxyl group in 99mTc renal function agents affecting the renal excretion rate? J Labelled Compds Radiopharm. 1991;30:86–88.
  36. 36.↵
    Hansen L, Taylor A, Marzilli LG. Influence of stereoisomerism on structure and renal clearance [abstract]. J Nucl Med. 1999;40(suppl):320P.
    OpenUrl
  37. 37.↵
    Rattat D, Terwinghe C, Verbruggen A. Comparison of ‘classic’ 99mTc-DTPA, 99mTc(CO)3-DTPA and 99mTc(CO)2(NO)-DTPA. Tetrahedron. 2005;61:9563–9568.
    OpenUrlCrossRef
  38. 38.↵
    Rattat D, Eraets K, Cleynhens B, Knight H, Fonge H, Verbruggen A. Comparison of tridentate ligands in competition experiments for their ability to form a [99mTc(CO)3] complex. Tetrahedron Lett. 2004;45:2531–2534.
    OpenUrl
  • Received for publication February 13, 2006.
  • Accepted for publication February 22, 2006.
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