Acute and subacute toxicity studies of CMICE-013, a novel iodinated rotenone-based myocardial perfusion tracer, in Sprague Dawley rats and Gottingen minipigs.

PURPOSE: Extensive acute and subacute toxicities studies are required to evaluate the toxicological profile of the novel cardiac perfusion imaging tracer (123)I-CMICE-013 to support applications for clinical trials. METHODS: Sprague-Dawley rats and Gottingen minipigs received injections of non-radioactive 127I-CMICE-013 at two dosage levels of 1 and 5 µg/kg, and vehicle buffer as control. In the acute toxicity studies, each animal was injected on two occasions 24 h apart and then underwent a 14-day recovery period; in the subacute study, animals received daily injections for 14 days continuously. The health status and mortality of test animals were monitored daily and body weight, food consumption, physiological and biochemical parameters were measured at various time points during the study. Animals were euthanized at the end of the studies and dissected for pathologic examination of organs and tissues. RESULTS: The acute and subacute administrations of injections of the non-radioactive CMICE-013 in rats and minipigs were well tolerated. Little to no dosing-related adverse effects were observed in animal body and organ weights, hematology, coagulation, clinical chemistry, urinalysis, ophthalmoscopy, electrocardiograms, heart rates, blood pressure, macroscopic and microscopic examination of the preserved animal tissues including the brain. CONCLUSION: The lack of adverse effects from acute and subacute dosing suggest that the CMICE-013 injection solution has a reasonable safety margin within the designed concentration range to be utilized in imaging applications. The dosage level of 5 µg/kg was considered the no adverse effect level for both rats and minipigs based on our acute and subacute studies.

Flow-Dependent Uptake of ¹²³I-CMICE-013, a Novel SPECT Perfusion Agent, Compared with Standard Tracers.

UNLABELLED: Rotenone derivatives have shown promise in myocardial perfusion imaging (MPI). CMICE-013 is a novel (123)I-labeled rotenone derivative developed for SPECT MPI. The objective of this study was to assess the image quality of CMICE-013 and compare its uptake with tetrofosmin, sestamibi, and (201)Tl in vivo in a porcine model of stress-induced myocardial ischemia. METHODS: Microspheres were injected simultaneously with the radiotracer injections at rest and stress to measure blood flow. Mimicking a 1-d tetrofosmin protocol, stress imaging used 3 times as much activity and occurred 1 h after the rest injection. SPECT images were obtained at both rest and stress. After imaging, the heart was sectioned into 44-50 pieces. In each heart sample, the tracer uptake was measured in a γ counter. The images were aligned, and the decay-corrected ratio of the signals at rest and stress was used to separate the well-counter signal into rest and stress components. The uptake at rest and stress was compared with microsphere flow measurements. RESULTS: The CMICE-013 images showed good contrast between the heart and surrounding organs, with heart-to-liver and heart-to-lung uptake ratios similar to those of the standard tracers. Uptake of CMICE-013 was 1.5% of the injected dose at rest and increased more rapidly with increased blood flow than did the standard SPECT tracers. The percentage injected dose of CMICE-013 taken up by the heart was greater (P < 0.05) than (201)Tl, tetrofosmin, or sestamibi at flows greater than 1.5 mL/min/g. CONCLUSION: CMICE-013 is a promising new SPECT MPI agent.

Biodistribution and radiodosimetry of a novel myocardial perfusion tracer 123I-CMICE-013 in healthy rats.

BACKGROUND: 123I-CMICE-013 is a novel radiotracer previously reported to have promising characteristics for single-photon emission computed tomography (SPECT) myocardial perfusion imaging. We evaluated the biokinetics and radiodosimetry of this rotenone-like 123I-labeled tracer in a microSPECT imaging-based study. METHODS: 37 to 111 MBq of 123I-CMICE-013 was synthesized and administered intravenously to 14 healthy rats. Images were acquired with a microSPECT/CT camera at various time intervals and reconstructed to allow activity quantification in the tissues of interest. Radiation dosage resulted from the injection of 123I-CMICE-013 was estimated base on the biodistribution data. Tissue uptake values from image analysis were verified by gamma-counting dissected organs ex vivo. RESULTS: The heart/stomach and heart/intestine uptake ratios peaked shortly after the injection of 123I-CMICE-013, meanwhile the heart/liver ratio reached 2 as early as at 23 min post-injection. Little activity was observed in the lung and overnight clearance was significant in most of the measured tissues. The radiation dosimetry analysis based on the time-activity curves provided an estimate of the effective human dose of 6.99E-03 mSv/MBq using ICRP 60 and 7.15E-03 mSv/MBq using ICRP 103, which is comparable to the popular myocardium perfusion imaging (MPI) agents such as 99mTc-tetrofosmin and 99mTc-sestamibi, as well as other 123I-based radiotracers. CONCLUSIONS: 123I-CMICE-013 demonstrated desirable characteristics in its biokinetic and radiodosimetric profiles, supporting its potential application as a novel myocardial perfusion imaging agent.

Characterization of the four isomers of (123)I-CMICE-013: a potential SPECT myocardial perfusion imaging agent.

UNLABELLED: Myocardial perfusion imaging (MPI) with single photon emission computed tomography (SPECT) is widely used in the assessment of coronary artery disease (CAD). We have developed (123)I-CMICE-013 based on rotenone, a mitochondrial complex I (MC-1) inhibitor, as a promising new MPI agent. Our synthesis results in a mixture of four species of (123)I-CMICE-013 A, B, C, D. In this study, we separated the four species and evaluated their biodistribution and imaging properties. The cold analogs (127)I-CMICE-013 A, B, C, D were isolated and characterized and their chemical structures proposed. METHODS: (123)I-CMICE-013 was synthesized by radiolabeling rotenone with Na(123)I in trifluoroacetic acid (TFA) with iodogen as the oxidizing agent at 60°C for 45min, and the four species were separated by RP-HPLC. The cold analogs (127)I-CMICE-013 A, B, C and D were isolated with a similar procedure and characterized by NMR and mass spectrometry. Biodistribution and microSPECT imaging studies were carried out on normal rats. RESULTS: We propose the mechanism of the rotenone iodination and the structures of the four species. First, I(+) forms an intermediate three-membered ring with 6' and 7' carbons. Second, the lone electron pair of the water molecule attacks the 6' or 7'-carbon, following by the formation of 6'-OH, and 7'-I bonds as in major products C and D, or 6'-I and 7'-OH bonds as in minor products A and B. The weaker 6'-I bond in the intermediate prompts the nucleophilic attachment of water at the favorable 6'-carbon to generate C and D. MicroSPECT images of (123)I-CMICE-013 A, B, C, D in rats showed clear visualization of myocardium and little interference from lung and liver. The imaging time activity curves and biodistribution data showed complex profiles for the four isomers, which is not expected from the structure activity relationship theory. CONCLUSION: (123/127)I-CMICE-013 A and B are constitutional isomers with C and D, while A and C are diastereomers of B and D, respectively. Overall, the biological characteristics of the four species are not correlated perfectly with their molecular structures.

Toxicological evaluation of a rotenone derivative in rodents for clinical myocardial perfusion imaging.

Myocardial perfusion scintigraphy is a valuable clinical tool for assessing coronary blood flow deficits in patients. We recently synthesized a new iodinated compound ((123)I-CMICE-013) based on rotenone and showed that it has excellent performance as a radiotracer for myocardial perfusion imaging. Here, we describe the cellular toxicity and subacute toxicity of CMICE-013 in rats. Cultured hepatocytes displayed sensitivity to rotenone but not CMICE-013 at equimolar concentrations. Following i.v. injection of CMICE-013 for 14 days, body weight, ambulation, behavior, grooming, guarding (abdominal, muscular), pale conjunctivae, and food intake were observed. Biochemical, hematological, and histopathological changes in tissues (heart, liver, kidney, spleen, lung, and brain) and echocardiography at pre- and post-dosing were also examined. All animals responded well to the daily injections of CMICE-013 and showed no mortality or adverse reactions with respect to the parameters above. Subacute i.v. injections at high- (5 µg/kg) and low (1 µg/kg)-dose levels did not result in any significant changes to either biochemical or hematological parameters and no detectable changes in histopathology compared to the vehicle or untreated animals. Echocardiographic analyses, including the measurements of cardiac function and anatomy (wall thickness, left atrial size, and left ventricular mass), were not different at pre- versus post-dose measures and were not different compared to the vehicle or untreated animals. Our observations in small animals reveal that CMICE-013 induces minimal toxicity when delivered intravenously for 14 days.

Synthesis and characterization of 123I-CMICE-013: a potential SPECT myocardial perfusion imaging agent.

UNLABELLED: Coronary artery disease (CAD) is a major cause of death in Canada and the United States. Single photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) is a useful diagnostic test in the management of patients with CAD. The widely used SPECT MPI agents, (99m)Tc sestamibi and (99m)Tc tetrofosmin, exhibit less than ideal pharmacokinetic properties with decreasing uptake with higher flows. (123)I has a similar energy as (99m)Tc, an ideal half life, and is readily available from cyclotrons. The objective of this study was to develop an (123)I labeled MPI agent based on rotenone, a mitochondrial complex I inhibitor, as an alternative to currently available SPECT MPI agents. METHODS: (123)I-CMICE-013 was synthesized by radiolabeling rotenone with (123)I in trifluoroacetic acid (TFA) with iodogen as the oxidizing agent at 60 °C for 45 min, followed by RP-HPLC purification. The product was formulated in 5% EtOH in 10 mM NaOAc pH 6.5. The inactive analog (127)I-CMICE-013 was isolated and characterized by NMR and mass spectrometry, and the structure determined. Micro-SPECT imaging studies were carried out in normal and infarcted rats. Biodistribution studies were performed in normal rats at 2 h (n=6) and 24 h (n=8) post injection (p.i.). RESULTS: (123)I-CMICE-013 was isolated with >95% radiochemical purity and high specific activity (14.8-111 GBq/µmol; 400-3000 mCi/µmol). Structural analysis showed that rotenone was iodinated at 7'-position, with removal of the 6',7'-double bond, and addition of a hydroxy group at 6'-position. MicroSPECT images in normal rats demonstrated homogeneous and sustained myocardial uptake with minimal interference from lung and liver. Absent myocardial perfusion was clearly identified in rats with permanent left coronary artery ligation and ischemia-reperfusion injury. In vivo biodistribution studies in normal rats at 2 h p.i. showed significant myocardial uptake (2.01±0.48%ID/g) and high heart to liver (2.98±0.93), heart to lung (4.11±1.04) and heart to blood (8.37±3.97) ratios. At 24 h p.i., the majority of (123)I-CMICE-013 was cleared from tissues, and a significant amount of tracer was found in the thyroid, indicating in vivo deiodination of the tracer. CONCLUSION: (123)I-CMICE-013 is a promising new radiotracer for SPECT MPI with high myocardial uptake, very good target to background ratios and favorable biodistribution characteristics.

Pharmacokinetic modulation of radiolabeled chelates facilitated by phosphonate ester coordinating groups.

Chelates are an important part of metal based radiopharmaceuticals. This study examines the possible application of phosphonate ester moieties incorporated into chelates, where the ester group can be changed to modulate the pharmacokinetics of the radiopharmaceutical, while the phosphonate stably binds the metal radioisotope. Two phosphonate ester containing chelates, PCTMB and PCTM(F)E, were compared to the carboxylate containing analogue, p-Bn-PCTA, with respect to radiochemistry with several radionuclides (In-111, Ga-68, Ga-67, Cu-64). The phosphonate ester derivatives were similar to p-Bn-PCTA with respect to efficient radiolabeling with each of the radiometals under mild, aqueous conditions. Each of the radiolabeled phosphonate esters was also shown to be stable under physiological conditions in vitro. The phosphonate ester moieties did exhibit a propensity to degrade under more acidic conditions. Biodistribution studies in mice with the In-111 radiolabeled versions of PCTMB, PCTM(F)E and p-Bn-PCTA demonstrated the ability of the phosphonate ester functionalities to change the pharmacokinetics of the BFCs. With increasing lipophilicity, the phosphonate ester derivatives showed increasing hepatic clearance; but no significant increase in background tissue uptake (bone, muscle) was observed, and all the In-111 radiolabeled BFCs were substantially cleared within 24 h. The substitution of phosphonate ester for carboxylate functional groups in chelates may be an effective strategy to assist in optimizing the pharmacokinetics of radiopharmaceuticals through varying of the ester group.

Comparison of bifunctional chelates for (64)Cu antibody imaging.

PURPOSE: Improved bifunctional chelates (BFCs) are needed to facilitate efficient (64)Cu radiolabeling of monoclonal antibodies (mAbs) under mild conditions and to yield stable, target-specific agents. The utility of two novel BFCs, 1-Oxa-4,7,10-triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,10-triacetic acid (p-SCN-Bn-Oxo-DO3A) and 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-4-S-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (p-SCN-Bn-PCTA), for mAb imaging with (64)Cu were compared to the commonly used S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-tetraacetic acid (p-SCN-Bn-DOTA). METHODS: The BFCs were conjugated to trastuzumab, which targets the HER2/neu receptor. (64)Cu radiolabeling of the conjugates was optimized. Receptor binding was analyzed using flow cytometry and radioassays. Finally, PET imaging and biodistribution studies were done in mice bearing either HER2/neu-positive or HER2/neu-negative tumors. RESULTS: (64)Cu-Oxo-DO3A- and PCTA-trastuzumab were prepared at room temperature in >95% radiochemical yield (RCY) in <30 min, compared to only 88% RCY after 2 h for the preparation of (64)Cu-DOTA-trastuzumab under the same conditions. Cell studies confirmed that the immunoreactivity of the mAb was retained for each of the bioconjugates. In vivo studies showed that (64)Cu-Oxo-DO3A- and PCTA-trastuzumab had higher uptake than the (64)Cu-DOTA-trastuzumab at 24 h in HER2/neu-positive tumors, resulting in higher tumor to background ratios and better tumor images. By 40 h all three of the (64)Cu-BFC-trastuzumab conjugates allowed for clear visualization of the HER2/neu-positive tumors but not the negative control tumor. CONCLUSION: The antibody conjugates of PCTA and Oxo-DO3A were shown to have superior (64)Cu radiolabeling efficiency and stability compared to the analogous DOTA conjugate. In addition, (64)Cu-PCTA and Oxo-DO3A antibody conjugates may facilitate earlier imaging with greater target to background ratios than the analogous (64)Cu-DOTA antibody conjugates.

Evaluation of bifunctional chelates for the development of gallium-based radiopharmaceuticals.

Ga radioisotopes, including the generator-produced positron-emitting isotope (68)Ga (t1/2 = 68 min), are of increasing interest for the development of new radiopharmaceuticals. Bifunctional chelates (BFCs) that can be efficiently radiolabeled with Ga to yield complexes with good in vivo stability are needed. To this end, we undertook a systematic comparison of four BFCs containing different chelating moieties: two novel BFCs, p-NO2-Bn-Oxo (1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid) and p-NO2-Bn-PCTA (3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), and two more commonly used BFCs, p-NO2-Bn-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and p-NO2-Bn-NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid). Each BFC was compared with respect to radiolabeling conditions, radiochemical yield, stability, and in vivo clearance properties. p-NO2-Bn-PCTA, p-NO2-Bn-Oxo, and p-NO2-Bn-NOTA were all more efficiently radiolabeled with Ga compared to p-NO2-Bn-DOTA. p-NO2-Bn-DOTA required longer reaction time, higher concentrations of BFC, or heating to obtain equivalent radiochemical yields. Better stability was observed for p-NO2-Bn-NOTA and p-NO2-Bn-PCTA compared to p-NO2-Bn-DOTA and p-NO2-Bn-Oxo, especially with respect to transmetalation to transferrin. Ga-radiolabled p-NO2-Bn-Oxo was found to be kinetically labile and therefore unstable in vivo. Ga-radiolabeled p-NO2-Bn-NOTA and p-NO2-Bn-PCTA were relatively inert, while Ga-radiolabeled p-NO2-Bn-DOTA had intermediate stability, losing >20% of Ga in less than one hour when incubated with apo-transferrin. Similar stability differences were seen when incubating at pH 2. In vivo PET imaging and biodistribution studies in mice showed that (68)Ga-radiolabeled p-NO2-Bn-PCTA, p-NO2-Bn-NOTA, and p-NO2-Bn-DOTA all cleared through the kidneys. While there was no statistical difference in the biodistribution results of (68)Ga-radiolabeled p-NO2-Bn-PCTA and p-NO2-Bn-DOTA, (68)Ga-radiolabeled p-NO2-Bn-NOTA cleared more rapidly from blood and muscle tissue but retained at up to 5 times higher activity in the kidneys.

Evaluation of novel bifunctional chelates for the development of Cu-64-based radiopharmaceuticals.

BACKGROUND: Currently available bifunctional chelates (BFCs) for attaching Cu-64 to a targeting molecule are limited by either their radiolabeling conditions or in vivo stability. With the goal of identifying highly effective BFCs, we compared the properties of two novel BFCs, 1-oxa-4,7,10-triazacyclododecane-S-5-(4-nitrobenzyl)-4,7,10-triacetic acid (p-NO(2)-Bn-Oxo) and 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-S-4-(4-nitrobenzyl)-3,6,9-triacetic acid (p-NO(2)-Bn-PCTA), with the commonly used S-2-(4-nitrobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid (p-NO(2)-Bn-DOTA). METHODS: p-NO(2)-Bn-DOTA, p-NO(2)-Bn-Oxo and p-NO(2)-Bn-PCTA were each radiolabeled with Cu-64 under various conditions to assess the reaction kinetics and robustness of the radiolabeling. Stability of each Cu-64 BFC complex was evaluated at low pH and in serum. Small animal positron emission tomography imaging and biodistribution studies in mice were undertaken. RESULTS: p-NO(2)-Bn-Oxo and p-NO(2)-Bn-PCTA possessed superior reaction kinetics compared to p-NO(2)-Bn-DOTA under all radiolabeling conditions; >98% radiochemical yields were achieved in <5 min at room temperature even when using near stoichiometric amounts of BFC. Under nonideal conditions, such as low or high pH, high radiochemical yields were still achievable with the novel BFCs. The radiolabeled compounds were stable in serum and at pH 2 for 48 h. The imaging and biodistribution of the Cu-64-radiolabeled BFCs illustrated differences between the BFCs, including preferential clearance via the kidneys for the p-NO(2)-Bn-PCTA Cu-64 complex. CONCLUSIONS: The novel BFCs facilitated efficient Cu-64 radiolabeling under mild conditions to produce stable complexes at potentially high specific activities. These BFCs may find wide utility in the development of Cu-64-based radiopharmaceuticals.

Ring-Opening Cycloaddition of Aziridines to Ketenimines.

The Lewis acid-catalyzed addition of aziridines to ketenimines gave substituted pyrrolidonimines in 47-87% yields. The hard Lewis acid LiClO(4) proved to be superior to the soft [(PhCN)(2)PdCl(2)], affording higher yields under milder conditions. The reaction is regioselective and occurs with complete stereoselectivity using [(PhCN)(2)PdCl(2)] and with a small amount of racemization in the case of LiClO(4).

Inner Coordination Sphere Control of Metal-Metal Superexchange in Ruthenium Dimers.

The dinuclear Ru(III) complexes trans-[{(NH(3))(4)Ru(py)}(2)(&mgr;-L)][PF(6)](4), where py represents pyridine and L represents 1,4-dicyanamidobenzene dianion (dicyd(2)(-)) derivatives dicyd(2)(-) (1), Me(2)dicyd(2)(-) (2), Cl(2)dicyd(2)(-) (3), and Cl(4)dicyd(2)(-) (4), have been prepared and characterized by electronic absorption spectroscopy and cyclic voltammetry. A crystal structure of the complex trans-[{(NH(3))(4)Ru(py)}(2)(&mgr;-dicyd)][PF(6)](4).(1)/(2)H(2)O showed the dicyd(2)(-) ligand to be approximately planar with the cyanamido groups in a syn configuration. Crystal structure data are space group P2(1), with a, b, and c = 7.826(3), 20.455(7), and 14.428(5) Å, respectively, beta = 95.76 (3) degrees, V = 2296.7(14) Å(3), and Z = 2. The structure was refined by using 3292 reflections with I > 2.5sigma(I) to an R factor of 0.069. Solid state magnetic susceptibility measurements of the Ru(III)-Ru(III) dimers showed diamagnetic behavior at room temperature, and this is suggested to be due to strong antiferromagnetic superexchange via the HOMO of the dicyd(2)(-) ligand. The bridging ligand dependence of metal-metal coupling in the Ru(III)-Ru(II) complexes of 1, 2, 3, and 4 in acetonitrile solution was demonstrated by the trend in comproportionation constants, 1.5 x 10(6), 5.7 x 10(6), 1.4 x 10(4), and 1.1 x 10(3), respectively. In addition, comparison to the analogous pentaammineruthenium dimers showed that the magnitude of metal-metal superexchange could be controlled by the nature of the spectator ligand. Spectroelectrochemical methods were used to acquire the absorption spectra of the mixed-valence complexes, and the intervalence band properties were modeled with PKS theory. Metal-metal coupling in the Ru(III)-Ru(II) complexes of 1, 2, 3, and 4 was analyzed by using Hush and CNS theories.