The renin–angiotensin system (RAS) plays a very important role in the regulation of blood pressure in humans (1-4). Renin is an enzyme produced in the kidneys, and it cleaves circulating angiotensinogen (a protein produced in the liver) to yield angiotensin I, an inactive decapeptide. The angiotensin-converting enzyme (ACE), found primarily in the lung, converts angiotensin I to angiotensin II, an active vasoconstrictor octapeptide. Angiotensin II also stimulates the production of aldosterone from the adrenal glands, which promotes retention of sodium and water. ACE is also responsible for inactivating bradykinin, a vasodilator (5). RAS and angiotensin II also play a role in interstitial fibrosis, cardiac remodeling and fibrosis, and heart failure (6, 7). It has been shown that RAS operates in the heart, and an upregulation of this system is correlated to heart failure (8-10). Inhibition of ACE in patients with heart failure has often resulted in a favorable outcome for the patient (11), although individuals treated with ACE inhibitors have been reported to respond differently as a result of genetic variations in the enzyme; a meta-analysis of data obtained from children of different races who had been treated with these inhibitors has shown the children reacted differently to the treatment (12, 13). In addition, the proper timing, optimal dosing, and monitoring of ACE inhibitor therapy for the individualized treatment of patients has not been fully worked out. Also, ACE appears to have a role in several other pathological conditions including myeloid leukemia, atherosclerosis, and diabetes (in the kidneys of patients) because an increased level of either the protein or its mRNA has been reported under these conditions (14-16).
In a preliminary radioautographic study, Dilsizian et al. showed that ACE expression correlated with the extent of the disease in patients with congestive heart failure and suggested that monitoring ACE expression may help monitor molecular remodeling of the heart (3). On the basis of this observation, Femia et al. envisioned that a radiotracer designed for imaging the RAS would be helpful for the development of individualized ACE inhibitor therapy (17). In this regard, a compound with high affinity for ACE, such as lisinopril or its derivative, would be best suited to study the expression of a radiotracer. Several derivatives of lisinopril that can incorporate rhodium or palladium while maintaining high affinity for ACE were reported and could probably be used to synthesize radiolabeled analogs of the compound (18). Using a single amino acid chelator technology based on the di(pyridylmethyl)amine chelator, Femia et al. synthesized a technetium-carbonyl (Tc(CO)3) core derivative of lisinopril and evaluated for the in vivo imaging of ACE expression in rats (17).
Several lisinopril derivatives were synthesized as described by Femia et al. (17). Various, commercially available, ε-amino acids were converted to their di(pyridylmethylamino)alkanoic (D) acids through reductive amination using standard procedures (19). The ε-amino acid derivatives were converted to their lisinopril analogs as detailed by Femia et al. (17). The final products respectively contained 3 to 7 methylene units in the alkyl chain to form the D(C4)lisinopril, D(C5)lisinopril, or D(C8)lisinopril compounds. The various lisinopril derivatives (D(CX)L) had yields from 34% to 40% of the starting materials and were respectively characterized using nuclear magnetic resonance and by electrospray mass spectroscopy (17).
99mTc-(CO)3D(C8)lisinopril was prepared with 99mTc-(CO)3(H2O)3 using a commercially available kit as described by Femia et al. (17). Sodium [99mTc]pertechnetate in saline was added to the radiolabeling kit, and the vial was heated in an oil bath at 100°C for 45 min. The reaction was stopped by the addition of 1 N HCl, and the product was added to another vial containing D(C8)lisinopril in methanol. The mixture was heated at 80°C for 1 h, and the product was purified on a C18 Sep-Pak plus cartridge activated with ethanol and washed with water. The loaded column was washed with water to remove all unreacted 99mTc (which could include Tc(CO)3+ and TcO4-). The labeled compound was eluted from the cartridge with ethanol, and the solvent was removed under a stream of nitrogen. The radiochemical was subsequently reconstituted in 10% ethanol/saline. The radiochemical purity of the 99mTc product was reported to be at least 98% with a yield of at least 88% as determined with high-performance liquid chromatography (HPLC). The specific activity of labeled lisinopril was reported to be 38,000 MBq/μm0l (1,027 mCi/μmol). The stability of the radiotracer was not reported (17).
Various non-radioactive rhenium (Re) complexes of D(Cx)lisinopril were also synthesized to determine the in vitro activity of the compounds (17). For this, D(Cx)lisinopril dissolved in methanol was mixed with [Re(CO)3(H2O)3]Br and heated at 80°C for 4 h. The solvent was then removed, and the product was purified with HPLC using a Dynamax C-18 reversed-phase column. The structure of the final product was determined with nuclear magnetic resonance and electrospray mass spectroscopy. The reaction yield of the Re-lisinopril complexes were not reported (17).
In Vitro Studies: Testing in Cells and Tissues
The Re complexes of lisinopril containing the different methylene spacer units (C4, C5, or C8), as Re(CO)3D(Cx)lisinopril, were evaluated in vitro against rabbit lung ACE (17). The activity of these compounds was reported to vary directly with the number of methylene spacers in the complex. The 50% inhibitory concentration (IC50) of Re(CO)3D(C8)lisinopril was 3 nM, followed by 144 nM for Re(CO)3D(C5)lisinopril and 1,146 nM for Re(CO)3D(C4)lisinopril. Similar IC50 values were obtained for the respective free D(Cx)lisinopril compounds. The IC50 for the Re(CO)3D(C8)lisinopril analog was similar to the parent lisinopril (IC50 = 4 nM) compound (17).
The biodistribution of 99mTc-(CO)3D(C8)lisinopril was studied in Sprague-Dawley rats after an intravenous injection of the radiochemical (17). To demonstrate specificity of the radiotracer, some animals were injected with nonradioactive lisinopril 5 min before the administration of 99mTc-(CO)3D(C8)lisinopril. The animals were euthanized by asphyxiation with carbon dioxide at 10, 30, 60, and 120 min after the injection (n = 5 animals per time point). Varying levels of the radiolabel were detected in the different tissue, and the levels decreased over time. The highest uptake was noticed in the lungs, which had 15.2% of the injected dose/g tissue (% ID/g) at 10 min and 3.39% ID/g remaining at 2 h; the lungs exhibit high ACE expression (17). Clearance of the radioactivity was mainly through the hepatobiliary route because an accumulation of the label in the intestine was reported to increase over time. Pretreatment of the animals with nonradioactive lisinopril before the administration of 99mTc-(CO)3D(C8)lisinopril resulted in a markedly reduced (0.17% ID/g) accumulation of the label in the lungs, indicating that the label was highly specific for the targeted ACE (17).
Whole-body imaging was performed with 99mTc-(CO)3D(C8)lisinopril in animals with or without pretreatment of nonradioactive lisinopril (n = 3 animals per group) (17). Images were obtained starting at 5 min after administration of the radioactivity for up to 60 min. The regions of interest were drawn over the lungs, liver, intestines, and the soft tissue (for background), and the regions of interest were quantified by counts after normalization to the background at the same time point. Quantitation of the radioactivity from the images reflected the same trends as obtained during the biodistribution study with uptake of the label primarily in the lungs and the intestines. Pretreatment of the animals with nonradioactive lisinopril was reported to block accumulation of radioactivity in the lungs.
Other Non-Primate Mammals
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Some studies reported in this chapter were supported by NIH, National Heart, Lung and Blood Institutes grant 1R43HL075918-01.
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Created: June 25, 2008; Last Update: July 29, 2008.
National Center for Biotechnology Information (US), Bethesda (MD)
Chopra A. Tricarbonyl[99mTc]technetium(I)-((S)-6-(8-(bis(pyridin-2-ylmethyl)amino)octanamido)-2-((S)-1-carboxy-3-phenylpropylamino)hexanoyl)pyrrolidine-2-carboxylic acid. 2008 Jun 25 [Updated 2008 Jul 29]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.