Curr Cardiol Rep (2014) 16:466 DOI 10.1007/s11886-014-0466-7
NUCLEAR CARDIOLOGY (V DILSIZIAN, SECTION EDITOR)
Novel Molecular Angiotensin Converting Enzyme and Angiotensin Receptor Imaging Techniques Jamshid Shirani & Vasken Dilsizian
# Springer Science+Business Media New York 2014
Abstract Angiotensin II (AII), an octapeptide member of the renin-angiotensin system (RAS), is formed by the enzyme angiotensin converting enzyme (ACE) and exerts adverse cellular effects through an interaction with its type 1 receptor (AT1R). Both ACE inhibitors and angiotensin receptor blockers (ARB) mitigate the vasoconstrictive, proliferative, proinflammatory, proapoptotic, and profibrotic effects of AII and are widely used as effective anti-remodeling agents in clinical practice. Prediction of individual response to these agents, however, remains problematic and is influenced by many factors including race, gender, and genotype. In addition, systemic and tissue RAS activity do not correlate closely. This report summarizes the results of on-going attempts to noninvasively determine tissue ACE activity and AT1R expression using novel nuclear tracers. It is hoped that the availability of such imaging techniques improve treatment of heart failure through more selective pharmacologic intervention and better dose titration of available drugs.
Keywords Molecular imaging . Cardiovascular imaging . Angiotensin II . Angiotensin converting enzyme . Angiotensin receptors . Left ventricular remodeling . Left ventricular systolic dysfunction . Congestive heart failure . Angiotensin converting enzyme inhibitors . Angiotensin receptor blockers . Imaging
This article is part of the Topical Collection on Nuclear Cardiology J. Shirani (*) Department of Cardiology, St. Luke’s University Health Network, 801 Ostrum Street, Bethlehem, PA 18015, USA e-mail: [email protected] V. Dilsizian Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, MD, USA
Abbreviations ACE Angiotensin Converting Enzyme AII Angiotensin II ARB Angiotensin Receptor Blocker(s) AT1R Angiotensin II Type-1 Receptor CHF Congestive Heart Failure CT Computerized Tomography LV Left Ventricle (Ventricular) PET Positron Emission Tomography RAS Renin-Angiotensin System SPECT Single Photon Emission Computed Tomography
Introduction Activation of the systemic and particularly of the tissue components of the renin-angiotensin system (RAS) is a major contributor to the pathogenesis and progression of left ventricular (LV) remodeling and congestive heart failure (CHF) [1••, 2••]. Much of the adverse influence of RAS on LV remodeling is mediated through the interaction of the primary effector molecule of RAS, angiotensin II (AII), with its type-1 receptor (AT1R) at the cellular level [1••, 2••]. Pharmacologic interventions that reduce the production of AII (angiotensinconverting-enzyme [ACE] inhibitors) or inhibit its interaction with AT1R (angiotensin receptor blockers [ARB]) have been shown to ameliorate LV remodeling and improve outcome of patients with LV systolic dysfunction in randomized clinical trials [3••]. However, individual responses to these medications have varied widely and have been influenced by many factors including race [4], gender [5•], and genotype [6]. In addition, the presence of a complex array of receptors, byproducts of AII metabolism, and alternative pathways of AII production makes prediction of the AII activity in individual patient difficult. Inadequate RAS suppression is
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associated with adverse outcomes [7•] and while some patients may benefit from combined ACE inhibitor and ARB therapy such a regimen is associated with higher risk of side effects [8]. For all these reasons, accurate assessment of tissue ACE activity and AT1R expression is of utmost clinical interest and has stimulated a concerted effort for developing noninvasive imaging techniques that target these clinically relevant components of RAS (Fig. 1). This historical perspective reviews the milestones in development of novel ACE and AT1R imaging techniques.
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domains with different biologic functions. Binding of ACE to vascular endothelium is through its C terminal hydrophobic transmembrane region while ACE inhibitors bind to the extracellular binding sites of the enzyme. Serum ACE activity is in general measured by tripeptide substrates, most commonly the hippuryl-his-leu; however, nearly 90 % of ACE activity occurs within the tissue component that is not accessible to such assays. The study of the distribution and magnitude of ACE expression in various organs has been greatly facilitated by the development of radiolabeled ACE inhibitors (Table 1). Radiolabeled Captopril
Imaging ACE Molecular Structure and Function of ACE ACE (kininase II) is a zinc-dependent (2 zinc atoms per molecule) polypeptide dicarboxypeptidase that produces AII by cleaving the carbonyl-terminal histidyl-leucine of its precursor angiotensin I [9•]. AII is the primary effector peptide of RAAS that is responsible for the vasoconstrictive, profibrotic, proinflammatory, proapoptotic, and proliferative actions of RAS. ACE is highly expressed on vascular endothelium and is locally produced in many organs including the heart. Although alternative pathways (such as chymase) to AII production exist, ACE is responsible for more than 90 % of AII produced locally in the heart in the absence of ACE inhibitors. The enzyme contains 2 homologous extracellular catalytic
Fig. 1 Diagrammatic representation of myocardial cell and potential targets of radiotracer imaging and mapping of the surface renin-angiotensin system. ACE angiotensin-converting enzyme, AGT angiotensinogen, Ang II angiotensin II, AT1R angiotensin II type 1 receptor, AT2R angiotensin II type 2 receptor, mRNA messenger RNA. Reproduced with permission from: Schindler TH, Dilsizian V. Cardiac positron emission tomography/ computed tomography imaging of the renin-angiotensin system in humans holds promise for imageguided approach to heart failure therapy. J Am Coll Cardiol. 2012;5:1269–84 [48]
The first attempts at radiolabeling angiotensin-converting enzyme (ACE) inhibitors were made to study selective in vitro binding to membrane-bound ACE using liquid scintillation chromatography. Early studies using [3H]-captopril demonstrated that regional distribution of the radiotracer paralleled closely those of the ACE enzymatic activity in various tissues including the heart [10]. Further in vitro studies demonstrated that the high affinity ligand binding occurred atone [3H]captopril site per ACE molecule [11]. The radiotracer was then used to study the distribution and activity of vascular endothelial (pulmonary and mesenteric) and tissue ACE in various models of systemic hypertension in rats [12]. Importantly, it was shown that vascular and tissue enzyme activity did not always parallel each other [12]. The tracer was eventually abandoned because it was unstable and was found to
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Table 1 Molecular radiotracers that target the renin-angiotensin system Angiotensin converting enzyme
Angiotensin II type 1 receptor
18
125
F-fluorocaptopril 11 C-zofenoprilat 125 I-MK351A 125 I-iodotyrosyl-lisinopril 18 F-fluorobenzoyl-lisinopril 99m
Tc-(CO)3D(C8)-lisinopril
I-[Sar1, Ile8]AII 11 C-MK-996 11 C-L-159884 11 C-KR31173 99m Tc-losartan-Leu-DiglycoloylPEG(4)-Tetraamine 11 C-losartan 11 C-methyl-candesartan
attach substantially to sites other than ACE. The first positronlabelled ACE-inhibitor that was tested in vitro was the fluorine-18-fluorocaptopril (18FCAP) [13]. In vivo biodistribution was first assessed in rats and showed high uptakes in organs known to have high ACE activity such as lungs, kidneys, and the aorta [13]. It was also noted that the clearance of the radiotracer was faster for lungs and kidneys, compared with the aorta. Competitive inhibition of ACE by co-injecting unlabeled 4-cis-fluorocaptopril (SQ 25750) resulted in marked decrease in 18FCAP uptake in the lungs and the kidneys [13].18FCAP was also administered to a healthy human volunteer, and displaceable uptake was observed in the lungs and the kidneys. The results demonstrated the feasibility of probing ACE in vivo using PET. In another human study of healthy volunteers and 5 patients with pulmonary hypertension,18FCAP PET was able to safely and reproducibly measure total mass of pulmonary ACE and determine the efficacy of treatment with the ACE inhibitor enalapril [14]. These landmark proof-of-concept observations demonstrated that radiolabeled ACE inhibitors could be used in vivo (in rats and humans) to monitor ACE. However, 18FCAP had several shortcomings. Captopril contained a sulfhydryl group that tended to form captopril disulfide dimers or mixed dimers of captopril and endogenous sulfhydryl compounds (such as L-cysteine, glutathione, and methionine) producing high nonspecific binding [15]. Also, in vivo conformational changes resulted in cis and trans isomers of 18FCAP with potentially different kinetic rate constants [16]. Finally, captopril was shown to have a higher affinity for vascular compared with tissue ACE and thus, was thought to be less suited for examination of tissue ACE activity [17] compared with more tissue specific ACE inhibitors such as lisinopril. Radiolabeled Zofenoprilat Zofenopril is another sulfhydryl containing ACE inhibitor pro-drug that requires systemic conversion to its active metabolite, zofenoprilat. The drug is much more potent than captopril as an ACE inhibitor. A radioligand of zofenoprilat
was synthesized after labeling with 11C [18]. The positron emission tomographic tracer, [11C]-zofenoprilat was then tested in human volunteers and was shown to accumulate in organs known to have high ACE levels such as lungs and kidneys [18]. Activity was also noted in the heart. Radiolabeled MK351A The next group of radiolabeled ACE inhibitors belonged to the decarboxylase group (including lisinopril) that had no sulfhydryl moiety and had more affinity for tissue ACE. A tyrosyl derivative of enalaprilic acid, MK351A, was labeled with 125I and was used to measure serum ACE activity in patients with sarcoidosis with reliable and reproducible results compared with standard enzyme kinetic assay [19]. The technique was then used as a binding assay to measure ACE activity in tissue preparations from rats [20]. This was advantageous over the available enzyme kinetic assays that failed to assess tissue ACE activity and could allow establishing pharmacokinetic and pharmacodynamics profiles of ACE inhibitors [21]. Using quantitative in vitro autoradiography, detailed distribution of myocardial ACE was demonstrated [22] and an increased expression of myocardial ACE following acute myocardial infarction was shown in rats [23]. Also, the effects of vasopeptidase inhibitor omapatrilat on tissue ACE and neutral endopeptidase activity could be evaluated in rats using [125I]-MK351A and [125I]-RB104, a neutral endopeptidase inhibitor ligand [24, 25]. Vasopeptidase inhibitors simultaneously inhibit ACE and neutral endopeptidase, the major enzyme that degrades natriuretic peptides. Although [125I]MK351A was not used in any human study, it did establish itself as the standard radioligand for ACE affinity assays. Radiolabeled Lisinopril The next dicarboxylate ACE inhibitor used as a radioligand was radiolabeled lisinopril. It had several important advantages over 18FCAP, [11C]-zofenoprilat and [125I]-MK351A: (1) it was an ACE inhibitor in wide clinical use and with an established safety record for human use; (2) although a lysine analog of enalapril, unlike the parent drug did not require systemic conversion to an active metabolite for its pharmacologic effect; (3) exhibited relatively high affinity for tissue ACE with more predictable dissociation compared with lipophilic avid ligands such as quinapril and ramipril; and that the (4) binding of lisinopril to the active site of ACE was well characterized [26]. The higher affinity of lisinopril for tissue ACE was also felt to improve image resolution compared with captopril. Finally, a unique feature of lisinopril was that the molecule could be modified substantially during the labeling process without compromising its pharmacologic properties [27]. Thus, [18F]fluorobenzoyl-lisinopril (18FBL) was developed by investigators at the National Institutes of Health by
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radiolabeling benzoic acid active ester with 18F and reacting that with the epsilon-amino group of lisinopril for positron emission tomography [28]. The radioligand was then tested for determining the magnitude and distribution of tissue ACE in myocardial sections of explanted human hearts of patients with ischemic cardiomyopathy [29••]. In vitro autoradiography demonstrated excellent concordance between 18FBL uptake and immunohistochemical evidence of ACE distribution with the highest uptake in the periphery of the infarcted myocardial segments [29••]. The study provided direct evidence for specific binding of the radioligand to myocardial ACE. It was felt that high resolution and the highly specific binding of the radioligand to myocardial ACE could potentially be used to assess enzyme activity in vivo with the potential to improve risk to benefit ratio of RAS modulation, monitor drug efficacy, and titrate dosage in heart failure. One of the major potential limitations of 18FBL for use in human is the lack of wide spread availability of PET. Other imaging techniques, such as single-positron emission computed tomography (SPECT) or SPECT-computed tomography (CT) [SPECT-CT] may be more readily available and, thus, parallel efforts were made to also develop technetium (Tc)based radiotracers for use in conjunction with SPECT imaging [30]. One such tracer, 99mTc-Lisinopril, was studied for in vivo imaging of myocardial ACE activity in transgenic rat model [31••]. Micro SPECT-CT imaging was performed at 10, 30, 60, and 120 minutes after radiotracer injection. In vivo and ex-vivo specific binding of 99mTc-Lisinopril to myocardial ACE was verified with pre-treatment with unlabeled lisinopril, enzyme-linked immunosorbent assay for ACE activity and with ACE messenger ribonucleic acid expression (Fig. 2). Overall, at 120 minutes, 99mTc-Lisinopril myocardial uptake was 5-fold higher in transgenic rats compared with control animals. This study provided a new evidence for the potential role of imaging in studying pharmacokinetics and pharmacodynamics of ACE inhibition in the intact animal with a potential for human application [32].
Imaging the AT1R Molecular Structure and Function of AT1R AT1R is a 359 amino acid G protein-coupled receptor that is the principal mediator of the adverse cellular effects of AII [33]. The receptor is activated in the presence of AII by transmembrane displacement that allows binding of AII to specific extracellular binding sites. Receptor activation then leads to a cascade of intracellular events that culminates in production of growth factors and proinflammatory cytokines. AT1R has, thus, been the target of research that has led to the development of a highly effective class of anti-remodeling agents, the angiotensin-receptor blockers (ARB). Similar to
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ACE, characterization of the structure and function of AT1R has been highly facilitated by the development of selective radiolabeled ligands (Table 1). Radiolabeled AII The initial effort characterize AII binding sites included in vitro receptor binding assays using [125I]-AII [34]. The early studies demonstrated that AII had 2 distinct receptors (initially designated as A and B) that could be identified by competitive displacement of the radioligand by specific nonlabeled antagonists [35]. Both [3H]-AII and [125I]-AII have been used to study AII-AT1R interaction in vitro [36] and for identification of new potent antagonists of AT1R [37]. Radiolabeled ARB The discovery and synthesis of selective ARB and their perceived significance in treatment of systemic hypertension heightened the interest in the study of AT1R receptor. The significance of in vivo imaging of these receptors was recognized early and lead to the application of [Sar1, Ile8]AII, a peptide antagonist of AT1R, as a radiotracer for in vivo imaging of rat and Rhesus monkeys [38]. Although the experiment was successful in demonstrating high uptake of the radiotracer in the liver and the kidney, image resolution was poor, and the high uptake by the liver and subsequent biliary excretion obscured imaging of the kidneys [38]. In general, the short duration of action and the partial agonist properties of peptide antagonists were thought to be limiting to their use as effective radioligands. The introduction of the nonpeptide ARB presented an opportunity for development of new tracers for selective AT1R imaging. The first such radiotracer was the [11C]-MK-996 for PET imaging [39]. The radiotracer was effectively used in dogs but was abandoned in favor of its methoxy analog, L-159884, due to difficulties in synthesis of the former [40]. In vivo imaging demonstrated binding of the [11C]-L-159884 to AT1R in mouse kidneys, lungs, and the heart [41] as well as the renal cortex in a canine model [42]. Concerns, however, existed regarding the usefulness of [11C]L-159884 as a clinical imaging tool due to its rapid metabolism and potential for nonspecific binding. The search for optimal candidates for human studies led to the development of radiolabeled 2-Butyl-5-methoxymethyl-6-(1-oxopyridin-2-yl)3-[[2’-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo [4,5-b]pyridine {[11C] KR31173}from the potent AT1R antagonist SK-1080 [43]. The radiotracer was produced by coupling a tetrazole-protected hydroxyl precursor with [11C]methyl iodide and subsequently removing the protecting group by acid hydrolysis [43]. The presence and time course of regional myocardial upregulation of AT1R was then studied using in vivo micro-PET [11C]-KR31173 imaging in a rat model of myocardial ischemia-reperfusion [44]. Injection of
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Fig. 2 Noninvasive micro-SPECT/micro-CT Imaging of ACE-1 activity. Micro SPECT-CT imaging provides simultaneous scintigraphic and morphologic localization of Tc-Lis uptake, 60 minutes after tracer administration, in a control animal (left), ACE-1 overexpressing transgenic animal (middle) and a transgenic animal after coldlisinopril administration (right). White arrowhead demonstrates intense lung uptake and yellow arrows point to myocardial ACE-1 activity. White arrows suggest a substantial reduction in tracer uptake after
pretreatment with nonradiolabeled lisinopril administration. Despite the several-fold lower lung-to-heart ratio in the transgenic animals when compared with the control animals, the lung uptake may still be visualized, and more importantly may contribute spill over counts to the myocardium, which can be corrected for. Reproduced with permission from: Dilsizian V, et al. Molecular imaging of human ACE-1 expression in transgenic rats. J Am Coll Cardiol Img. 2012;5:409–418 [31••]
the radioligand following 20–25 minutes of coronary ligation resulted in detectable increase in uptake of the tracer in the infarct area. The specific binding of the tracer to AT1R was validated with ex vivo autoradiography and with immunohistochemical staining [44]. Comprehensive evaluation of this radiotracer has also shown the feasibility and suitability of this agent for in vivo imaging using PET and PET-CT in larger animals [45, 46]. It was shown that the uptake and retention of the radioligand correlated with the AT1R distribution (high in the adrenal gland, kidney, lung, and heart) with a high organ to
blood ratio [45]. Importantly, the myocardium was shown to demonstrate high specific binding (96 %), which appeared promising for the future application of this tracer in cardiac imaging [46]. The quality of PET images was also found to be excellent in the studied dogs [45] and pigs [46] with clear visualization of various organs including kidneys and the heart. In 2012, Fukushima et al presented the first-in-man application of receptor ligand [11C]-KR31173 combined with PET/ CT that confirmed the presence of local tissue RAS in human hearts, proved to be safe, and that the signal was high enough
Fig. 3 PET/CT of Myocardial AT1R in a Healthy human patient. Transaxial fusion images through the mid-cardiac region are shown. Baseline images show regionally homogeneous uptake of the angiotensin II type 1 receptor (AT1R) ligand [11C]-KR31173 in left ventricular myocardium (left). Repeat imaging 3 hours after an oral (p.o.) dose of 40 mg olmesartan for specific blocking of AT1R shows complete absence
of myocardial [11C]-KR31173 uptake, confirming tracer specificity for the receptor. Tracer is only present in the blood pool of atria and ventricles. Reproduced with permission from: Fukushima K, et al. Molecular hybrid positron emission tomography/computed tomography imaging of cardiac angiotensin II type 1 receptors. J Am Coll Cardiol. 2012:60:2527–34 [47••]
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to allow external imaging with PET (Fig. 3) [47••]. Myocardial retention of [11C]-KR31173 was visually detectable in 4 healthy volunteers, which was homogenously distributed in the myocardium and stable over time. However, myocardial retention of KR31173 in these healthy human subjects was significantly lower (nearly 4-fold) than those in normal healthy pigs, and only 54 % of the receptors were blocked after pretreatment with olmesartan (AT1R blocking agent), suggesting limited specificity [48]. Additional experiments performed in young farm pigs under healthy conditions and 3–4 weeks after myocardial infarction showed AT1R upregulation in the infarcted area when compared with remote myocardium [47••]. Moreover, the retention of KR31173 in infarcted and remote myocardium was significantly higher than in the myocardium of healthy pigs. In postmortem immunohistochemistry analysis, anti-AT1R antibody binding was localized to spindleshaped cells, presumably myofibroblasts, in the infarct region, while there was also significant binding to cardiomyocytes in remote areas [48]. These findings in pigs are similar to those observed in experimental mouse model of postinfarction heart failure where in vivo AT1R imaging was accomplished with Tc-99 m losartan micro SPECT-CT and immunohistochemical analysis showed binding of the radiotracer almost exclusively in the myofibroblast rather than cardiomyocytes [49]. Thus, while the [11C]-KR31173 imaging signal identifies AT1R expression in the myocardium, it does not differentiate between myofibroblast and cardiomyocyte cell types and with 54 % of the receptors blocked after pre-treatment with an AT-1 blocker, it has limited specificity [48]. Losartan, the first ARB that was used clinically, has also been radiolabeled and used in experimental settings. The chelate-coupled losartan-Leu-Diglycoloyl-PEG(4)-tetraamine, with higher affinity for AT1R compared with the parent drug, was radiolabeled with 99mTc and used for cardiac imaging in a mouse model of acute myocardial infarction [49]. In vivo micro SPECT-CT showed an increase uptake within the infarct region 3 weeks after coronary ligation. The specific binding of the tracer to AT1R receptor was verified with immunohistochemical studies. The potential use of 99mTc-losartan in clinical studies is encouraged by the fact that the radiotracer can be synthesized rapidly and effectively at a high radiochemical yield and high uptake in the heart when studied in the mouse [50]. Radiolabeling of losartan for PET imaging ([11C]losartan) has also been successfully done at high radiochemical purity [51]. Finally, candesartan, a more lipophilic ARB than the prototype drug losartan, has been successfully labeled with [11C] as a potential PET tracer and is shown to specifically and competitively bind to AT1R [52]. The radiotracer was originally made with the hope that the high lipophilicity would allow the agent to cross the blood brain barrier and provide a means for imaging intracerebral AT1R. However, in practice, it did not cross into the brain in sufficient amount to allow
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detection of the receptors in the brain. Preliminary evaluation of this PET radioligand ([11C]-methyl-candesartan) has shown promising results for in vivo detection of changes in AT1R receptor density in the rat kidney [53].
Conclusions and Future Directions RAS is a system of ever-increasing complexity with numerous biologically active components often with opposing cellular effects [54••]. In clinical management of patients with left ventricular systolic dysfunction, however, the activities of several components of the system appear to have the most profound effects on systemic and tissue response to injury and persistent abnormal stimuli [55•]. Among these, AII and aldosterone appear to be the most influential and have been the primary targets of established and novel pharmacologic interventions aimed at amelioration of adverse cardiac remodeling [55•, 56, 57]. RAS blockade in heart failure, however, remains challenging due to the absence of reliable clinical markers of adequate target inhibition and potential for serious side effects that may limit clinical benefits and affect outcomes [58]. Dose titration, dual RAS blockade, alternative therapy, and identification of reactivation of the system through alternative pathways are some of the important clinical questions that are largely unanswered at this time [59]. Molecular imaging is well poised to provide important insights into these clinically relevant questions through application of advanced imaging techniques and sophisticated molecular probes [60, 61]. Combined assessment and serial evaluation of myocardial ACE activity and AT1R holds great promise for the most efficacious modulation of RAS in patients with left ventricular systolic function. Compliance with Ethics Guidelines Conflict of Interest Jamshid Shirani declares that he has no conflict of interest. Vasken Dilsizian declares that he has no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.
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NUCLEAR CARDIOLOGY (V DILSIZIAN, SECTION EDITOR)
Novel Molecular Angiotensin Converting Enzyme and Angiotensin Receptor Imaging Techniques Jamshid Shirani & Vasken Dilsizian
# Springer Science+Business Media New York 2014
Abstract Angiotensin II (AII), an octapeptide member of the renin-angiotensin system (RAS), is formed by the enzyme angiotensin converting enzyme (ACE) and exerts adverse cellular effects through an interaction with its type 1 receptor (AT1R). Both ACE inhibitors and angiotensin receptor blockers (ARB) mitigate the vasoconstrictive, proliferative, proinflammatory, proapoptotic, and profibrotic effects of AII and are widely used as effective anti-remodeling agents in clinical practice. Prediction of individual response to these agents, however, remains problematic and is influenced by many factors including race, gender, and genotype. In addition, systemic and tissue RAS activity do not correlate closely. This report summarizes the results of on-going attempts to noninvasively determine tissue ACE activity and AT1R expression using novel nuclear tracers. It is hoped that the availability of such imaging techniques improve treatment of heart failure through more selective pharmacologic intervention and better dose titration of available drugs.
Keywords Molecular imaging . Cardiovascular imaging . Angiotensin II . Angiotensin converting enzyme . Angiotensin receptors . Left ventricular remodeling . Left ventricular systolic dysfunction . Congestive heart failure . Angiotensin converting enzyme inhibitors . Angiotensin receptor blockers . Imaging
This article is part of the Topical Collection on Nuclear Cardiology J. Shirani (*) Department of Cardiology, St. Luke’s University Health Network, 801 Ostrum Street, Bethlehem, PA 18015, USA e-mail: [email protected] V. Dilsizian Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, MD, USA
Abbreviations ACE Angiotensin Converting Enzyme AII Angiotensin II ARB Angiotensin Receptor Blocker(s) AT1R Angiotensin II Type-1 Receptor CHF Congestive Heart Failure CT Computerized Tomography LV Left Ventricle (Ventricular) PET Positron Emission Tomography RAS Renin-Angiotensin System SPECT Single Photon Emission Computed Tomography
Introduction Activation of the systemic and particularly of the tissue components of the renin-angiotensin system (RAS) is a major contributor to the pathogenesis and progression of left ventricular (LV) remodeling and congestive heart failure (CHF) [1••, 2••]. Much of the adverse influence of RAS on LV remodeling is mediated through the interaction of the primary effector molecule of RAS, angiotensin II (AII), with its type-1 receptor (AT1R) at the cellular level [1••, 2••]. Pharmacologic interventions that reduce the production of AII (angiotensinconverting-enzyme [ACE] inhibitors) or inhibit its interaction with AT1R (angiotensin receptor blockers [ARB]) have been shown to ameliorate LV remodeling and improve outcome of patients with LV systolic dysfunction in randomized clinical trials [3••]. However, individual responses to these medications have varied widely and have been influenced by many factors including race [4], gender [5•], and genotype [6]. In addition, the presence of a complex array of receptors, byproducts of AII metabolism, and alternative pathways of AII production makes prediction of the AII activity in individual patient difficult. Inadequate RAS suppression is
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associated with adverse outcomes [7•] and while some patients may benefit from combined ACE inhibitor and ARB therapy such a regimen is associated with higher risk of side effects [8]. For all these reasons, accurate assessment of tissue ACE activity and AT1R expression is of utmost clinical interest and has stimulated a concerted effort for developing noninvasive imaging techniques that target these clinically relevant components of RAS (Fig. 1). This historical perspective reviews the milestones in development of novel ACE and AT1R imaging techniques.
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domains with different biologic functions. Binding of ACE to vascular endothelium is through its C terminal hydrophobic transmembrane region while ACE inhibitors bind to the extracellular binding sites of the enzyme. Serum ACE activity is in general measured by tripeptide substrates, most commonly the hippuryl-his-leu; however, nearly 90 % of ACE activity occurs within the tissue component that is not accessible to such assays. The study of the distribution and magnitude of ACE expression in various organs has been greatly facilitated by the development of radiolabeled ACE inhibitors (Table 1). Radiolabeled Captopril
Imaging ACE Molecular Structure and Function of ACE ACE (kininase II) is a zinc-dependent (2 zinc atoms per molecule) polypeptide dicarboxypeptidase that produces AII by cleaving the carbonyl-terminal histidyl-leucine of its precursor angiotensin I [9•]. AII is the primary effector peptide of RAAS that is responsible for the vasoconstrictive, profibrotic, proinflammatory, proapoptotic, and proliferative actions of RAS. ACE is highly expressed on vascular endothelium and is locally produced in many organs including the heart. Although alternative pathways (such as chymase) to AII production exist, ACE is responsible for more than 90 % of AII produced locally in the heart in the absence of ACE inhibitors. The enzyme contains 2 homologous extracellular catalytic
Fig. 1 Diagrammatic representation of myocardial cell and potential targets of radiotracer imaging and mapping of the surface renin-angiotensin system. ACE angiotensin-converting enzyme, AGT angiotensinogen, Ang II angiotensin II, AT1R angiotensin II type 1 receptor, AT2R angiotensin II type 2 receptor, mRNA messenger RNA. Reproduced with permission from: Schindler TH, Dilsizian V. Cardiac positron emission tomography/ computed tomography imaging of the renin-angiotensin system in humans holds promise for imageguided approach to heart failure therapy. J Am Coll Cardiol. 2012;5:1269–84 [48]
The first attempts at radiolabeling angiotensin-converting enzyme (ACE) inhibitors were made to study selective in vitro binding to membrane-bound ACE using liquid scintillation chromatography. Early studies using [3H]-captopril demonstrated that regional distribution of the radiotracer paralleled closely those of the ACE enzymatic activity in various tissues including the heart [10]. Further in vitro studies demonstrated that the high affinity ligand binding occurred atone [3H]captopril site per ACE molecule [11]. The radiotracer was then used to study the distribution and activity of vascular endothelial (pulmonary and mesenteric) and tissue ACE in various models of systemic hypertension in rats [12]. Importantly, it was shown that vascular and tissue enzyme activity did not always parallel each other [12]. The tracer was eventually abandoned because it was unstable and was found to
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Table 1 Molecular radiotracers that target the renin-angiotensin system Angiotensin converting enzyme
Angiotensin II type 1 receptor
18
125
F-fluorocaptopril 11 C-zofenoprilat 125 I-MK351A 125 I-iodotyrosyl-lisinopril 18 F-fluorobenzoyl-lisinopril 99m
Tc-(CO)3D(C8)-lisinopril
I-[Sar1, Ile8]AII 11 C-MK-996 11 C-L-159884 11 C-KR31173 99m Tc-losartan-Leu-DiglycoloylPEG(4)-Tetraamine 11 C-losartan 11 C-methyl-candesartan
attach substantially to sites other than ACE. The first positronlabelled ACE-inhibitor that was tested in vitro was the fluorine-18-fluorocaptopril (18FCAP) [13]. In vivo biodistribution was first assessed in rats and showed high uptakes in organs known to have high ACE activity such as lungs, kidneys, and the aorta [13]. It was also noted that the clearance of the radiotracer was faster for lungs and kidneys, compared with the aorta. Competitive inhibition of ACE by co-injecting unlabeled 4-cis-fluorocaptopril (SQ 25750) resulted in marked decrease in 18FCAP uptake in the lungs and the kidneys [13].18FCAP was also administered to a healthy human volunteer, and displaceable uptake was observed in the lungs and the kidneys. The results demonstrated the feasibility of probing ACE in vivo using PET. In another human study of healthy volunteers and 5 patients with pulmonary hypertension,18FCAP PET was able to safely and reproducibly measure total mass of pulmonary ACE and determine the efficacy of treatment with the ACE inhibitor enalapril [14]. These landmark proof-of-concept observations demonstrated that radiolabeled ACE inhibitors could be used in vivo (in rats and humans) to monitor ACE. However, 18FCAP had several shortcomings. Captopril contained a sulfhydryl group that tended to form captopril disulfide dimers or mixed dimers of captopril and endogenous sulfhydryl compounds (such as L-cysteine, glutathione, and methionine) producing high nonspecific binding [15]. Also, in vivo conformational changes resulted in cis and trans isomers of 18FCAP with potentially different kinetic rate constants [16]. Finally, captopril was shown to have a higher affinity for vascular compared with tissue ACE and thus, was thought to be less suited for examination of tissue ACE activity [17] compared with more tissue specific ACE inhibitors such as lisinopril. Radiolabeled Zofenoprilat Zofenopril is another sulfhydryl containing ACE inhibitor pro-drug that requires systemic conversion to its active metabolite, zofenoprilat. The drug is much more potent than captopril as an ACE inhibitor. A radioligand of zofenoprilat
was synthesized after labeling with 11C [18]. The positron emission tomographic tracer, [11C]-zofenoprilat was then tested in human volunteers and was shown to accumulate in organs known to have high ACE levels such as lungs and kidneys [18]. Activity was also noted in the heart. Radiolabeled MK351A The next group of radiolabeled ACE inhibitors belonged to the decarboxylase group (including lisinopril) that had no sulfhydryl moiety and had more affinity for tissue ACE. A tyrosyl derivative of enalaprilic acid, MK351A, was labeled with 125I and was used to measure serum ACE activity in patients with sarcoidosis with reliable and reproducible results compared with standard enzyme kinetic assay [19]. The technique was then used as a binding assay to measure ACE activity in tissue preparations from rats [20]. This was advantageous over the available enzyme kinetic assays that failed to assess tissue ACE activity and could allow establishing pharmacokinetic and pharmacodynamics profiles of ACE inhibitors [21]. Using quantitative in vitro autoradiography, detailed distribution of myocardial ACE was demonstrated [22] and an increased expression of myocardial ACE following acute myocardial infarction was shown in rats [23]. Also, the effects of vasopeptidase inhibitor omapatrilat on tissue ACE and neutral endopeptidase activity could be evaluated in rats using [125I]-MK351A and [125I]-RB104, a neutral endopeptidase inhibitor ligand [24, 25]. Vasopeptidase inhibitors simultaneously inhibit ACE and neutral endopeptidase, the major enzyme that degrades natriuretic peptides. Although [125I]MK351A was not used in any human study, it did establish itself as the standard radioligand for ACE affinity assays. Radiolabeled Lisinopril The next dicarboxylate ACE inhibitor used as a radioligand was radiolabeled lisinopril. It had several important advantages over 18FCAP, [11C]-zofenoprilat and [125I]-MK351A: (1) it was an ACE inhibitor in wide clinical use and with an established safety record for human use; (2) although a lysine analog of enalapril, unlike the parent drug did not require systemic conversion to an active metabolite for its pharmacologic effect; (3) exhibited relatively high affinity for tissue ACE with more predictable dissociation compared with lipophilic avid ligands such as quinapril and ramipril; and that the (4) binding of lisinopril to the active site of ACE was well characterized [26]. The higher affinity of lisinopril for tissue ACE was also felt to improve image resolution compared with captopril. Finally, a unique feature of lisinopril was that the molecule could be modified substantially during the labeling process without compromising its pharmacologic properties [27]. Thus, [18F]fluorobenzoyl-lisinopril (18FBL) was developed by investigators at the National Institutes of Health by
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radiolabeling benzoic acid active ester with 18F and reacting that with the epsilon-amino group of lisinopril for positron emission tomography [28]. The radioligand was then tested for determining the magnitude and distribution of tissue ACE in myocardial sections of explanted human hearts of patients with ischemic cardiomyopathy [29••]. In vitro autoradiography demonstrated excellent concordance between 18FBL uptake and immunohistochemical evidence of ACE distribution with the highest uptake in the periphery of the infarcted myocardial segments [29••]. The study provided direct evidence for specific binding of the radioligand to myocardial ACE. It was felt that high resolution and the highly specific binding of the radioligand to myocardial ACE could potentially be used to assess enzyme activity in vivo with the potential to improve risk to benefit ratio of RAS modulation, monitor drug efficacy, and titrate dosage in heart failure. One of the major potential limitations of 18FBL for use in human is the lack of wide spread availability of PET. Other imaging techniques, such as single-positron emission computed tomography (SPECT) or SPECT-computed tomography (CT) [SPECT-CT] may be more readily available and, thus, parallel efforts were made to also develop technetium (Tc)based radiotracers for use in conjunction with SPECT imaging [30]. One such tracer, 99mTc-Lisinopril, was studied for in vivo imaging of myocardial ACE activity in transgenic rat model [31••]. Micro SPECT-CT imaging was performed at 10, 30, 60, and 120 minutes after radiotracer injection. In vivo and ex-vivo specific binding of 99mTc-Lisinopril to myocardial ACE was verified with pre-treatment with unlabeled lisinopril, enzyme-linked immunosorbent assay for ACE activity and with ACE messenger ribonucleic acid expression (Fig. 2). Overall, at 120 minutes, 99mTc-Lisinopril myocardial uptake was 5-fold higher in transgenic rats compared with control animals. This study provided a new evidence for the potential role of imaging in studying pharmacokinetics and pharmacodynamics of ACE inhibition in the intact animal with a potential for human application [32].
Imaging the AT1R Molecular Structure and Function of AT1R AT1R is a 359 amino acid G protein-coupled receptor that is the principal mediator of the adverse cellular effects of AII [33]. The receptor is activated in the presence of AII by transmembrane displacement that allows binding of AII to specific extracellular binding sites. Receptor activation then leads to a cascade of intracellular events that culminates in production of growth factors and proinflammatory cytokines. AT1R has, thus, been the target of research that has led to the development of a highly effective class of anti-remodeling agents, the angiotensin-receptor blockers (ARB). Similar to
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ACE, characterization of the structure and function of AT1R has been highly facilitated by the development of selective radiolabeled ligands (Table 1). Radiolabeled AII The initial effort characterize AII binding sites included in vitro receptor binding assays using [125I]-AII [34]. The early studies demonstrated that AII had 2 distinct receptors (initially designated as A and B) that could be identified by competitive displacement of the radioligand by specific nonlabeled antagonists [35]. Both [3H]-AII and [125I]-AII have been used to study AII-AT1R interaction in vitro [36] and for identification of new potent antagonists of AT1R [37]. Radiolabeled ARB The discovery and synthesis of selective ARB and their perceived significance in treatment of systemic hypertension heightened the interest in the study of AT1R receptor. The significance of in vivo imaging of these receptors was recognized early and lead to the application of [Sar1, Ile8]AII, a peptide antagonist of AT1R, as a radiotracer for in vivo imaging of rat and Rhesus monkeys [38]. Although the experiment was successful in demonstrating high uptake of the radiotracer in the liver and the kidney, image resolution was poor, and the high uptake by the liver and subsequent biliary excretion obscured imaging of the kidneys [38]. In general, the short duration of action and the partial agonist properties of peptide antagonists were thought to be limiting to their use as effective radioligands. The introduction of the nonpeptide ARB presented an opportunity for development of new tracers for selective AT1R imaging. The first such radiotracer was the [11C]-MK-996 for PET imaging [39]. The radiotracer was effectively used in dogs but was abandoned in favor of its methoxy analog, L-159884, due to difficulties in synthesis of the former [40]. In vivo imaging demonstrated binding of the [11C]-L-159884 to AT1R in mouse kidneys, lungs, and the heart [41] as well as the renal cortex in a canine model [42]. Concerns, however, existed regarding the usefulness of [11C]L-159884 as a clinical imaging tool due to its rapid metabolism and potential for nonspecific binding. The search for optimal candidates for human studies led to the development of radiolabeled 2-Butyl-5-methoxymethyl-6-(1-oxopyridin-2-yl)3-[[2’-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo [4,5-b]pyridine {[11C] KR31173}from the potent AT1R antagonist SK-1080 [43]. The radiotracer was produced by coupling a tetrazole-protected hydroxyl precursor with [11C]methyl iodide and subsequently removing the protecting group by acid hydrolysis [43]. The presence and time course of regional myocardial upregulation of AT1R was then studied using in vivo micro-PET [11C]-KR31173 imaging in a rat model of myocardial ischemia-reperfusion [44]. Injection of
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Fig. 2 Noninvasive micro-SPECT/micro-CT Imaging of ACE-1 activity. Micro SPECT-CT imaging provides simultaneous scintigraphic and morphologic localization of Tc-Lis uptake, 60 minutes after tracer administration, in a control animal (left), ACE-1 overexpressing transgenic animal (middle) and a transgenic animal after coldlisinopril administration (right). White arrowhead demonstrates intense lung uptake and yellow arrows point to myocardial ACE-1 activity. White arrows suggest a substantial reduction in tracer uptake after
pretreatment with nonradiolabeled lisinopril administration. Despite the several-fold lower lung-to-heart ratio in the transgenic animals when compared with the control animals, the lung uptake may still be visualized, and more importantly may contribute spill over counts to the myocardium, which can be corrected for. Reproduced with permission from: Dilsizian V, et al. Molecular imaging of human ACE-1 expression in transgenic rats. J Am Coll Cardiol Img. 2012;5:409–418 [31••]
the radioligand following 20–25 minutes of coronary ligation resulted in detectable increase in uptake of the tracer in the infarct area. The specific binding of the tracer to AT1R was validated with ex vivo autoradiography and with immunohistochemical staining [44]. Comprehensive evaluation of this radiotracer has also shown the feasibility and suitability of this agent for in vivo imaging using PET and PET-CT in larger animals [45, 46]. It was shown that the uptake and retention of the radioligand correlated with the AT1R distribution (high in the adrenal gland, kidney, lung, and heart) with a high organ to
blood ratio [45]. Importantly, the myocardium was shown to demonstrate high specific binding (96 %), which appeared promising for the future application of this tracer in cardiac imaging [46]. The quality of PET images was also found to be excellent in the studied dogs [45] and pigs [46] with clear visualization of various organs including kidneys and the heart. In 2012, Fukushima et al presented the first-in-man application of receptor ligand [11C]-KR31173 combined with PET/ CT that confirmed the presence of local tissue RAS in human hearts, proved to be safe, and that the signal was high enough
Fig. 3 PET/CT of Myocardial AT1R in a Healthy human patient. Transaxial fusion images through the mid-cardiac region are shown. Baseline images show regionally homogeneous uptake of the angiotensin II type 1 receptor (AT1R) ligand [11C]-KR31173 in left ventricular myocardium (left). Repeat imaging 3 hours after an oral (p.o.) dose of 40 mg olmesartan for specific blocking of AT1R shows complete absence
of myocardial [11C]-KR31173 uptake, confirming tracer specificity for the receptor. Tracer is only present in the blood pool of atria and ventricles. Reproduced with permission from: Fukushima K, et al. Molecular hybrid positron emission tomography/computed tomography imaging of cardiac angiotensin II type 1 receptors. J Am Coll Cardiol. 2012:60:2527–34 [47••]
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to allow external imaging with PET (Fig. 3) [47••]. Myocardial retention of [11C]-KR31173 was visually detectable in 4 healthy volunteers, which was homogenously distributed in the myocardium and stable over time. However, myocardial retention of KR31173 in these healthy human subjects was significantly lower (nearly 4-fold) than those in normal healthy pigs, and only 54 % of the receptors were blocked after pretreatment with olmesartan (AT1R blocking agent), suggesting limited specificity [48]. Additional experiments performed in young farm pigs under healthy conditions and 3–4 weeks after myocardial infarction showed AT1R upregulation in the infarcted area when compared with remote myocardium [47••]. Moreover, the retention of KR31173 in infarcted and remote myocardium was significantly higher than in the myocardium of healthy pigs. In postmortem immunohistochemistry analysis, anti-AT1R antibody binding was localized to spindleshaped cells, presumably myofibroblasts, in the infarct region, while there was also significant binding to cardiomyocytes in remote areas [48]. These findings in pigs are similar to those observed in experimental mouse model of postinfarction heart failure where in vivo AT1R imaging was accomplished with Tc-99 m losartan micro SPECT-CT and immunohistochemical analysis showed binding of the radiotracer almost exclusively in the myofibroblast rather than cardiomyocytes [49]. Thus, while the [11C]-KR31173 imaging signal identifies AT1R expression in the myocardium, it does not differentiate between myofibroblast and cardiomyocyte cell types and with 54 % of the receptors blocked after pre-treatment with an AT-1 blocker, it has limited specificity [48]. Losartan, the first ARB that was used clinically, has also been radiolabeled and used in experimental settings. The chelate-coupled losartan-Leu-Diglycoloyl-PEG(4)-tetraamine, with higher affinity for AT1R compared with the parent drug, was radiolabeled with 99mTc and used for cardiac imaging in a mouse model of acute myocardial infarction [49]. In vivo micro SPECT-CT showed an increase uptake within the infarct region 3 weeks after coronary ligation. The specific binding of the tracer to AT1R receptor was verified with immunohistochemical studies. The potential use of 99mTc-losartan in clinical studies is encouraged by the fact that the radiotracer can be synthesized rapidly and effectively at a high radiochemical yield and high uptake in the heart when studied in the mouse [50]. Radiolabeling of losartan for PET imaging ([11C]losartan) has also been successfully done at high radiochemical purity [51]. Finally, candesartan, a more lipophilic ARB than the prototype drug losartan, has been successfully labeled with [11C] as a potential PET tracer and is shown to specifically and competitively bind to AT1R [52]. The radiotracer was originally made with the hope that the high lipophilicity would allow the agent to cross the blood brain barrier and provide a means for imaging intracerebral AT1R. However, in practice, it did not cross into the brain in sufficient amount to allow
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detection of the receptors in the brain. Preliminary evaluation of this PET radioligand ([11C]-methyl-candesartan) has shown promising results for in vivo detection of changes in AT1R receptor density in the rat kidney [53].
Conclusions and Future Directions RAS is a system of ever-increasing complexity with numerous biologically active components often with opposing cellular effects [54••]. In clinical management of patients with left ventricular systolic dysfunction, however, the activities of several components of the system appear to have the most profound effects on systemic and tissue response to injury and persistent abnormal stimuli [55•]. Among these, AII and aldosterone appear to be the most influential and have been the primary targets of established and novel pharmacologic interventions aimed at amelioration of adverse cardiac remodeling [55•, 56, 57]. RAS blockade in heart failure, however, remains challenging due to the absence of reliable clinical markers of adequate target inhibition and potential for serious side effects that may limit clinical benefits and affect outcomes [58]. Dose titration, dual RAS blockade, alternative therapy, and identification of reactivation of the system through alternative pathways are some of the important clinical questions that are largely unanswered at this time [59]. Molecular imaging is well poised to provide important insights into these clinically relevant questions through application of advanced imaging techniques and sophisticated molecular probes [60, 61]. Combined assessment and serial evaluation of myocardial ACE activity and AT1R holds great promise for the most efficacious modulation of RAS in patients with left ventricular systolic function. Compliance with Ethics Guidelines Conflict of Interest Jamshid Shirani declares that he has no conflict of interest. Vasken Dilsizian declares that he has no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.
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