Kinetics of the norepinephrine analog [76Br]-metabromobenzylguanidine in isolated working rat heart

doi:10.1016/S0969-8051(97)00141-8

Nuclear Medicine and Biology

Volume 25, Issue 1, January 1998, Pages 1-16

https://doi.org/10.1016/S0969-8051(97)00141-8 Get rights and content

Abstract

A related set of kinetic studies of the norepinephrine analog [⁷⁶Br]-meta-bromobenzylguanidine (MBBG) were performed with an isolated working rat heart preparation. A series of constant infusion studies over a wide range of MBBG concentrations allowed estimation of the Michaelis-Menten constants for transport by the neuronal norepinephrine transporter (uptake₁) and the extraneuronal uptake system (uptake₂). Pharmacological blocking studies with inhibitors of uptake₁, uptake₂ and vesicular uptake were performed to delineate the relative importance of these norepinephrine handling mechanisms on the kinetics of MBBG in the rat heart. Bolus injection studies were done to assess the ability of compartmental modeling techniques to characterize the kinetics of MBBG. These studies demonstrate that MBBG shares many of the same uptake mechanisms as norepinephrine in the rat heart. PET imaging studies with MBBG would be useful for assessing sympathetic nerve status in the living human heart.

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Guanidine units are important molecular classes well-known to be wildly present in natural products, biologically active pharmaceuticals, proteins, peptides, and peptidomimetics [1]. Moreover, radiopharmaceuticals and molecular probes containing guanidine moiety, which in many cases are very useful as radiotracers (Fig. 1, Fig. 2) for positron emission tomography (PET) [2–11] and single photon emission computed tomography (SPECT) [12–17], have been utilized for exploratory preclinical applications and clinical studies [18–25]. For example, [123I]-meta-iodobenzylguanidine ([123I]MIBG) has been originally developed for cardiac sympathetic innervation and adrenal medulla, which is still popular for cardiac imaging to determine the prognosis of the disease and to assess the response to therapy in several applications, as well as being extensively used for staging of adrenergic tumors [13].
We analyze the effects of protecting group(s) on the efficiency of aromatic ¹⁸F-labeling of guanidine-containing radiopharmaceuticals by quantum chemical methods. We elucidate the origin of experimental observations: The fully protected N,N’,N’’,N’’-tetrakis-Boc guanidine group exhibits remarkably enhanced reactivity and improved selectivity in contrast to both N,N’-bis-Boc protected guanidine and its isomer in the absence of hydrogen bonding with fluoride ion. We find that this very intriguing observation of reaction rates highly depending on the position of protection by –Boc is due to the effects of strong Coulombic interactions among the ionic species, which allow an optimal position (just above the electropositive carbon and the leaving group) of F⁻ in the reaction involving the fully protected N,N’,N’’,N’’-tetrakis-Boc guanidine group.
Synthesis and evaluation of 4-[18F]fluoropropoxy-3-iodobenzylguanidine ([18F]FPOIBG): A novel 18F-labeled analogue of MIBG
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MIBG contains an iodine substituent and thus it is enigmatic why, contrary to our expectations, FPOIBG behaved poorly compared with its bromine-substituted analogue, FPOBBG. With regard to bromine-substituted MIBG analogues, [76Br]MBBG has been evaluated in PC-12 rat pheochromocytoma cells in vitro, isolated rat hearts ex vivo, and animal models in vivo [14,48–50]. The neuronal uptake of MIBG is mediated by two mechanisms—active and passive (see above).
Radioiodinated meta-iodobenzylguanidine (MIBG), a norepinephrine transporter (NET) substrate, has been extensively used as an imaging agent to study the pathophysiology of the heart and for the diagnosis and treatment of neuroendocrine tumors. The goal of this study was to develop an ¹⁸F-labeled analogue of MIBG that like MIBG itself could be synthesized in a single radiochemical step. Towards this end, we designed 4-fluoropropoxy-3-iodobenzylguanidine (FPOIBG).
Standards of FPOIBG and 4-fluoropropoxy-3-bromobenzylguanidine (FPOBBG) as well as their tosylate precursors for labeling with ¹⁸F, and a tin precursor for the preparation of radioiodinated FPOIBG were synthesized. Radiolabeled derivatives were synthesized by nucleophilic substitution and electrophilic iododestannylation from the corresponding precursors. Labeled compounds were evaluated for NET transporter recognition in in vitro assays using three NET-expressing cell lines and in biodistribution experiments in normal mice, with all studies performed in a paired-label format. Competitive inhibition of [¹²⁵I]MIBG uptake by unlabeled benzylguanidine compounds was performed in UVW-NAT cell line to determine IC₅₀ values.
[¹⁸F]FPOIBG was synthesized from the corresponding tosylate precursor in 5.2 ± 0.5% (n = 6) overall radiochemical yields starting with aqueous fluoride in about 105 min. In a paired-label in vitro assay, the uptake of [¹⁸F]FPOIBG at 2 h was 10.2 ± 1.5%, 39.6 ± 13.4%, and 13.3 ± 2.5%, in NET-expressing SK-N-SH, UVW-NAT, and SK-N-BE(2c) cells, respectively, while these values for [¹²⁵I]MIBG were 57.3 ± 8.1%, 82.7 ± 8.9%, and 66.3 ± 3.6%. The specificity of uptake of both tracers was demonstrated by blocking with desipramine. The ¹²⁵I-labeled congener of FPOIBG gave similar results. On the other hand, [¹⁸F]FPOBBG, a compound recently reported in the literature, demonstrated much higher uptake, albeit less than that of co-incubated [¹²⁵I]MIBG. IC₅₀ values for FPOIBG were higher than those obtained for MIBG and FPOBBG. Unlike the case with [¹⁸F]FPOBBG, the heart uptake [¹⁸F]FPOIBG in normal mice was significantly lower than that of MIBG.
Although [¹⁸F]FPOIBG does not appear to warrant further consideration as an ¹⁸F-labeled MIBG analogue, analogues wherein the iodine in it is replaced with a chlorine, fluorine or hydrogen might be worth pursuing.
An ¹⁸F-labeled analogue of the well-known radiopharmaceutical MIBG could have significant impact, potentially improving imaging of NET related disease in cardiology and in the imaging of neuroendocrine tumors. Although ¹⁸F-labeled analogues of MIBG have been reported including LMI1195, we undertook this work hypothesizing that based on its greater structural similarity to MIBG, FPOIBG might be a better analogue than LMI1195.
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For example, the transport rate constant Kup = Vmax/Km was measured to be 0.60 ± 0.20 ml/min/g wet for EPI [3], and 2.66 ± 0.39 ml/min/g wet for HED [2] (Table 2). Iversen's approach to measuring NET transport kinetics has successfully been applied to [131I]metaiobenzylguanidine (MIBG) [15] and [76Br]bromobenzylguanidine (MBBG) [16], which exhibit similar kinetic properties (Table 2). While it is possible to determine the NET transport constants in this way, such studies are challenging and time consuming to perform, requiring a large number of perfusion studies to yield estimates of Km and Vmax.
Most radiotracers for imaging of cardiac sympathetic innervation are substrates of the norepinephrine transporter (NET). The goal of this study was to characterize the NET transport kinetics and binding affinities of several sympathetic nerve radiotracers, including [¹¹C]-(−)-meta-hydroxyephedrine, [¹¹C]-(−)-epinephrine, and a series of [¹¹C]-labeled phenethylguanidines under development in our laboratory. For comparison, the NET transport kinetics and binding affinities of some [³H]-labeled biogenic amines were also determined.
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K_m, V_max and K_I values were determined for each NET substrate with a high degree of reproducibility. Interestingly, C6-hNET transport rates for ‘tracer concentrations’ of substrate, given by the ratio V_max/K_m, were found to be highly correlated with neuronal transport rates measured previously in isolated rat hearts (r² = 0.96). This suggests that the transport constants K_m and V_max measured using the C6-hNET cells accurately reflect in vivo transport kinetics.
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Analogs of the SPECT tracer I-123 MIBG have also been investigated as potential targets for labeling with positron-emitting isotopes. F-18 para-fluorobenzylguanidine, bromine 76 meta-bromobenzylguanidine, and F-18 fluoroiodobenzylguanidine have been synthesized successfully and tested in experimental settings.24-26 Kinetics of these tracers seemed to be comparable to that of I-123 MIBG, but affinity for uptake-1 was considerably lower than for classical PET tracers such as C-11 HED and F-18 fluorodopamine.
The autonomic nervous system plays a key role for regulation of cardiac performance, and the importance of alterations of innervation in the pathophysiology of various heart diseases has been increasingly emphasized. Nuclear imaging techniques have been established that allow for global and regional investigation of the myocardial nervous system. The guanethidine analog iodine 123 metaiodobenzylguanidine (MIBG) has been introduced for scintigraphic mapping of presynaptic sympathetic innervation and is available today for imaging on a broad clinical basis. Not much later than MIBG, positron emission tomography (PET) has also been established for characterizing the cardiac autonomic nervous system. Although PET is methodologically demanding and less widely available, it provides substantial advantages. High spatial and temporal resolution along with routinely available attenuation correction allows for detailed definition of tracer kinetics and makes noninvasive absolute quantification a reality. Furthermore, a series of different radiolabeled catecholamines, catecholamine analogs, and receptor ligands are available. Those are often more physiologic than MIBG and well understood with regard to their tracer physiologic properties. PET imaging of sympathetic neuronal function has been successfully applied to gain mechanistic insights into myocardial biology and pathology. Available tracers allow dissection of processes of presynaptic and postsynaptic innervation contributing to cardiovascular disease. This review summarizes characteristics of currently available PET tracers for cardiac neuroimaging along with the major findings derived from their application in health and disease.

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^☆: This work was supported by a fellowship grant from the Commissariat à l'Energie Atomique (CEA), Saclay, France.

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Kinetics of the norepinephrine analog [76Br]-metabromobenzylguanidine in isolated working rat heart☆

Abstract

J. Am. Coll. Cardiol.

J. Am. Coll. Cardiol.

Nucl. Med. Biol.

J. Am. Coll. Cordiol.

Nucl. Med. Biol.

Am. Heart. J.

J. Am. Coll. Cardiol.

Extraneuronal uptake of noradrenaline in human tissue (uptake2)

Heart Vessels

Uptake and retention kinetics of para-fluorine-18-fluorobenzylguanidine in isolated rat heart

J. Nucl. Med.

Analysis of the compartments involved in the extraneuronal storage and metabolism of isoprenaline in the perfused rat heart

Naunyn-Schmiedeberg's Arch. Pharmacol.

Further studies on the extraneuronal uptake and metabolism of isoprenaline in the perfused rat heart

Naunyn-Schmiedeberg's Arch. Pharmacol.

Uptake of 14C-tyramine and release of extravesicular 3H-noradrenline in isolated perfused rabbit heart

Naunyn-Schmiedeberg's Arch. Pharmacol.

The mechanism of action of indirectly acting sympathomimetic amines

Scintigraphic assessment of MIBG uptake in globally denervated human and canine hearts—implications for clinical studies

J. Nucl. Med.

Myocardial kinetics of carbon-11-meta-hydroxyephedrine: Retention mechanisms and effects of norepinephrine

J. Nucl. Med.

The effect of inhibitors of extraneuronal uptake on the distribution of 3H-(±)-noradrenaline in nerve-free rabbit aortic strips

Naunyn-Schmiedeberg's Arch. Pharmacol.

Disposition of endogenous adrenaline compared to noradrenaline released by cardiac sympathetic nerves in the anaesthetized dog

Naunyn-Schmiedeberg's Arch. Pharmacol.

The kinetic constants for the extraneuronal uptake and metabolism of 3H-(−)-noradrenaline in the perfused rat heart

Naunyn-Schmiedeberg's Arch. Pharmacol.

Absence of MIBG uptake in the denervated rabbit heart [abstract]

J. Nucl. Med.

Iodine-123 metaiodobenzylguanidine imaging of the heart in idiopathic congestive cardiomyopathy and cardiac transplants

J. Nucl. Med.

Evaluation of metaiodobenzylguanidine heart and lung extraction fraction by first-pass analysis in pigs

J. Nucl. Med.

Evaluation of metaiodobenzylguanidine uptake by the norepinephrine, dopamine, and serotonin transporters

J. Nucl. Med.

Cardiac studies with metaiodobenzylguanidine: A critique of methods and interpretation of results

J. Nucl. Med.

Saturation kinetics of the adrenergic neurone uptake system in the perfused rabbit heart: A new method for determination of initial rates of amine uptake

Naunyn-Schmiedeberg's Arch. Pharmacol.

The disposition of 3H-(−)-noradrenaline in the perfused cat and rabbit heart

Naunyn-Schmiedeberg's Arch. Pharmacol.

The substrate specificity of uptake2 in the rat heart

Naunyn-Schmiedeberg's Arch. Pharmacol.

The recovery of α-adrenoceptor function and binding sites after phenoxybenzamine: An index of receptor turnover?

Naunyn-Schmiedeberg's Arch. Pharmacol.

Abnormal I-123 metaiodobenzylguanidine myocardial washout and distribution may reflect adrenergic derangement in patients with congestive cardiomyopathy

Circulation

The uptake of noradrenaline by the isolated perfused rat heart

Br. J. Pharmacol.

Kinetics of the norepinephrine analog [⁷⁶Br]-metabromobenzylguanidine in isolated working rat heart☆

Extraneuronal uptake of noradrenaline in human tissue (uptake₂)

Uptake of ¹⁴C-tyramine and release of extravesicular ³H-noradrenline in isolated perfused rabbit heart

The effect of inhibitors of extraneuronal uptake on the distribution of ³H-(±)-noradrenaline in nerve-free rabbit aortic strips

The kinetic constants for the extraneuronal uptake and metabolism of ³H-(−)-noradrenaline in the perfused rat heart

The disposition of ³H-(−)-noradrenaline in the perfused cat and rabbit heart

The substrate specificity of uptake₂ in the rat heart