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In Vitro and in Vivo Characterization of Cholinergic Receptors in the Rat Heart

In Vitro and in Vivo Characterization of Cholinergic Receptors in the Rat Heart

In Vitro and In Vivo Characterization of 44 1251]Iododexetimide Binding to Muscarinic Receptors in the Rat Heart

Kaname Matsumura, Yoshihiro Uno, Ursula Scheffel, Alan A. Wilson, Robert F. Dannals, and Henry N. Wagner, Jr.

Division ofNuclear and Radiation Health Sciences, The Johns Hopkins Medical Institutions, Baltimore, Maryland

A recent study of cardiac mAChR in man using 4-[1251]iododexetimidebinding to muscarinic cholinergic carbon-i 1-(‘‘C)-labeledmethiodide quinuclidinyl ben receptors(mAChR)was evaluatedin the rat heart.4-[125l] zilate ([‘‘C]MQNB)reported that the physiologically iododexetimide displayed high in vitro affinity (Kd = 14.0 active form of the mAChR could be evaluated by PET nM) for rat myocardialmAChR.In vivo, there was high (5). Although radioiodinated 3-quinuclidinyl benzilate accumulation of 4-[125ljiododexetimide in the rat atrium (4-['231]IQNB) (6) has been used to visualize mAChR and ventriclewhichcouldbe blockedby ‘@-60%by prein in the (CNS) (7), it is not known jectionof .In contrast,accumulationof the radio whether this agent binds to cardiac mAChR. SPECT labeled stereoisomer, 4-[125I]iodolevetimide,was 63% lowerthan 4-[125I]iododexetimideand was not blockedby imaging of mAChR in the human heart has not been atropine. The blood clearance of 4-[125I]iododexetimide demonstrated. was rapid, providing heart-to-blood ratios of up to 14:1; Tritium-labeled has been shown to bind however,heart-to-lungandheart-to-liverratioswere below to mAChR in rat brain in vitro (8) and in vivo (9) with unity.The data indicatethat 4-[125I]iododexetimidebinds high affinity, low nonspecific binding, and slow disso potentlyto ratmAChR.However,sincenonspecificbinding ciation rate. The accumulation of dexetimide in the is relativelyhigh, it is not clear whether iododexetimide CNS was reported to be stereospecific, saturable, and labeledwith 1231will be usefulin SPECTimagingstudies displaceable. In contrast, the stereoisomer levetimide of myocardial mAChR. Further studies in humans are did not show preferential uptake in regions known to indicated. have high concentrations of mAChR, and its affinity J NucI Med 1991; 32:76—80 was more than a thousand times lower (4,8,10). This suggested that levetimide could be used for assessing nonspecific binding. Dexetimide was labeled with ‘‘C (11) and an analog has been radiolabeled with iodine uscarinic cholinergic receptors (mAChR) of the 125 (1251)and iodine-123 (@23I)(12). The in vivo binding heart are important for cardiac function and have been of 4-['25I]iododexetimide in the mouse brain was eval reported to be altered in various conditions and dis uated and shown to be highly specific, saturable, and eases, such as aging (1), diabetes (2), and congestive stereoselective. It was concluded that this ligand may heart disease (3). The widely used anti-arrhythmic be useful for imaging mAChR in the living human agents, , and , exhibit brain with SPECT (12). interactions with cardiac mAChR (4). The localization In the present study, 4-['251]iododexetimide (Fig. 1A) and quantitation of mAChR in the living human heart was investigated as a ligand for cardiac mAChR in the using a noninvasive method such as positron emission rat to assess the feasibility of using this ligand for the tomography (PET) or single-photon emission com delineation of myocardial mAChR in vivo. The in vitro puted tomography (SPECT) may provide valuable in and the in vivo binding of 4-['251]iododexetimide to formation about changes in various disease cardiac mAChR was evaluated. In this study, 44@25I] states and may be useful in monitoring interven iodolevetimide (Fig. 1B) was used to determine nonspe tions. cific binding.

ReceivedFeb.26,1990;revisionacceptedJul.6, 1990. MATERIALSAND METHODS For reprints contact: 1@suIaScheffel, ScD, Divisionof Nuclear Medicine and RadiatiOnHealth Sciences, The Johns Hopkins Medical Institutions, Iodine- 125-labeled 4-iododexetimide (4-['251]iododexetim 615NorthWolfeSt.,Baltimore,MD21205-2179. ide) and 4-iodolevetimide (4-['25I]iodolevetimide) were syn

76 The Journalof NuclearMedicine•Vol. 32 •No. 1 •January1991 A. 4-[1@Iododexebmide tion of the tracer. Hearts were rapidly removed and dissected into the atrium and ventricle. The lungs and livers were also excised. The radioactivity concentration in tissue samples and an aliquot of the injectate was measured in an automated gamma counter. A second series of rats (n = 4) received 10 @Ciof4-['25I]iodolevetimide and was killed in similar fashion. Blocking experiments (n = 4) were carried out by i.v. injection of atropine sulfate(20 mg/kg)at 5 mm beforeinjectionof 4- B. 4-['@I]Iodolevetimide [‘25lJiododexetimide.The influence of on 4@[l25I] iododexetimide binding was determined by injection of 10 mg/kg imipramine at 30 mm before tracer administration (n=4). Statistics Data are presented as means ±s.d. Differencesbetween groups were analyzed using an unpaired t-test. Probability levels of 0.05 or smaller were considered significant. FIGURE 1 Chemicalstructures of (A) 4-[1251]iododexetimideand (B) 4- RESULTS [125I]iodolevetimide. In Vitro Binding of 4{125I]iododexetimide to Cardiac mAChR thesized as previously described (12). The specific activity of Figure 2A shows the total and specific binding of 4- these ligands varied from 300 to 600 Ci/mmol. Atropine [‘25I]iododexetimideto homogenates of rat ventricle as sulfate and imipramine hydrochloride were obtained from a function of increasing concentrations of unlabeled Sigma Chemical Co., St. Louis, MO. iododexetimide. Figure 2B shows an example ofa Scat In Vitro Experiments chard plot of 4-['25I]iododexetimide binding to ventri Homogenate of ventricle was prepared by excising the rat cle. Nonspecific binding in the assay, estimated at a heart immediately after killing the animal, mincing it with concentration of2O nM unlabeled iododexetimide, was scissors,and rapidly homogenizingthe tissue using a Brink high (76% of total binding). A summary of the in vitro mann Polytron (setting 6, 30 s) in 50 mM phosphate-buffered binding data is given in Table 1. The dissociation saline (PBS) (pH = 7.4, 37°C).After filtration through four constants (K@,s)for 4-['25ljiododexetimide binding to rat layers of cheese cloth, the homogenate was centrifuged for 15 atrium versus that to the ventricle were not significantly mm at 50,000 g, and the pellet was washed with PBS, recen different from each other (p = 0.3). On the other hand, trifuged, and stored at —80°C.Homogenate of rat atrium was also prepared in similar fashion. Ten minutes before the assay, the Bmaxofthe atrium was significantly higher than that the tissuehomogenatesweredilutedto 8 mgoftissue/mI with of the ventricle (p < 0.005). phosphate buffer. Triplicate samples of 0.05 ml of 4@[l25IJ Biodistributlon in Rats iododexetimide (final concentration ofO.Ol nM), and 0.05 ml Time-activity curves for 4-['251]iododexetimide and ofincreasing concentrations ofunlabeled iododexetimide (0.5 4-['25lJiodolevetimide in rat atrium and ventricle at nM-32 nM) were incubated in microfuge tubes with 0.5 ml of the diluted tissue homogenate for 30 mm at 37°Cin a various times after injection are shown in Figures 3A- shaking water bath. The final volume was 1.0 ml. Samples prepared in the presence of 1.0 mM atropine defined nonspe A B 12 0.4 cificbinding. TOtalbirdng

Following the incubation period, the samples were centri 0 fuged at 10,000 g for 10 mm and the pellets were washed with 0.3 Kd- 9.6nM ice cold PBS. The radioactivity concentration in each sample @ wasdeterminedusingstandardtechniques.Net specificbind 6 02 ing was determined by subtracting nonspecific binding (sam S@fIc binding ples with atropine) from total binding. Scatchard analysis (13) 0.1 was performed and the dissociation constant (K@,)and the H: number of binding sites (B,,,,,,@)were calculated using EBDA 0@2@3@ 40 I 2 3 (14) and LIGAND (15) programs. Each of the tissues was @dodexe@m@e(nM) B(pmoL/mgprotein) analyzed in triplicate on four separate days. The protein concentration was measured using the method by Lowry (16). FIGURE 2 (A) In vitro total ( • ) and specific(—0-—)4-[1251]iodo In Vivo Experiments dexetimidebindingas a functionof increasingamountsof iododexetimide in the homogenate of rat ventricle. Data shown Male Sprague-Dawley rats (n = 4 per time point; 180—250 are the mean of four separateexperimentsperformedin g) were injected intravenously via tail vein with 10 zCi of 4- triplicate. Errors shown are standard deviations. (B) Repre [‘25ljiododexetimide.The animals were killed by guillotine at sentativeScatchardplot of 4-[1251]iododexetimidebindingto various times (15, 30, 60, and 120 mm) after i.v. administra thehomogenateof rat ventricle.

4-[125ljlododexetimideinthe RatHeart•Matsumuraet al 77 TABLE I Dissociation Constant (1(d)and Maximum Concentration of Binding Sites (Bmax)of 4-[125l]lododexetimide Binding to Rat Atrium and Ventride (pmoVmgiç Bmax (nM) protein) 3 0 Lungs

1.0(CV)t(9.2—27.2)(6.2—21.2)Ventricle14.0±4.43.7±1.2*(cv)(15.4Atrium1 1.3 ±1.77.1 ± 2

—23.8)(11.1 —19.5) Blood

. All values represent means ± s.d. for four separate expen 20 40 60 80 100 120 mentsperformedin [email protected] mm andLIGANDprograms. FIGURE 4 t cv = ranges of coefficients of variation (%) calculated for each Average time-activity curves of 4-[125ljiododexetimidein rat KdandBmax. lungs,liver,and blood. tp<0.005.

enantiomer, were also determined. At all times after B. Between 15 and 120 mm after injection, 4@[l25IJ injection, the distribution of 4-['25ljiodolevetimide in iododexetimide concentrations decreased from 2.7% to rat lungs and liver was similar to that of 4-['251]iodo 1.9 % of the injected dose per gram of tissue (%ID/g) dexetimide (data not shown). These results indicate in the atrium and from 1.9 to 1.0 % ID/g in the that, although accumulation of 4-['251]iododexetimide ventricle. In the blood, the 4-['25ljiododexetimide con in lungs and liver is high, the binding does not appear centration was relatively low at all time points (0.13— to be specific. 0. 18% ID/g) (Fig. 4). At 30 mm, the atrium-to-blood In Vivo Blocking Study ratio was 14.2:1, the ratio for ventricle-to-blood was To verify the specificity of 4-['251]iododexetimide 10.4:1. In contrast to 4-['25I]iododexetimide, the 4@[l25I] binding to cardiac mAChR, inhibition by atropine was iodolevetimide concentration in both the atrium and studied. As shown in Table 2, the accumulation of 4- ventricle was significantly lower and decreased only [‘25I]iododexetimidein the rat atrium and ventricle was slightly during the time ofobservation (15 to 120 mm). blocked by prior i.v. administration of a high dose (20 For comparison, 4-['25I]iododexetimide concentra mg/kg) of atropine. The highest inhibition (%ID/g of tions were also followed in the lungs and liver. In both 4-['25I]iododexetimide minus %ID/g of 4-['251Jiodo organs (Fig. 4), 4-['251]iododexetimide concentrations dexetimide with preadministration of atropine) oc were comparatively high (4.6% ID/g and 2.2% ID/g, curred in the atrium (65.6% at 30 mm after injection respectively, at 15 mm and 2. 1% ID/g and 1.5% ID/g, of tracer). In the ventricle, inhibition by atropine aver respectively, at 120 mm after injection). Because of the aged 55%. high lung uptake, heart-to-lung ratios for 4-['25I]iodo Previous in vitro studies have shown that cardiac dexetimide were relatively low (0.4—0.9:1).Heart-to mAChR are blocked by the antidepressant imipramine liver ratios were also low (0.7—1.3:1). (1 7). In this investigation, the effect of preadministra To evaluate whether 4-['25ljiododexetimide accu tion of imipramine on the in vivo 4-['25I]iododexetim mulation in lungs and liver was mAChR-specific, time ide binding was determined. Imipramine (10 mg/kg) activity curves of 4-['25lliodolevetimide, the inactive was injected intravenously 30 mm before administra tion of the ligand. The dose of 10 mg/kg imipramine

A Atrkjrn B Ventricle constitutes approximately twice the maximum dose administered to patients (18). Imipramine reduced 4- [‘251]iododexetimideaccumulation in the rat atrium by 2 59.5%, in the ventricle by 45.8% (Table 2).

DISCUSSION In this investigation, the in vitro and in vivo binding 0 0 20 40 60 60 100 120 20 40 60 $0 100 120 of4-['25I]iododexetimide to rat heart mAChR was stud mm mm ied. In vitro, the 4-['251]iododexetimide binding was FIGURE 3 found to be potent (I@ = 11.3 and 14 nM in atrium Averagetime-activitycurves of 4-[125I]iododexetimideand 4- and ventricle, respectively). In vivo distribution studies [125l)iodolevetimidein rat atrium (A), ventricle (B), and blood (A).Valuesare expressedas means±S.D. of %ID/g. —0— with 4-['25I]iododexetimide in rats demonstrated that = 4-['25ljiododexetimide, . 4-[125ljiodolevetimide. this localizes in the heart and

78 The Journal of Nuclear Medicine •Vol. 32 •No. 1 •January 1991 TABLE 2 4-[1251]lododexetimideand4-[125I]lodolevetimideAccumulationin the Rat Heart (mean 4-[125l]lododexetimide(mean%ID/gtissue)4-[125ljlodolevetimide tissu&)ControlsAtropineImiprammneControls %lD/g Atropine

Atrium2.620.20Ventricle1.79 ±0.340.90± O.l3@± [email protected] ±0.130.91 ± ±0.160.81± [email protected] ±[email protected] ±0.070.80 ±0.09

.Resultsareexpressedasmean%ID/gtissue±s.d.at30mmaftertracerinjection;n=4rats.Atropine(20mg/kg)wasinjected intravenously5 mm before administration of the radiolabeledligand. Imipramine(10 mg/kg) was injected intravenously 30 mm before administrationof 4-[125l]iododexetimide. t Significantly different from controls, p < 0.001.

that large target-to-blood ratios are achieved. Kinetic preadministration of imipramine at a dose (10 mg/kg) studies with 4-['251]iododexetimide in the rat heart approximately twice the maximum dose administered showed that peak tracer concentrations were reached to patients. A number of neuroleptics such as chlor early (within the first 15 mm) after i.v. injection. There also interact with cardiac mAChR (4). after, the radioactivity decreased slowly with an approx Therefore, monitoring of cardiac mAChR during drug imate half-time of 150 mm. Evidence for the specificity intervention could provide a valuable tool in the man of the interaction of 4-['251]iododexetimide with agement and treatment ofpatients with not only cardiac mAChR in vivo comes from the results of two experi disease but also with psychiatric disease. ments: (a) the accmumulation of 4-['25ljiododexetimide 4-['251]iododexetimide has a high lipophilicity which in the heart was significantly higher than that of the contributes to the high accumulation in the lungs. In inactive enantiomer (4-['251]iodolevetimide, and (b) the present investigation, the clearance of 4-['251]iodo 4-['251]iododexetimide accumulation in the heart was dexetimide was found to be slow, and the lung activity inhibited by the muscarinic antagonist atropine. In was more than twice that of the ventricle. High lung contrast, 4-['251]iodolevetimide was not affected by uptake was also reported for other iodinated ligands atropine. such as 4-['251]IQNB (22) and [‘311]iodopindolol for In in vivo distribution studies, “specific―binding of beta-adrenoreceptor (23), and may become a major 4-['251]iododexetimide was defined as the difference obstacle to the use of these ligands for cardiac receptor between total 4-['25ljiododexetimide accumulation and imaging. In addition, some pharmaceuticals, such as the radioactivity remaining after blockade with a high imipramine, are known to affect uptake of ligands in dose of atropine. “Specific―binding of 4-['25ljiododex the lungs (24). This fact may have to be taken into etimide was significantly higher in the atrium than in consideration in quantitative in vivo receptor studies. the ventricle. This finding correlated with the in vitro For obtaining a more favorable heart-to-lung ratio, results (Bmaxdata shown in Table 1), as well as with hydrophilic compounds like MQNB may be more use previous results in the mouse heart (data not shown). ful (19). Indeed, [‘‘C]MQNBhas been used in in vivo The regional distribution ofmAChR seems to vary with PET scanning of the human heart, in which heart different species. In the rabbit heart, as in the rat, highest images could be obtained without the need for subtract concentrations of mAChR are found in the atrium (4, ing lung activity (5). Although 4-['251]iododexetimide 19), the distribution in The guinea pig and dog heart is exhibits high lung uptake, quantitative SPECT imaging diffuse (20). The organization ofmAChR in the human of cardiac mAChR with 4-['23I]iododexetimide should heart is not known for certain. PET studies with [‘‘C] be possible as long as overlying lung activity can clearly MQNB showed higher tracer concentrations in the be distinguished and separated from that in the atria ventricular septum and left ventricular wall whereas the and ventricles. atria was not visualized (5). Whether the distribution Similar considerations have to be made for the pos seen on the PET images is an accurate reflection of sible interference in cardiac imaging by radioactivity receptor densities is not known. Human autopsy data accumulated in the liver. 4-['251]iododexetimide accu on mAChR distribution in the heart are still outstand mulation in the rat liver was about unity with that in ing. the heart. Whether uptake of the ‘23I-labeledligand in Tricyclic antidepressants such as imipramine have the human liver would be of equal proportion has to been reported to block cardiac mAChR (17) and play a be explored. role in clinical cardiotoxicity (21). In in vivo experi The results presented in this study show a high affin ments reported here, the accumulation of 4-['251]iodo ity of 4-['25I]iododexetimide for cardiac mAChR. In dexetimide in the heart was significantly blocked by vivo inhibition studies with atropine, as well as com

4-[125l]Iododexetimidein the Rat Heart •Matsumura et al 79 parative studies with the inactive enantiomer, 4@[125I] 5. Syrota A, Comar D, Paillotin G, Ctal. Muscarinic cholinergic iodolevetimide, had indicated that @-40%of 4@[l25I] receptor in the human heart evidenced under physiological conditions by positron emission tomography. Proc NailAcad iododexetimide binding in the rat heart was nonspecific. Sci USA l985;82:584—588. In in vitro assays, nonspecific binding in the heart (in 6. RzeszotarskiWi, Eckelman WC, Francis BE,et al. Synthesis the presence of 20 nM cold iododexetimide) was even and evaluation of radioiodinated derivatives of 1-Azabicy more prominent, amounting to as much as 76% of total clo(2.2.2)oct-3-yl alpha-hydroxy-alpha-(4-iodophenyl)phen binding. A high nonspecific binding (50% oftotal bind ylacetate as potential radiopharmaceuticals. J Med Chem 1984:27:156—160. ing) in the myocardium has also been reported for 4- 7. Eckelman WC, Reba RC, Gibson RE, et al. External imaging [‘25IJIQNB(22), which has been attributed to (a) the of cerebral muscarinic receptors. Science high lipophilicity of the ligand (b) interactions with 1984;223:291—293. contractile cardiac proteins. Whether the same factors 8. Laduron PM, Verwimp M, Leysen JE. Stereospecific in vitro or others are responsible for the relatively high nonspe binding of [3H]dexetimideto brain muscarinic receptors. J Neurochem 1979:32:42 1—427. cific portion of 4-['251]iododexetimide binding in the 9. Laduron PM, Janssen PFM. Characterization and subcellular myocardium is not known at present. localization of brain muscarinic receptors labelled in vivo by [3Hldexetimide.J Neurocheml979;33:1223—1231. CONCLUSIONS 10. Soudijn W., Van Wijngaarden I, AriënsEl. Dexetimide, a usefultool in acetylcholine-receptorlocalization. Eur J Phar 4-['231]iododexetimide may not necessarily be an macol l973;24:43—48. ideal ligand for imaging of mAChR of heart because of 11. Dannals RF, LãngstromB, Ravert HT, et al. Synthesis of its high nonspecific binding and low heart-to-lung and radiotracers for studying muscarinic cholinergic receptors in heart-to-liver ratios. Nonetheless, an advantage of 4- the livinghuman brain using positron emission tomography: [‘251]iododexetimide is the availability of its labeled [I ‘C]dexetimide and [‘‘Cjlevetimide. In! J App/ Radial Isot 1988;39:291—295. inactive enantiomer. Blocking studies for an estimation 12. Wilson AA, Dannals RF, Ravert HT, et al. Synthesis and of nonspecific binding of mAChR in imaging studies of biological evaluation of [1251]and [‘231]4-iododexetimide,a human heart are supposed to be not totally viable, since potent muscarinic cholinergic . J Med mAChR antagonists have pronounced pharmacological Chem 1989;32:1057—1062. action which affects cardiac function and may preclude 13. Scatchard 0. The attractions of proteins for small molecules and ions. Ann NYAcadSci l949;5l:660—672. blocking studies. The use of 4-['23ljiodolevetimide 14. McPherson GD. A practical computer-based approach to the should allow an estimation of nonspecific binding in analysis of radioligand binding experiments. Computer Prog cardiac mAChR imaging studies and measurement of InBiomed l983;17:l07—l14. its binding should closely approximate the nonspecific 15. Munson PJ, Rodbard D. LIGAND: a versatile computerized binding component of its enantiomer, 4-['23ljiododex approach for the characterization of ligand binding systems. AnalBiochem1980;107:220—239. etimide. Since SPECT should allow an in vivo analysis 16. LowryOH, Rosebrough NJ, Farr AL, et al. Protein measure of mAChR in the heart under sympathetic and para ment with the Folin phenol reagent. J Bio/ Chem sympathetic physiologic control, it is worth considering I951:193:265—275. that this method might represent the more ideal ap 17. Snyder SH, Yamamura HI. Antidepressants and the musca proach to research of the physiologially active form of rinic . Arch Gen ps.vchiatr.v1977:34: 236—239. mAChR. Whether or not successful imaging of mAChR 18. Davis JM, Glassman AH. Antidepressant drags. In: Kaplan with 4-['23I]iododexetimide will be possible, will de HI, Sadock BJ, eds. Comprehensivetextbook of psychiatry, pend, in large part, on the kinetics and binding of the 5th edition. Baltimore: Wiliams & Wilkins; 1989:1627—1655. tracer in the human heart. 19. Gibson RE, Eckelman WC, Vieras F, et al. The distribution of the muscarinic acetylcholine receptor antagonists, quinu clidinyl benzilate and quinuclidinyl benzilate methiodide ACKNOWLEDGMENT (both tritiated), in rat, guinea pig, and rabbit. J Nuc/ Med This work was supported in part by D.H.H.S. grant number l979;20:865—870. CA-32845. 20. Wei JW, Sulakhe PV. Regional and subcellular distribution of myocardial muscarinic cholinergic receptors. Eur J Phar REFERENCES maco/ l978;52:235—238. 21. Dumovic P. Burrows GD, Vohra J, et al. The effect of tricyclic 1. Narayanan N, Derby JA. 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80 The Journal of Nuclear Medicine •Vol. 32 •No. 1 •January 1991

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