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Autoradiographic Evidence That 3-Quinuclidinyl-4-fluorobenzilate (FQNB) Displaysin VivoSelectivity for the m2 Subtype
NEUROIMAGE ARTICLE NO.
3, 35–39 (1996) 0004
Autoradiographic Evidence That 3-Quinuclidinyl-4-fluorobenzilate (FQNB) Displays in Vivo Selectivity for the m2 Subtype S. F. BOULAY,* V. K. SOOD,* M. R. RAYEQ,* V. I. COHEN,* B. R. ZEEBERG,*
AND
R. C. REBA*,†
*Section of Radiopharmaceutical Chemistry, Walter G. Ross Hall, George Washington University Medical Center, 2300 Eye St., N.W., Washington, DC 20037 and †Department of Radiology, Nuclear Medicine Section, University of Chicago, 5841 S. Maryland Avenue, MC 2026, Chicago, IL 60637 Received June 13, 1995
1985; Weinberger et al., 1990, 1991; Kim et al., 1990) have recently attempted to determine whether single photon emission computed tomographic (SPECT) imaging of the distribution of (R)-3-quinuclidinyl (S)-4iodobenzilate ((R,S)-[123I]IQNB)1 could be useful for detecting pathological changes in muscarinic neuroreceptor concentration in AD. These studies are subject to the limitation that the m2 subtype constitutes only about 19% of the total mAChR in the posterior parietal cortex (Li et al., 1991). Even a complete loss of the m2 receptors would correspond to a small relative change in the observed (R,S)-[123I]IQNB concentration. Thus, it is essential to develop a radioligand which can penetrate the blood–brain barrier and which has high in vivo selectivity for the m2 subtype. We have previously reported the results of autoradiographic studies of the in vivo inhibition of radioiodinated (R,S)-[125I]IQNB binding by unlabeled QNB, from which we had concluded that QNB is m2-selective in vivo (McRee et al., 1995). However, radioiodinated derivatives of QNB appear to lose the in vivo m2 selectivity (McRee et al., 1995). We suspect that the loss of the in vivo m2 selectivity experienced by IQNB is the result of the bulky steric properties of the iodo- group. Therefore, we have been studying a series of derivatives of QNB in which the phenyl ring contains a bromo- or fluoro-substituent. We report here on the autoradiographic demonstration that, in contrast to IQNB, FQNB retains in vivo m2 selectivity.
Alzheimer’s disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood–brain barrier. We now demonstrate the in vivo m2 selectivity of a fluorine derivative of QNB (FQNB), by studying autoradiographically the in vivo inhibition of radioiodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) binding by unlabeled FQNB. In the absence of FQNB, (R,S)-[125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 30.0 nmol of racemic FQNB, (R,S)-[125I]IQNB labeling in those brain regions containing predominantly the m2 subtype is reduced to background levels. We conclude that FQNB is m2-selective in vivo and that [18F]FQNB or a closely related analogue may be of potential use in positron emission tomographic study of the loss of m2 receptors in AD. © 1996 Academic Press, Inc.
INTRODUCTION Alzheimer’s disease (AD) appears to involve selective loss of m2 subtype neuroreceptors in cortical and hippocampal regions of the human brain, as determined by the use of in vitro subtype-selective pharmacological characterization (Mash et al., 1985; Wang et al., 1987; Araujo et al., 1988; Quirion et al., 1989; Aubert et al., 1992) or, more recently, by molecular characterization (Flynn et al., 1995). Several studies (Holman et al.,
MATERIALS AND METHODS Radiopharmaceuticals and Chemicals The racemic mixture of FQNB was synthesized as previously described (Cohen et al., 1992). The (R,S)-diastereomer of IQNB, (R,S)-[125I]IQNB, was radioiodinated by the triazene method (Eckelman et al., 1985; Rzeszotarski et al., 1984) and characterized according to the method of Rzeszotarski et al.
1 “(R,S)-IQNB” has been incorrectly designated as “(R,R)-IQNB” in the literature. Thus, when we now refer to “(R,S)-IQNB,” we are referring to the same diastereoisomer that was previously called “(R,R)-IQNB.” This issue will be addressed in detail (Kiesewetter et al., 1996; Zeeberg et al., submitted for publication). To avoid unnecessary confusion, the corrected designation has been employed here and when referring to previous publications.
35 1053-8119/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
36
BOULAY ET AL.
FQNB DISPLAYS IN VIVO m2 SELECTIVITY
37
Note. Computed from the data reported in (Li et al., 1991; Wall et al., 1991a,b; Yasuda et al., 1993). Abbreviations used in this table and elsewhere are: left corpus striatum (csl), right corpus striatum (csr), hippocampus (hipp), left frontal cortex (fcl), right frontal cortex (fcr), left thalamus (thl), right thalamus (thr), medulla (med), pons (pons), cerebellum (cb), inferior colliculus (ic), and superior colliculus (sc).
(1984). (R,S)-[125I]IQNB was obtained by the reaction of an (R,S)-QNB triazene precursor with Na125I. The product was purified by high-performance liquid chromatography (HPLC) using a Z-module C-18 Bondapak reversed-phase cartridge, eluted with MeOH:water (60:40) containing 10 mM formic acid and 1% octane sulfonic acid as an ion pair agent to improve resolution. Final radiochemical purity was $95% as determined by thin-layer chromatography (TLC). The specific activity of (R,S)-[125I]IQNB (averaging approximately 900 Ci/mmol) was determined by comparing the radioactivity specifically bound to a caudate nucleus homogenate to that bound when using [3H]QNB of known specific activity (Cohen et al., 1989): A saturation analysis is performed using caudate nucleus homogenate in the presence of eight different concentrations of the [3H]QNB of known specific activity. In this analysis, eight identical samples are studied in the presence of atropine to provide a subtraction for nonspecific binding. Based upon the known specific activity of the [3H]QNB and a computer fit of the specific binding of the [3H]QNB to a single binding site, the concentration of muscarinic binding sites is computed. Simultaneously, an identical saturation experiment is performed using the (R,S)-[125I]IQNB of unknown specific activity. In this case, the computer fit results in a saturating specific binding expressed in terms of disintegrations per minute (dpm). Dividing this saturating radioactivity value determined with the (R,S)-[125I]IQNB of unknown specific activity by the concentration of muscarinic binding sites determined with the [3H]QNB of known specific activity provides the specific activity of the (R,S)-[125I]IQNB. In Vivo Studies Male Sprague–Dawley rats weighing 200–250 g were used in the experiments. Animals were anesthe-
tized with ketamine:xylazine (100:10 mg/kg im) and the right jugular vein was exposed for intravenous injection. One hundred sixty-five microcuries of (R,S)[125I]IQNB in 0.4 ml of normal saline containing up to 50% ethanol was injected alone or coinjected with 30.0 nmol of the racemic mixture of FQNB. Animals were maintained under anesthesia until time of sacrifice at 2 h postinjection. Animals were sacrificed by decapitation and the brains were rapidly removed, blotted free of excess blood, immediately frozen on dry ice, and stored at −70°C until sliced. Autoradiography The frozen brains were sliced into 20-mm-thick coronal sections and thaw mounted onto chrome-alum/ gelatin-coated slides. The slides along with 125I standards (Amersham) were then apposed to hyperfilm MP (Amersham) for 1 to 3 weeks, depending upon the amount of radioactivity within a typical section. The film was developed using an automated x-ray film developer (Kodak RP X-OMAT film processor, Model M6B). The images were digitized using a RAS 3000 imaging system (Loats, Inc.) and analysis was performed on a Macintosh IIci computer using the public domain NIH Image program (written by Wayne Rasband at the U.S. National institutes of Health and available from the Internet by anonymous ftp from zippy.nimh.nih.gov or on a floppy disk from NTIS, 5285 Port Royal Rd., Springfield, VA 22161, part number PB93-504868). Data Analysis For each film, all digitized images were converted to optical density (OD) values using a program developed within our group. Brain regions to be measured were
FIG. 1. Rats were injected in the jugular vein with 165 mCi of (R,S)-[125I]IQNB in the absence (A–C) or presence (D–F) of 30.0 nmol of coinjected racemic FQNB. Those regions that are enriched in the m2 subtype (Table 1) are preferentially blocked by FQNB. Representative coronal slices shown are through the anteroventral nucleus of the thalamus (A, D), the hippocampus (B, E), and the pontine nuclei (C, F). The profiles demonstrate the regional (R,S)-[125I]IQNB distribution in the absence (darker lines) and in the presence (lighter lines) of coinjected FQNB. The profiles (with approximate horizontal axis positions given in parentheses) for A and D traversed parietal cortex (15 and 225), caudate putamen (45 and 200), anteroventral thalamic nucleus, ventrolateral (100 and 150), and anteroventral thalamic nucleus, dorsomedial (110 and 140). The profiles for B and E traversed frontal cortex (20), hippocampus (50), thalamic nuclei (90), and hypothalamic nuclei (130). The profiles for C and F traversed occipital cortex (10), subiculum (35), superior colliculus (45), and pons (110).
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BOULAY ET AL.
selected by reference to coronal sections displayed in a standard rat brain atlas (Paxinos and Watson, 1986). Because of interanimal variability in percentage dose/g for (R,S)-[125I]IQNB binding, we now express data as ratios relative to a control region, which has been shown to be highly reproducible from animal to animal (Gitler et al., 1995). Thus, the color scales used for displaying and comparing the autoradiographic images have been adjusted to provide an approximately equivalent intensity for the control regions, which constitute corpus striatum or hippocampus (if these are present in the slice) or cortex. Profiles were also normalized so as to maintain these control regions at a value of approximately unity. RESULTS AND DISCUSSION Despite the fact that QNB exhibits little or no subtype selectivity in in vitro assays (Zeeberg et al., 1991), QNB exhibits substantial m2 selectivity in in vivo studies (Gitler et al., 1994, 1995; McRee et al., 1995). Similar discrepancies in mouse brain between in vitro and in vivo subtype selectivity for the opiate receptor ligand diprenorphine have been reported by Frost et al. (1984). Frost et al. found that “. . . although diprenorphine appears to bind to the opiate receptor subtypes with approximately equal affinity in vitro, it may bind to only one subtype in vivo, possibly the m receptor . . . .” FQNB also exhibits little or no subtype selectivity in in vitro assays (Eckelman et al., 1984). On the other hand, as does QNB, FQNB exhibits substantial m2 selectivity in in vivo studies. The in vivo autoradiographic results of competition studies of FQNB against (R,S)-[125II]IQNB (Fig. 1) demonstrate the in vivo m2 selectivity of FQNB. Those regions that are enriched in the m2 subtype (Table 1) are preferentially blocked by FQNB. The regional pattern of competition of FQNB is very similar to that previously exhibited by QNB (McRee et al., 1995). The profiles in the right column (Fig. 1) are measured along straight lines that were selected to traverse some m2-rich and some m2-poor regions in order to provide a quantitative sense of the differential effect upon the receptor subtypes. Some of the limitations of this method are that a quantitative measure of the in vivo selectivity of FQNB is not available. To obtain a quantitative measure would require a rather cumbersome mathematical analysis such as that which we had performed for [3H]QNB (Gitler et al., 1994). Another limitation is that, although the competition studies indicate in vivo selectivity of FQNB, direct studies using [18F]FQNB may indicate that the in vivo selectivity of FQNB is not as great as that shown by [3H]QNB (Gitler et al., 1994) or that there is excessive nonspecific binding of [18F]FQNB which would render it useless for imaging studies.
We conclude that FQNB is m2-selective in vivo and that [18F]FQNB is potentially a suitable m2 selective radioligand for positron emission tomographic study of the loss of m2 receptors in AD. However, before this goal can be achieved, the racemic mixture of FQNB should be resolved to determine which of the four diastereomers exhibits the highest degree of in vivo m2 selectivity, and a precursor for the radiofluorination of this diastereomer or of a closely related analogue (Boulay et al., 1995) would need to be synthesized. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (NS22215) and the Department of Energy (DE FG05 88ER60649).
REFERENCES Araujo, D. M., Lapchak, P. A., Robitaille, Y., Gauthier, S., and Quirion, R. 1988. Differential alteration of various cholinergic markers in cortical and subcortical regions of human brain in Alzheimer’s disease. J. Neurochem. 50: 1914–1923. Aubert, I., Araujo, D. M., Cécyre, D., Robitaille, Y., Gauthier, S., and Quirion, R. 1992. Comparative alterations of nicotinic and muscarinic binding sites in Alzheimer’s and Parkinson’s diseases. J. Neurochem. 58: 1529–1541. Boulay, S. F., Sood, V. K., Rayeq, M. R., McRee, R. C., Cohen, E. I., Cohen, V. I., Zeeberg, B. R., and Reba, R. C. 1995. Autoradiographic evidence that quinuclidinyl 4-(bromophenyl)-2thienylglycolate (QBPTG) displays in vivo selectivity for the m2 subtype. NeuroImage 2: 209–213. Cohen, V. I., Gibson, R. E., Fan, L. H., De La Cruz, R., Gitler, M. S., Hariman, E., and Reba, R. C. 1992. Synthesis and receptor affinities of new 3-quinuclidinyl a-heteroaryl-a-aryl-a-hydroxyacetates. J. Pharm. Sci. 81: 326–329. Cohen, V. I., Rzeszotarski, W. J., Gibson, R. E., Fan, L. H., and Reba, R. C. 1989. Preparation and properties of (R)-(−)-1-azabicyclo[2.2.2]oct-3-yl-(R)-(+)-a-hydroxy-a-(4-[125I]iodo-phenyl)-a-phenyl acetate and (R)-(−)-1-azabicyclo[2.2.2]oct-3-yl-(S)-(−)-a-hydroxy-a(4-[125I]iodophenyl)-a-phenyl acetate as potential radiopharmaceuticals. J. Pharm. Sci. 78: 833–836. Eckelman, W. C., Eng, R., Rzeszotarski, W. J., Gibson, R. E., Francis, B., and Reba, R. C. 1985. Use of 3-quinuclidinyl 4-iodobenzilate as a receptor binding radiotracer. J. Nucl. Med. 26: 637–642. Eckelman, W. C., Grissom, M., Conklin, J., Rzeszotarski, W. J., Gibson, R. E., Francis, B. E., Jagoda, E. M., Eng, R., and Reba, R. C. 1984. In vivo competition studies with analogues of 3-quinuclidinyl benzilate. J. Pharm. Sci. 73: 529–534. Flynn, D. D., Ferrari-DiLeo, G., Mash, D. C., and Levey, A. I. 1995. Differential regulation of molecular subtypes of muscarinic receptors in Alzheimer’s Disease. J. Neurochem. 64: 1888–1891. Frost, J. J., Dannals, R. F., Duelfer, T., Burns, H. D., Ravert, H. T., Langstrom, B., Balasubramanian, V., and Wagner JR., H. N. 1984. In vivo studies of opiate receptors. Ann. Neurol. 15 (Suppl.):S85– S92. Gitler, M. S., Boulay, S. F., Sood, V. K., McPherson, D. W., Knapp, F. F., Zeeberg, B. R., and Reba, R. C. 1995. Characterization of in vivo brain muscarinic acetylcholine receptor subtype selectivity by competition studies against (R,S)-[125I]IQNB. Brain Res. 687: 71– 78. Gitler, M. S., de la Cruz, R., Zeeberg, B. R., and Reba, R. C. 1994.
FQNB DISPLAYS IN VIVO m2 SELECTIVITY [3H]QNB displays in vivo selectivity for the m2 subtype. Life Sci. 55: 1493–1508. Holman, B. L., Gibson, R. E., Hill, T. C., Eckelman, W. C., Albert, M., and Reba, R. C. 1985. Muscarinic acetylcholine receptors in Alzheimer’s disease in vivo imaging with iodine 123-labeled 3-quinuclidinyl-4-iodobenzilate and emission tomography. JAMA 254: 3063–3066. Kiesewetter, D. O., Paik, C. H., Flippen-Anderson, J. L., and Eckelman, W. C. 1995. The stereochemistry of (R,R)-IQNB. J. Label. Comp. Radiogpharm., 37: 686–688. Kim, H. J., Zeeberg, B. R., Gibson, R. E., Hosain, P., Wesley, R., and Reba, R. C. 1990. SPECT study of the localization of R,R[123I]IQNB in Alzheimer’s patients and normals. J. Nucl. Med. 31: 729. Li, M., Yasuda, R. P., Wall, S. J., Wellstein, A., and Wolfe, B. 1991. Distribution of m2 muscarinic receptors in rat brain using antisera selective for m2 receptors. Mol. Pharmacol. 40: 28–35. Mash, D. C., Flynn, D. D., and Potter, L. T. 1985. Loss of M2 muscarinic receptors in the cerebral cortex in Alzheimer’s disease and experimental cholinergic denervation. Science 228: 1115–1117. McRee, R. C., Boulay, S. F., Sood, V. K., Cohen, E. I., Cohen, V. I., Gitler, M. S., Zeeberg, B. R., Gibson, R. E., and Reba, R. C. 1995. Autoradiographic evidence that QNB displays in vivo selectivity for the m2 subtype. NeuroImage 2: 55–62. Paxinos, G., and Watson, C. 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, Inc., New York. Quirion, R., Aubert, I., Lapchak, P. A., Schaum, R. P., Teolis, S., Gauthier, S., and Araujo, D. M. 1989. Muscarinic receptor subtypes in human neurodegenerative disorders: Focus on Alzheimer’s disease. Trends Pharmacol. Sci. Suppl. IV: 80–84. Rzeszotarski, W. J., Eckelman, W. C., Francis, B. E., Simms, D. A., Gibson, R. E., Jagoda, E. M., Grissom, M. P., Eng, R. R., Conklin, J. J., and Reba, R. C. 1984. Synthesis and evaluation of radioiodinated derivatives of 1-azabicyclo[2.2.2]ocy-3-yl a -hydroxy- a phenylacetate as potential radiopharmaceuticals. J. Med. Chem. 27: 156–160. Wall, S. J., Yasuda, R. P., Hory, F., Flagg, S., Martin, B. M., Ginns,
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E. I., and Wolfe, B. B. 1991a. Production of antisera selective for m1 muscarinic receptors using fusion proteins: distribution of m1 receptors in rat brain. Mol. Pharmacol. 39: 643–649. Wall, S. J., Yasuda, R. P., Li, M., and Wolfe, B. B. 1991b. Development of an antiserum against m3 muscarinic receptors: Distribution of m3 receptors in rat tissues and clonal cell lines. Mol. Pharmacol. 40: 783–789. Wang, J. X., Roeske, W. R., Mei, L., Malatynska, E., Wang, W., Perry, E. K., Perry, R. H., and Yamamura, H. I. 1987. Nicotinic and muscarinic M2 receptor alteration in the cerebral cortex of patients with senile dementia of the Alzheimer’s type (SDAT). In Pharmacology, Proceedings of the Xth IUPHAR, Sydney, Australia (M. J. Rand and C. Raper, Eds.), pp. 83–86. Elsevier, New York. Weinberger, D. R., Gibson, R. E., Coppola, R., Jones, D. W., Molchan, S., Sunderland, T., Berman, K. F., and Reba, R. C. 1991. The distribution of cerebral muscarinic acetylcholine receptors in vivo in patients with dementia. A controlled study with 123IQNB and single photon emission computed tomography. Arch. Neurol. 48: 169–176. Weinberger, D. R., Mann, U., Gibson, R. E., Coppola, R., Jones, D. W., Braun, A. R., Berman, K. F., Sunderland, T., Reba, R. C., and Chase, T. N. 1990. Cerebral muscarinic receptors in primary degenerative dementia as evaluated by SPECT with iodine-123labeled QNB. Adv. Neurol. 51: 147–150. Yasuda, R. P., Ciesla, W., Flores, L. R., Wall, S. J., Li, M., Satkus, S. A., Weisstein, J. S., Spagnola, B. V., and Wolfe, B. B. 1993. Development of antisera selective for m4 and m5 muscarinic cholinergic receptors: Distribution of m4 and m5 receptors in rat brain. Mol. Pharmacol. 43: 149–157. Zeeberg, B. R., Boulay, S. F., Gitler, M. S., Sood, V. K., and Reba, R. C., Correction of the stereochemical assignment of the benzilic acid center in (R)-(−)-3-quinuclidinyl (S)-(+)-4-iodobenzilate ((R,S)4-IQNB). Submitted for publication. Zeeberg, B. R., Gitler, M. S., Baumgold, J., de la Cruz, R. A., and Reba, R. C. 1991. Binding of radioiodinated SPECT ligands to transfected cell membranes expressing single muscarinic receptor subtypes. Biochem. Biophys. Res. Commun. 179: 768–775.
3, 35–39 (1996) 0004
Autoradiographic Evidence That 3-Quinuclidinyl-4-fluorobenzilate (FQNB) Displays in Vivo Selectivity for the m2 Subtype S. F. BOULAY,* V. K. SOOD,* M. R. RAYEQ,* V. I. COHEN,* B. R. ZEEBERG,*
AND
R. C. REBA*,†
*Section of Radiopharmaceutical Chemistry, Walter G. Ross Hall, George Washington University Medical Center, 2300 Eye St., N.W., Washington, DC 20037 and †Department of Radiology, Nuclear Medicine Section, University of Chicago, 5841 S. Maryland Avenue, MC 2026, Chicago, IL 60637 Received June 13, 1995
1985; Weinberger et al., 1990, 1991; Kim et al., 1990) have recently attempted to determine whether single photon emission computed tomographic (SPECT) imaging of the distribution of (R)-3-quinuclidinyl (S)-4iodobenzilate ((R,S)-[123I]IQNB)1 could be useful for detecting pathological changes in muscarinic neuroreceptor concentration in AD. These studies are subject to the limitation that the m2 subtype constitutes only about 19% of the total mAChR in the posterior parietal cortex (Li et al., 1991). Even a complete loss of the m2 receptors would correspond to a small relative change in the observed (R,S)-[123I]IQNB concentration. Thus, it is essential to develop a radioligand which can penetrate the blood–brain barrier and which has high in vivo selectivity for the m2 subtype. We have previously reported the results of autoradiographic studies of the in vivo inhibition of radioiodinated (R,S)-[125I]IQNB binding by unlabeled QNB, from which we had concluded that QNB is m2-selective in vivo (McRee et al., 1995). However, radioiodinated derivatives of QNB appear to lose the in vivo m2 selectivity (McRee et al., 1995). We suspect that the loss of the in vivo m2 selectivity experienced by IQNB is the result of the bulky steric properties of the iodo- group. Therefore, we have been studying a series of derivatives of QNB in which the phenyl ring contains a bromo- or fluoro-substituent. We report here on the autoradiographic demonstration that, in contrast to IQNB, FQNB retains in vivo m2 selectivity.
Alzheimer’s disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood–brain barrier. We now demonstrate the in vivo m2 selectivity of a fluorine derivative of QNB (FQNB), by studying autoradiographically the in vivo inhibition of radioiodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) binding by unlabeled FQNB. In the absence of FQNB, (R,S)-[125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 30.0 nmol of racemic FQNB, (R,S)-[125I]IQNB labeling in those brain regions containing predominantly the m2 subtype is reduced to background levels. We conclude that FQNB is m2-selective in vivo and that [18F]FQNB or a closely related analogue may be of potential use in positron emission tomographic study of the loss of m2 receptors in AD. © 1996 Academic Press, Inc.
INTRODUCTION Alzheimer’s disease (AD) appears to involve selective loss of m2 subtype neuroreceptors in cortical and hippocampal regions of the human brain, as determined by the use of in vitro subtype-selective pharmacological characterization (Mash et al., 1985; Wang et al., 1987; Araujo et al., 1988; Quirion et al., 1989; Aubert et al., 1992) or, more recently, by molecular characterization (Flynn et al., 1995). Several studies (Holman et al.,
MATERIALS AND METHODS Radiopharmaceuticals and Chemicals The racemic mixture of FQNB was synthesized as previously described (Cohen et al., 1992). The (R,S)-diastereomer of IQNB, (R,S)-[125I]IQNB, was radioiodinated by the triazene method (Eckelman et al., 1985; Rzeszotarski et al., 1984) and characterized according to the method of Rzeszotarski et al.
1 “(R,S)-IQNB” has been incorrectly designated as “(R,R)-IQNB” in the literature. Thus, when we now refer to “(R,S)-IQNB,” we are referring to the same diastereoisomer that was previously called “(R,R)-IQNB.” This issue will be addressed in detail (Kiesewetter et al., 1996; Zeeberg et al., submitted for publication). To avoid unnecessary confusion, the corrected designation has been employed here and when referring to previous publications.
35 1053-8119/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
36
BOULAY ET AL.
FQNB DISPLAYS IN VIVO m2 SELECTIVITY
37
Note. Computed from the data reported in (Li et al., 1991; Wall et al., 1991a,b; Yasuda et al., 1993). Abbreviations used in this table and elsewhere are: left corpus striatum (csl), right corpus striatum (csr), hippocampus (hipp), left frontal cortex (fcl), right frontal cortex (fcr), left thalamus (thl), right thalamus (thr), medulla (med), pons (pons), cerebellum (cb), inferior colliculus (ic), and superior colliculus (sc).
(1984). (R,S)-[125I]IQNB was obtained by the reaction of an (R,S)-QNB triazene precursor with Na125I. The product was purified by high-performance liquid chromatography (HPLC) using a Z-module C-18 Bondapak reversed-phase cartridge, eluted with MeOH:water (60:40) containing 10 mM formic acid and 1% octane sulfonic acid as an ion pair agent to improve resolution. Final radiochemical purity was $95% as determined by thin-layer chromatography (TLC). The specific activity of (R,S)-[125I]IQNB (averaging approximately 900 Ci/mmol) was determined by comparing the radioactivity specifically bound to a caudate nucleus homogenate to that bound when using [3H]QNB of known specific activity (Cohen et al., 1989): A saturation analysis is performed using caudate nucleus homogenate in the presence of eight different concentrations of the [3H]QNB of known specific activity. In this analysis, eight identical samples are studied in the presence of atropine to provide a subtraction for nonspecific binding. Based upon the known specific activity of the [3H]QNB and a computer fit of the specific binding of the [3H]QNB to a single binding site, the concentration of muscarinic binding sites is computed. Simultaneously, an identical saturation experiment is performed using the (R,S)-[125I]IQNB of unknown specific activity. In this case, the computer fit results in a saturating specific binding expressed in terms of disintegrations per minute (dpm). Dividing this saturating radioactivity value determined with the (R,S)-[125I]IQNB of unknown specific activity by the concentration of muscarinic binding sites determined with the [3H]QNB of known specific activity provides the specific activity of the (R,S)-[125I]IQNB. In Vivo Studies Male Sprague–Dawley rats weighing 200–250 g were used in the experiments. Animals were anesthe-
tized with ketamine:xylazine (100:10 mg/kg im) and the right jugular vein was exposed for intravenous injection. One hundred sixty-five microcuries of (R,S)[125I]IQNB in 0.4 ml of normal saline containing up to 50% ethanol was injected alone or coinjected with 30.0 nmol of the racemic mixture of FQNB. Animals were maintained under anesthesia until time of sacrifice at 2 h postinjection. Animals were sacrificed by decapitation and the brains were rapidly removed, blotted free of excess blood, immediately frozen on dry ice, and stored at −70°C until sliced. Autoradiography The frozen brains were sliced into 20-mm-thick coronal sections and thaw mounted onto chrome-alum/ gelatin-coated slides. The slides along with 125I standards (Amersham) were then apposed to hyperfilm MP (Amersham) for 1 to 3 weeks, depending upon the amount of radioactivity within a typical section. The film was developed using an automated x-ray film developer (Kodak RP X-OMAT film processor, Model M6B). The images were digitized using a RAS 3000 imaging system (Loats, Inc.) and analysis was performed on a Macintosh IIci computer using the public domain NIH Image program (written by Wayne Rasband at the U.S. National institutes of Health and available from the Internet by anonymous ftp from zippy.nimh.nih.gov or on a floppy disk from NTIS, 5285 Port Royal Rd., Springfield, VA 22161, part number PB93-504868). Data Analysis For each film, all digitized images were converted to optical density (OD) values using a program developed within our group. Brain regions to be measured were
FIG. 1. Rats were injected in the jugular vein with 165 mCi of (R,S)-[125I]IQNB in the absence (A–C) or presence (D–F) of 30.0 nmol of coinjected racemic FQNB. Those regions that are enriched in the m2 subtype (Table 1) are preferentially blocked by FQNB. Representative coronal slices shown are through the anteroventral nucleus of the thalamus (A, D), the hippocampus (B, E), and the pontine nuclei (C, F). The profiles demonstrate the regional (R,S)-[125I]IQNB distribution in the absence (darker lines) and in the presence (lighter lines) of coinjected FQNB. The profiles (with approximate horizontal axis positions given in parentheses) for A and D traversed parietal cortex (15 and 225), caudate putamen (45 and 200), anteroventral thalamic nucleus, ventrolateral (100 and 150), and anteroventral thalamic nucleus, dorsomedial (110 and 140). The profiles for B and E traversed frontal cortex (20), hippocampus (50), thalamic nuclei (90), and hypothalamic nuclei (130). The profiles for C and F traversed occipital cortex (10), subiculum (35), superior colliculus (45), and pons (110).
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selected by reference to coronal sections displayed in a standard rat brain atlas (Paxinos and Watson, 1986). Because of interanimal variability in percentage dose/g for (R,S)-[125I]IQNB binding, we now express data as ratios relative to a control region, which has been shown to be highly reproducible from animal to animal (Gitler et al., 1995). Thus, the color scales used for displaying and comparing the autoradiographic images have been adjusted to provide an approximately equivalent intensity for the control regions, which constitute corpus striatum or hippocampus (if these are present in the slice) or cortex. Profiles were also normalized so as to maintain these control regions at a value of approximately unity. RESULTS AND DISCUSSION Despite the fact that QNB exhibits little or no subtype selectivity in in vitro assays (Zeeberg et al., 1991), QNB exhibits substantial m2 selectivity in in vivo studies (Gitler et al., 1994, 1995; McRee et al., 1995). Similar discrepancies in mouse brain between in vitro and in vivo subtype selectivity for the opiate receptor ligand diprenorphine have been reported by Frost et al. (1984). Frost et al. found that “. . . although diprenorphine appears to bind to the opiate receptor subtypes with approximately equal affinity in vitro, it may bind to only one subtype in vivo, possibly the m receptor . . . .” FQNB also exhibits little or no subtype selectivity in in vitro assays (Eckelman et al., 1984). On the other hand, as does QNB, FQNB exhibits substantial m2 selectivity in in vivo studies. The in vivo autoradiographic results of competition studies of FQNB against (R,S)-[125II]IQNB (Fig. 1) demonstrate the in vivo m2 selectivity of FQNB. Those regions that are enriched in the m2 subtype (Table 1) are preferentially blocked by FQNB. The regional pattern of competition of FQNB is very similar to that previously exhibited by QNB (McRee et al., 1995). The profiles in the right column (Fig. 1) are measured along straight lines that were selected to traverse some m2-rich and some m2-poor regions in order to provide a quantitative sense of the differential effect upon the receptor subtypes. Some of the limitations of this method are that a quantitative measure of the in vivo selectivity of FQNB is not available. To obtain a quantitative measure would require a rather cumbersome mathematical analysis such as that which we had performed for [3H]QNB (Gitler et al., 1994). Another limitation is that, although the competition studies indicate in vivo selectivity of FQNB, direct studies using [18F]FQNB may indicate that the in vivo selectivity of FQNB is not as great as that shown by [3H]QNB (Gitler et al., 1994) or that there is excessive nonspecific binding of [18F]FQNB which would render it useless for imaging studies.
We conclude that FQNB is m2-selective in vivo and that [18F]FQNB is potentially a suitable m2 selective radioligand for positron emission tomographic study of the loss of m2 receptors in AD. However, before this goal can be achieved, the racemic mixture of FQNB should be resolved to determine which of the four diastereomers exhibits the highest degree of in vivo m2 selectivity, and a precursor for the radiofluorination of this diastereomer or of a closely related analogue (Boulay et al., 1995) would need to be synthesized. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (NS22215) and the Department of Energy (DE FG05 88ER60649).
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