Regional brain uptake of the muscarinic ligand, [¹⁸F]FP-TZTP, is greatly decreased in M2 receptor knockout mice but not in M1, M3 and M4 receptor knockout mice

Abstract

A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (fP-TZTP) radiolabeled with the positron emitting radionuclide ¹⁸F ([¹⁸F]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [¹⁸F]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [¹⁸F]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.3 to 61.4%; P<0.01) when compared to the wild-type (WT) mice in amygdala, brain stem, caudate putamen, cerebellum, cortex, hippocampus, hypothalamus, superior colliculus, and thalamus. In similar studies with M1KO, M3KO and M4KO compared to their WT mice, [¹⁸F]FP-TZTP uptakes in the same brain regions were not significantly decreased at P<0.01. However, in amygdala and hippocampus small decreases of 19.5% and 22.7%, respectively, were observed for M1KO vs WT mice at P<0.05. Given the fact that large decreases in [¹⁸F]FP-TZTP brain uptakes were seen only in M2 KO vs. WT mice, we conclude that [¹⁸F]FP-TZTP preferentially labels M2 receptors in vivo.

Introduction

Our goal is to develop receptor-specific ligands labeled with a positron emitting radionuclide in order to follow biochemical changes as a function of disease by external imaging using positron emission tomography (PET). Postmortem quantitation of muscarinic receptor subtypes in Alzheimer’s patients indicate a selective loss of M2 subtype in cortical regions while the M1 subtype was preserved (Aubert et al., 1992a, Quirion et al., 1989, Rodriguez-Puertas et al., 1997). Therefore, we sought to develop an M2 selective ligand labeled with a positron-emitting radionuclide since that would be most sensitive to M2 receptor subtype density changes in the living human brain. This, in turn, would allow the study of the progression of Alzheimer’s dementia, early diagnosis, and monitoring of drug therapies using non-invasive external imaging.

We have developed a radioligand that is a fluorinated analog of a compound that showed M2 selectivity in biological assays (Sauerberg et al., 1992). This compound, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methyl-pyridine (FP-TZTP) has been radiolabeled with F-18, a positron emitting radionuclide with a half life of 109.7 min (Kiesewetter et al., 1995). Ex vivo autoradiography of rat brain using no carrier added [¹⁸F]FP-TZTP confirmed a distribution of radioactivity in gray matter which was characteristic of M2 receptor density (Kiesewetter et al., 1999). This distribution of radioactivity is in good agreement with the distribution as described in immunocytochemical localization studies of M2 receptors (Levey, 1993). Intravenous co-injection of P-TZTP [3-(3-(propyl)thio)-1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methyl-pyridine] at 5, 50 and 500 nmol per rat inhibited [¹⁸F]FP-TZTP uptake in a dose-dependent manner in all gray matter regions which contain M2 receptors. Rats co-injected with 50 nmol P-TZTP and [¹⁸F]FP-TZTP showed 55% inhibition of uptake in the heart when sacrificed 5 min after injection compared to the uptake in the heart of [¹⁸F]FP-TZTP alone. The brain distribution of the agonist [¹⁸F]FP-TZTP was only weakly inhibited by coinjection of antagonists such as RS FMeQNB (Kiesewetter et al., 1995). At present, there are no subtype selective muscarinic agonists with high enough affinity to compete with the high affinity radioligand, [¹⁸F]FP-TZTP. The inability of known antagonists to block agonist binding may be a result of different binding characteristics and pharmacokinetics (Herschberg et al., 1995).

Although these data were consistent with FP-TZTP binding to the M2 receptor subtype, the degree to which other muscarinic receptor subtypes are labeled by FP-TZTP remains uncertain. Unfortunately, no highly subtype-selective muscarinic agonists or antagonists that freely cross the blood–brain barrier have been identified. As a result, selective pharmacological blockade of specific muscarinic receptor subtypes in vivo is not possible at present. However, the recent development of mutant mouse strains in which specific muscarinic receptor genes are inactivated by gene targeting techniques has offered new perspectives for studying the selectivity of muscarinic agonists and antagonists in vivo (Gomeza et al., 1999a, Gomeza et al., 1999b, Hamilton et al., 1997). In the present study, we have used homozygous M1^−/− (M1 KO), M2^−/− (M2 KO), M3^−/− (M3 KO) and M4^−/− (M4 KO) receptor mutant mice (Fisahn et al., 2002, Gomeza et al., 1999a, Gomeza et al., 1999b, Miyakawa et al., 2001, Yamada et al., 2001) to further examine the receptor subtype selectivity of the radiotracer, [¹⁸F]FP-TZTP, in vivo. Immunoprecipitation studies with receptor subtype-selective antibodies showed that the lack of an individual muscarinic receptor subtype had no significant effect on the expression levels of the remaining muscarinic receptor subtypes (Fisahn et al., 2002, Gomeza et al., 1999a, Gomeza et al., 1999b, Miyakawa et al., 2001, Yamada et al., 2001).

The goal of this study is to use mice that are deficient in muscarinic subtypes to show unequivocally that the distribution of [¹⁸F]FP-TZTP in vivo is controlled by the M2 subtype. If [¹⁸F]FP-TZTP can be validated as a probe of the M2 receptor using muscarinic receptor subtype KO mice, then this probe will be the first available to study a specific muscarinic subtype as a function of treatment in such diseases as Alzheimer’s disease by external imaging.