Comparative distribution of binding of the muscarinic receptor ligands pirenzepine, AF-DX 384, (R,R)-I-QNB and (R,S)-I-QNB to human brain

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Abstract

Quinuclidinyl benzilate (QNB) and its derivatives are being developed to investigate muscarinic receptor changes in vivo in Alzheimer's disease and dementia with Lewy bodies. This is the first study of [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB binding in vitro in human brain. We have compared the in vitro binding of the muscarinic ligands [3H]pirenzepine and [3H]AF-DX 384, which have selectivity for the M1 and M2/M4 receptor subtypes, respectively, to the binding of [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB. This will provide a guide to the interpretation of in vivo SPET images generated with [123I]-(R,R)-I-QNB and [123I]-(R,S)-I-QNB. Binding was investigated in striatum, globus pallidus, thalamus and cerebellum, and cingulate, insula, temporal and occipital cortical areas, which show different proportions of muscarinic receptor subtypes, in post-mortem brain from normal individuals. M1 receptors are of high density in cortex and striatum and are relatively low in the thalamus and cerebellum, while M4 receptors are mainly expressed in the striatum, and M2 receptors are most evident in the cerebellum and thalamus. [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB density distribution patterns were consistent with binding to both M1 and M4 receptors, with [125I]-(R,R)-I-QNB additionally binding to a non-cholinergic site not displaceable by atropine. This distribution can be exploited by in vivo imaging, developing ligands for both SPET and PET, to reveal muscarinic receptor changes in Alzheimer's disease and dementia with Lewy bodies during the disease process and following cholinergic therapy.

Introduction

In Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) cortical cholinergic neurotransmission is compromised, and this is thought to underlie cognitive decline and the neuropsychiatric symptoms experienced by these patients (Ballard et al., 2000, Perry et al., 1990a, Perry et al., 1990b, Perry and Perry, 1995, Perry et al., 1978). Acetylcholinesterase inhibitor treatment has recently been introduced in dementia, which by inhibiting the enzyme responsible for the breakdown of acetylcholine aims to maximize available transmitter. This therapy has proved efficacious especially for DLB patients. The symptoms reported to improve include cognition, delusions, apathy, agitation and hallucinations (Francis et al., 1999, McKeith et al., 2000).

Muscarinic acetylcholine receptors (mAChR) have been reported to be altered in AD and DLB. Muscarinic M1 receptors, visualised either by selective displacement of [3H]N-methylscopolamine (by the difference in binding in the presence and absence of 0.1 μM pirenzepine) or by [3H]pirenzepine binding, are elevated in DLB in temporal and parietal cortex post-mortem (Perry et al., 1990a, Perry et al., 1990b), especially in patients with persistent delusions (Ballard et al., 2000). M2 receptor density has been reported to be moderately reduced in cortex in AD (Roberson and Harrell, 1997), particularly in the hippocampus (Rodriguez-Puertas et al., 1997), and to be elevated in the striatum in AD (Rodriguez-Puertas et al., 1997). By immunoprecipitation with subtype-specific antibodies, proportions of [3H]QNB labeled muscarinic receptor subtypes differed between AD and DLB patients in temporal cortex, with a higher proportion of M1 in DLB cases, and a higher proportion of M2 in AD cases (Shiozaki et al., 1999). Coupling of M1 receptors to second messenger generating mechanisms is preserved in DLB (Perry et al., 1998), which suggests that the postsynaptic target neurons are intact, in contrast to AD in which a reduced density and coupling of M1 receptors is likely to reflect cortical neuron loss (Flynn et al., 1995, Ladner et al., 1995).

There is a need to visualise muscarinic receptors in vivo in patients suspected of suffering from AD and other dementias to specify where and when during disease progression muscarinic changes occur. In vivo imaging could provide important information as to how muscarinic receptor expression relates to symptomatology and influences the efficacy of anticholinesterase therapy (Owens et al., 2001). At present there is no ligand that will label individual muscarinic receptors with complete specificity, either in vitro or in vivo, although derivatives of QNB are being developed to demonstrate muscarinic receptor changes in vivo in dementia (Baumgold et al., 1991, Wyper et al., 1993). It should be noted that in previous reports about (R,R)-I-QNB and (R,S)-I-QNB the nomenclature of the isomers was reversed in error (Zeeberg et al., 1997), and that the correct terminology is used in the present paper.

The muscarinic receptor subtype specificity of the two isomers of [123I] QNB (R,R and R,S), suggested for use as in vivo ligands (Wyper et al., 1993, Owens et al., 2001), is investigated by comparison to the binding of [3H]pirenzepine and [3H]AF-DX 384, in post-mortem brain from elderly individuals. Different brain areas, which show varying proportions of muscarinic receptor subtypes, were compared.

[3H]Pirenzepine shows selectivity for the M1 receptor, which is of relatively high density in cortex and striatum and low density in the thalamus and cerebellum (Quirion et al., 1993). The affinity of pirenzepine for human brain M1 receptors has been reported as 2.0–2.3 nM (Perry et al., 1989) and as 5 nM (Dean et al., 1996), and as 2–8 nM in rat cortex (Watson et al., 1983). Cloned muscarinic receptor pirenzepine affinities have been found to be 18, 120, 130–180, and 660 nM for M1, M4, M3, and M2, respectively (Fukuda et al., 1989). Similarly, cloned muscarinic receptors in CHO cells have been reported to have relative IC50 values for pirenzepine for M1 as 6.3 times, 7 times, 13 times, and 17 times greater than M4, M5, M3 and M2, respectively (Buckley et al., 1989).

[3H]AF-DX 384 binds preferentially to M2 and M4 muscarinic receptor subtypes, which are expressed in striatum, cortex, thalamus and cerebellum (Quirion et al., 1993). The affinity of AF-DX 384 in human striatum was reported as 3.5 nM (Crook et al., 1999). In rat brainstem the affinity of AF-DX 384 to M2 receptors has been reported as 2.4 nM (Miller et al., 1991) and as 3–4 nM (Castoldi et al., 1991), and in rat cortex two sites have been described, with high (0.28 nM) and low (28 nM) affinity (Aubert et al., 1992). The affinity of AF-DX 384 to M2 receptors in rat brain and in cloned muscarinic receptors in CHO cells was reported to be 1.8, and 2.5 nM to M4 receptors, with lower affinity for M3 (15 nM), and M1 receptors (55 nM) (Miller et al., 1991). Binding of all four ligands was quantified in striatum, globus pallidus, thalamus, cerebellum, and cingulate, insula, temporal and occipital cortical areas.

Section snippets

Materials

[125I]-(R,S)-I-QNB and [125I]-(R,R)-I-QNB were prepared from the corresponding cold iodinated compounds using a copper(I) assisted nucleophillic exchange reaction as previously reported (Owens et al., 1992). Both the [125I]-(R,S)-I-QNB and [125I]-(R,R)-I-QNB produced were purified by high performance liquid chromatography, evaporated to dryness in-vacuo then reconstituted and stored at −20 °C in ethanol. The specific activities of [125I]-(R,S)-I-QNB and [125I]-(R,R)-I-QNB were estimated at

Results

Average specific binding in control tissue of [3H]pirenzepine, [3H]AF-DX 384, [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB, as fmol per mg tissue, in each area measured are shown in Table 1, Table 2, Table 3, Table 4, Table 5. The binding in different areas is illustrated in Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, comparing binding for all four ligands in sections from anterior striatum, post-commissural striatum, temporal and occipital cortex, cerebellum and anterior thalamus. Binding was

Pirenzepine binding

M1 receptors, as shown by [3H]pirenzepine binding, are expressed at high levels in both cortex (especially outer layers of visual and temporal cortex) and striatum, but not at all in cerebellum and at a very low level in the thalamus, which is a distribution consistent with findings in both human (Perry et al., 1989, Rodriguez-Puertas et al., 1997), and primate (Flynn and Mash, 1993) brain. Autoradiography at only one ligand concentration does not provide information about receptor affinity but

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