In vivo competition studies of Z-(-,-)-[125I]IQNP against 3-quinuclidinyl 2-(5-bromothienyl)-2-thienylglycolate (BrQNT) demonstrating in vivo m2 muscarinic subtype selectivity for BrQNT.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Until recently, emission tomographic study of the loss of m2 receptors in AD has been limited by the absence of available m2-selective radioligands that can penetrate the blood-brain barrier. We now demonstrate the in vivo m2 selectivity of an analog of (R)-QNB, 3-quinuclidinyl 2-(5-bromothienyl)-2-thienylglycolate (BrQNT), by dissection and autoradiographic studies of the in vivo inhibition of radioiodinated Z-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenyl-acetate (Z-(-,-)-[125I]IQNP) binding by unlabeled BrQNT in rat brain. In the absence of BrQNT, Z-(-,-)-[125I]IQNP labels brain regions containing muscarinic receptors, with an enhanced selectivity for the m2 subtype. In the presence of 60-180 nmol of co-injected racemic BrQNT, Z-(-,-)-[125I]IQNP labeling in those brain regions containing predominantly m2 subtype is reduced to background levels, while levels of radioactivity in areas not enriched in the m2 subtype do not significantly decrease. We conclude that BrQNT is m2-selective in vivo, and that [76Br]BrQNT, or a radiofluorinated analog, may be of potential use in positron emission tomographic (PET) study of the loss of m2 receptors in AD. In addition, a radioiodinated analog may be of potential use in single photon emission tomographic (SPECT) studies.

In vivo autoradiographic competition studies of isomers of [125I]IQNP against QNB demonstrating in vivo m2 muscarinic subtype selectivity for QNB.

(R,S)-[125I]IQNB has been used extensively in in vivo studies in rats, and has been of utility in demonstrating the in vivo subtype selectivity of nonradioactive ligands in competition studies. Because of the implications for the study of Alzheimer's disease (AD), those ligands that demonstrate m2 selectivity are of particular interest. Radiolabelled Z- and E-(-,-)-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (Z- and E-(-,-)-[125I]IQNP) are analogs of (R,S)-[125I]IQNB. Rat brain regional dissection studies and in vivo autoradiographic comparison of the time-courses of (R,S)-[125I]IQNB, Z-(-,-)-[125I]IQNP, and E-(-,-)-[125I]IQNP have indicated that Z- and E-(-,-)-[125I]IQNP, in general, are distributed similarly to (R,S)-[125I]IQNB. Z-(-,-)-[125I]IQNP binds to the muscarinic receptors in those brain regions enriched in the m2 subtype with approximately a two- to fivefold higher % dose/g compared with (R,S)-[125I]IQNB. Thus, as we show here autoradiographically, using QNB as the competing nonradioactive ligand in in vivo competition studies against Z-(-,-)-[125I]IQNP provides a sensitive and accurate probe for demonstrating the in vivo m2 selectivity of nonradioactive ligands.

In vivo autoradiography of radioiodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate [(R, S)-IQNB] and (R)-3-quinuclidinyl (R)-4-iodobenzilate [(R,R)-IQNB]. Comparison of the radiolabelled products of a novel tributylstannyl precursor with those of the established triazene and exchange methods.

Radioiodinated (R,S)-IQNB and (R,R)-IQNB are prepared either from a triazene precursor or using an exchange reaction. In both cases the radiochemical yield is low. The product of the exchange reaction also suffers from having a fairly low specific activity. A new method for preparing radioiodinated (R,S)-IQNB and (R,R)-IQNB from a tributylstannyl precursor has recently been developed. This method is more convenient and much faster than the triazene and exchange methods, and it reliably results in a high radiochemical yield of a high specific activity product. In rat brain, the in vivo properties of the radioiodinated products of the tributylstannyl method are identical to those of the corresponding radioiodinated (R,S)-IQNB and (R,R)-IQNB prepared using the triazene and exchange methods. Dissection studies of selected brain regions show that at 3 h post injection (R,S)-[125I]IQNB prepared by all three methods have indistinguishable % dose g-1 values in all brain regions studied. Autoradiographic comparison of coronal slices through the anteroventral nucleus of the thalamus, through the hippocampus and through the pons at 2 h post injection shows that (R,S)-[125I]IQNB prepared by the triazene and tributylstannyl methods have indistinguishable patterns of binding.

Autoradiographic evidence that (R)-3-quinuclidinyl (S)-4-fluoromethylbenzilate ((R,S)-FMeQNB) displays in vivo selectivity for the muscarinic m2 subtype.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Until recently, emission tomographic study of the loss of m2 receptors in AD has been limited by the absence of available m2-selective radioligands that can penetrate the blood-brain barrier. We now demonstrate the in vivo m2 selectivity of a fluorinated derivative of QNB, (R)-3-quinuclidinyl (S)-4-fluoromethylbenzilate ((R,S)-FMeQNB), by studying autoradiographically the in vivo inhibition of radioiodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) binding by unlabelled (R,S)-FMeQNB. In the absence of (R,S)-FMeQNB, (R,S)-[125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 75 nmol of (R,S)-FMeQNB, (R,S)-[125I]IQNB labelling in those brain regions containing predominantly m2 subtype is reduced to background levels. We conclude that (R,S)-FMeQNB is m2-selective in vivo, and that (R,S)-[18F]FMeQNB may be of potential use in positron emission tomographic (PET) study of the loss of m2 receptors in AD.

Autoradiographic evidence that 3-quinuclidinyl-4-fluorobenzilate (FQNB) displays in vivo selectivity for the m2 subtype.

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.

In vivo autoradiographic and dissection evaluation of isomers of 125I-labeled 1-azabicyclo[2.2.2] oct-3-yl-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (IQNP).

(R,S)-[125I]IQNB has been used extensively in in vivo studies in rats and has been of utility in demonstrating the in vivo subtype selectivity of nonradioactive ligands in competition studies. Radiolabeled Z- and E-(-,-)-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (Z- and E-[-,-]-[125I]IQNP) are analogs of (R,S)-[125I]IQNB. Preliminary rat brain regional dissection studies have indicated that Z- and E-(-,-)-[125I]IQNP, in general, are distributed similarly to (R,S)-[125I]IQNB. An important observation is that Z-(-,-)-[125I]IQNP binds to the muscarinic receptors in those brain regions enriched in the m2 subtype with approximately a two- to fivefold higher percent dose/g compared to (R,S)-[125I]IQNB. These observations are confirmed here by in vivo autoradiographic comparison of the time-courses of (R,S)-[125I]IQNB, Z-(-,-)-[125I]IQNP, and E-(-,-)-[125I]IQNP. Thus, in vivo competition studies against Z-(-,-)-[125I]IQNP would provide a potentially more sensitive and accurate probe for demonstrating the in vivo m2 selectivity of the nonradioactive ligands. In addition, Z-(-,-)-[123I]IQNP would potentially be useful for SPECT imaging of muscarinic receptor loss in neurodegenerative diseases.

Autoradiographic evidence that quinuclidinyl 4-(bromophenyl)-2-thienylglycolate (QBPTG) displays in vivo selectivity for the muscarinic m2 subtype.

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 an analogue of QNB, 4-(bromophenyl)-2-thienylglycolate (QBPTG), by studying autoradiographically the in vivo inhibition of radioiodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) binding by unlabeled QBPTG in rat brain. In the absence of QBPTG, (R,S)-[125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 37.5 nmol of racemic QBPTG, (R,S)-[125I]IQNB labeling in those brain regions containing predominantly the m2 subtype is reduced to background levels. We conclude that QBPTG is m2-selective in vivo and that [76Br]QBPTG, or a radiofluorinated analogue, may be of potential use in positron emission tomographic study of the loss of m2 receptors in AD. In addition, a radioiodinated analogue may be of potential use in single photon emission tomographic studies.