Stereoselective synthesis, in vitro, and initial in vivo evaluation of 1-methylpiperidin-4-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (IPIP): a novel radioiodinated molecular probe with high affinity for the muscarinic receptor.

1-Methylpiperidin-4-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (IPIP, Fig. 1) was investigated as a potential radioiodinated molecular probe targeted to the muscarinic receptor complex. The IPIP stereoisomers were synthesized via a chiral intermediate in >95% enantiomeric excess. The R-isomers demonstrated a M(1) to M(2) subtype selectivity of approximately 3 to 1 and the S-isomers demonstrated non-subtype selective binding in vitro. IPIP was radiolabeled with iodide-125 with an average radiochemical yield of 74.4% (+/-14.8, n = 5), specific activities >800 mCi/micromol, and radiochemical purities >97%. In vivo the Z-isomers demonstrated high uniform cerebral uptake suggesting non-subtype selective binding. In contrast, E-R-IPIP, after allowing a low uptake in M(2) rich areas to clear, demonstrated a retention of activity in M(1) and M(4) rich cerebral regions. In addition, the cerebral uptake of E-R-IPIP and Z-S-IPIP were inhibited by 70-90% via pretreatment with R-QNB, an established muscarinic antagonist. An ex vivo metabolism study demonstrated Z-S-IPIP was stable at the receptor site with an absence of radiolabeled metabolites.

SPET imaging of central muscarinic acetylcholine receptors with iodine-123 labelled E-IQNP and Z-IQNP.

1-Azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (IQNP) is a muscarinic acetylcholine receptor (mAChR) antagonist and the racemic ligand contains eight stereoisomers. In a single-photon emission tomography (SPET) study in monkeys we recently confirmed that [123I]E-(R,R)-IQNP ([123I]E-IQNP) is a radioligand with modest selectivity for the M1 and M4 subtypes, whereas [123I]Z-(R,R)-IQNP ([123I]Z-IQNP) is non-subtype selective. In the present SPET study, E- and Z-IQNP were examined in human subjects. SPET examination was performed on three male subjects after i.v. injection of [123I]E-IQNP and in another three after i.v. injection of [123I]Z-IQNP. The binding potential (BP) for [123I]E-IQNP was calculated using several quantitative approaches with the cerebellum as a reference region. High-performance liquid chromatography was used to measure radioligand metabolism in plasma. Following [123I]E-IQNP, the radioactivity was high in the neocortex and striatum, intermediate in the thalamus and low in the pons and cerebellum, which is consistent with the rank order for the regional density of M1 and M4 subtypes in vitro. For all regions, peak equilibrium was identified within the 48-h data acquisition. The simplified reference tissue approach using SPET data from 0 to 48 h was the most reliable in this limited series of subjects. Following injection of [123I]Z-IQNP, radioactivity was high in the neocortex and striatum, intermediate in the thalamus and pons and low in the cerebellum, which is in agreement with the density of M1, M2 and M4 subtypes as measured in vitro. Quantitative analyses provided indirect support for specific M2 binding of Z-IQNP in the cerebellum. The high selectivity of [123I]E-IQNP for M1 and M4 receptors allowed the use of cerebellum as a reference region devoid of specific binding, and may be advantageous for applied clinical studies of M1 and M4 receptors binding in man. [123I]Z-IQNP has potential for exploration of M2 receptor binding in the cerebellum.

Therapy with lesbian and gay parents and their children.

This article explores some of the social and clinical issues facing the many different kinds of gay and lesbian families that are becoming increasingly visible in the United States. Research findings are discussed that dispel popularly held myths and stereotypes concerning these families, gays and lesbians as parents, and their children. Clinical vignettes are presented to illustrate issues often encountered in the consulting room, some unique to gay and lesbian families and some common to all families.

Z-IQNP: a potential radioligand for SPECT imaging of muscarinic acetylcholine receptors in Alzheimer's disease.

RATIONALE: The density of the M2 subtype of muscarinic acetylcholine receptors (mAChR) has been shown to be reduced in the brain of patients with Alzheimer's disease (AD). It is therefore of interest to develop a brain imaging method for diagnostic purposes. Z-(R,R)-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo1-propen-3-yl)-alpha-phenylacetat e (Z-IQNP) is a muscarinic antagonist with high affinity for the M2 subtype. OBJECTIVE: The pharmacological characteristics and topographic distribution of radiolabelled Z-IQNP as a radioligand for the M2 mAChR subtype were examined in vitro and in vivo. METHODS: Z-IQNP was labelled with 1251 and 123I. Autoradiography was performed on whole-hemisphere cryosections from human post mortem brains. SPECT was performed in a cynomolgus monkey. RESULTS: Autoradiography showed binding of [125I]Z-IQNP in all brain regions, which was inhibited by the non-selective muscarinic antagonist scopolamine. The addition of BIBN 99, a compound with high affinity for the M2 subtype, inhibited [125I]Z-IQNP binding particularly in the cerebellum, which has a high density of the M2 subtype. SPECT demonstrated high uptake of [123I]Z-IQNP in all brain regions. The binding was markedly reduced in all brain regions after pretreatment with the non-selective muscarinic antagonist dexetimide and also the M1 antagonist biperiden. Dexetimide markedly inhibited [123I]Z-IQNP binding in the cerebellum, which is consistent with a high density of M2-receptors in this region. The sigma receptor binding compound DuP 734 had no effect on Z-IQNP binding either in vitro or in vivo. CONCLUSIONS: This study indicates that radiolabelled Z-IQNP has high specificity for mAChR with higher affinity for the M2 than the M1 subtype and negligible affinity for sigma recognition sites both in vitro and in vivo. [123I]Z-IQNP should be useful for future SPECT studies in AD for examination of the density of M2 receptors particularly in the cerebellum.

In vivo biodistribution of 125IPIP and internal dosimetry of 123IPIP radioiodinated agents selective to the muscarinic acetylcholinergic receptor complex.

The development of new radioiodinated ligands for imaging the muscarinic acetylcholinergic complex (mAChR) using single photon emission computed tomography (SPECT) requires the evaluation of human organ doses prior to approval for human use. Animal biodistribution and excretion data were obtained and evaluated for IPIP, a new mAChR agent. Preliminary biodistribution studies were performed on four different stereoisomers of IPIP. A biokinetic model of the Z-(S)-IPIP stereoisomer was constructed for the rat and used to estimate the internal absorbed dose in humans based on an extrapolation of the rat model. The thyroid is the critical organ for this radiopharmaceutical, with an absorbed dose estimate of 2.4 mGy/MBq for both males and females, when labeled with 123I. Even when blocked, the thyroid is still the critical organ, yet with a 90% dose reduction. The heart and brain receive the next highest doses in both males and females. Effective dose estimates for the use of pure 123I-PIP in humans are 0.16 mSv/MBq for males and 0.14 mSv/MBq for females. The biodistribution studies of the Z-(S)-IPIP stereoisomer showed the most promise as a successful agent for imaging muscarinic receptor sites in the heart and brain. IPIP also demonstrated potential as a therapeutic radiopharmaceutical for some colon carcinomas where muscarinic receptor sites are expressed in the tumor cells. These results provide preliminary data for use of IPIP in clinical studies on humans.

Evaluation of Z-(R,R)-IQNP for the potential imaging of m2 mAChR rich regions of the brain and heart.

Alterations in the function or density of the m2 muscarinic (mAChR) subtype have been postulated to play an important role in various dementias such as Alzheimer's disease. The ability to image and quantify the m2 mAChR subtype is of importance for a better understanding of the m2 subtype function in various dementias. Z-(R)-1-Azabicyclo[2.2.2]oct-3-y (R)-alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (Z-(R,R)-IQNP) has demonstrated significant uptake in cerebral regions that contain a high concentration of m2 mAChR subtype in addition to heart tissue. The present study was undertaken to determine if the uptake of Z-(R,R)-IQNP in these regions is a receptor mediated process and to identify the radiospecies responsible for binding at the receptor site. A blocking study demonstrated cerebral and cardiac levels of activity were significantly reduced by pretreatment (2-3 mg/kg) of (R)-3-quinuclidinyl benzilate, dexetimide and scopolamine, established muscarinic antagonists. A direct comparison of the cerebral and cardiac uptake of [I-125]-Z-(R,R)-IQNP and [I-131]-E-(R,R)-IQNP (high uptake in ml, m4 rich mAChR cerebral regions) demonstrated Z-(R,R)-IQNP localized to a higher degree in cerebral and cardiac regions containing a high concentration of the m2 mAChR subtype as directly compared to E-(R,R)-IQNP. In addition, a study utilizing [I-123]-Z-(R,R)-IQNP, [I-131]-iododexetimide and [I-125]-R-3-quinuclidinyl S-4-iodobenzilate, Z-(R,R)-IQNP demonstrated significantly higher uptake and longer residence time in those regions which contain a high concentration of the m2 receptor subtype. Folch extraction of global brain and heart tissue at various times post injection of [I-125]-Z-(R,R)-IQNP demonstrated that approximately 80% of the activity was extracted in the lipid soluble fraction and identified as the parent ligand by TLC and HPLC analysis. These results demonstrate Z-(R,R)-IQNP has significant uptake, long residence time and high stability in cerebral and cardiac tissues containing high levels of the m2 mAChR subtype. These combined results strongly suggest that Z-(R,R)-IQNP is an attractive ligand for the in vivo imaging and evaluation of m2 rich cerebral and cardiac regions by SPECT.

Dual-label studies with [125I]-3(R)/[131I]-3(S)-BMIPP show similar metabolism in rat tissues.

UNLABELLED: Biodistribution studies with the radioiodinated 3(R)- and 3(S)-isomers of 15-(p-iodophenyl)-3-methylpentadecanoic acid (BMIPP) in rats have shown that 3(R)-BMIPP has 20%-25% higher heart uptake than 3(S)-BMIPP (15-180 min). In contrast, the 3(S)-isomer has slightly higher liver uptake, and uptake in other tissues examined is similar. METHODS: To evaluate the possible differences in metabolic fate of the two isomers, a mixture of [125I]-3(R)/[131I]-3(S)-BMIPP was administered to fasted female Fisher rats. Groups of rats (3 per group) were killed 15, 60 and 180 min after administration. Urine and feces were collected from a fourth group (n = 3) over 7 d. Samples of blood, heart, liver, lungs, kidney and urine were Folch extracted. The distributions of 125I and 131I in the organic (lipid), aqueous and pellet samples were determined. The lipid samples as well as the organic fractions from base-hydrolyzed triglyceride (TG) fractions and acid-hydrolyzed urine samples were then analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). RESULTS: The relative distributions of 125I and 131I in the lipid, aqueous and pellet samples were similar for both isomers. Distribution of 125I and 131I in the various components of the lipid extracts observed by TLC (hexane:ether:HOAc, 70:30:1) was also similar, with principal incorporation into the free fatty acid (FFA) and TG pools. HPLC analyses (C18) of the FFA fraction showed similar 125I and 131I profiles, corresponding to BMIPP, and the alpha-methyl-C14 (14-(p-iodophenyl)-3-(R,S)-methyltetradecanoic acid) and C12, C10 and C6 carbon chain-length catabolites. By TLC, radioactive components of 125I and 131I in the urine had the same TLC mobility as hippuric acid. HPLC analyses (C18) of acid-hydrolyzed urine gave a single 125I/131I component with the same relative retention time as 2-(p-iodophenyl)acetic acid, which is the final alpha/beta-oxidative BMIPP catabolite. Unexpectedly, HPLC of lipids from base-hydrolyzed TG from the heart tissue showed 125I/131I components with the same retention times as shorter-chain fatty acids, similar to the FFA fraction, with only low levels of activity detected in BMIPP. CONCLUSION: These results show that 3(R)-BMIPP and 3(S)-BMIPP are metabolized similarly in rat tissues and that higher myocardial extraction observed for 3(R)-BMIPP may reflect differences in the relative membrane transport of the two isomers.

Improved radioiodination of 1,2-dipalmitoyl-3-IPPA via a tributyltin intermediate.

1,2-Palmitoyl-3-[15-(4-iodophenyl)pentadecan-3-oyl]-rac-glycerol (MIPAG) is a new agent for the clinical evaluation of pancreatic lipase activity and has demonstrated promise in preliminary clinical studies with patients affected with pancreatic insufficiency. Iodine-131-MIPAG was initially prepared via thallium-iodide displacement. Because of the need for a simple method which is amendable for the routine clinical use of MIPAG we have investigated the preparation and radioiodination of MIPAG utilizing the tributyltin precursor, 1,2-palmitoyl-3-[15-(4-tributylstannylphenyl)pentadecan-3-oy l]-rac-glycerol (TBT-MIPAG, 2). Compound 2 was prepared via the condensation of 1,2-palmitoyl-rac-glycerol with 15-(4-tributylstannylphenyl)pentadecanoic acid (TBT-PPA) prepared from 4-bromophenylacetylene. Electrophilic radioiodination using peracetic acid with sodium iodide-125 in ethanol at 80 degrees C for 60 min afforded I-125-MIPAG in 65.9% (+/- 11.5%) yield and radiochemical purity of 94% (+/- 3.0%) after C-18 Sep-Pak purification (n = 6). This improved method for radioiodination utilizing TBT-MIPAG now provides radioiodinated MIPAG for routine clinical evaluation.

A rapid and simple Sep Pak method for purification of radioiodinated IQNP, a high affinity ligand for the muscarinic receptor.

A simplified procedure for the purification of 1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (IQNP) stereoisomers utilizing a silica Sep Pak (SSP) is described. Iodine-131-E- and iodine-125-Z-(R,R)-IQNP were isolated after SSP purification in 80% and 75% radiochemical yields, respectively. The biodistribution of iodine-131-E-/iodine-125-Z-(R,R)-IQNP, purified either by SSP or high performance liquid chromatography (HPLC), was evaluated in female rats and demonstrated no significant differences in the uptake in various organs and cerebral regions. The utilization of SSP thus affords a simple and rapid method for the purification of IQNP for use in a variety of animal studies.

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.

Evaluation and metabolite studies of 125I- and 123I-labelled E-(R,R)-IQNP: potential radioligands for visualization of M1 muscarinic acetylcholine receptors in brain.

A new ligand for the M1 muscarinic receptor subtype, E-(R,R)-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (E-IQNP), was labelled with 125I and 123I for autoradiographic studies on human whole-brain cryosections and SPET studies, respectively, in Cynomolgus monkey. Autoradiography demonstrated E-[125I]IQNP binding in M1 receptor-rich regions such as the neocortex and the striatum. The binding was displaceable by the selective M1 antagonist biperiden. In vivo single photon emission tomography (SPET) studies with E-[123I]IQNP demonstrated a high accumulation of radioactivity in the monkey neocortex. Rapid hydrolysis of the quinuclidinyl ester to the free acid was found to be a major biotransformation route for E-[123I]IQNP. The free acid of E-[123I]IQNP does not pass the blood-brain barrier, but the plasma concentration was high as compared to the total radioactivity in brain. It is thus necessary to correct for the high concentration of radioactive metabolites in parenchymal blood (CBV) to obtain accurate values for E-[123I]IQNP binding in brain.

Effects of configuration on the myocardial uptake of radioiodinated 3(R)-BMIPP and 3(S)-BMIPP in rats.

UNLABELLED: Radioiodinated 3(R)-(+)- and 3(S)-(-)-15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (BMIPP) were prepared and evaluated in rats to investigate the effects of absolute configuration of the 3(beta)-methyl group on myocardial uptake and release kinetics. METHODS: The 3(R)-(+)-BMIPP analog was synthesized by initial acylation of a thiophene template with the acid chloride of ethyl 3(R)-methylglutarate. 3(S)-(-)-BMIPP was obtained by separation from the mixture of diastereomeric amides prepared from reaction of the acid chloride of racemic BMIPP with the S-(-)-alpha-methylbenzylamine. The amide of synthetic 3(R)-BMIPP prepared from S-(-)-alpha-methylbenzylamine was identical to the chromatographically more polar isomer. Free acids were obtained by acid hydrolysis of the amides, fully characterized and then converted to the radioiodinated BMIPP isomers. RESULTS: Biodistribution studies in rats with the dual-labeled [(131)I]-3(S)-BMIPP/[(125)I]-3(R)-BMIPP mixture demonstrated greater myocardial uptake of 3(R)-BMIPP compared with the 3(S)-BMIPP isomer [60 min: 3(R)-BMIPP = 4.37 %ID/g; 3(S)-BMIPP = 3.44; p < 0.05; 180 min, 2.31 and 1.78 %ID/G, respectively, p < 0.01], although both isomers had similar myocardial washout curves (5-180 min). Percent ID/g values for other tissues which were examined (blood, lungs, thyroid) were similar. CONCLUSION: Higher myocardial uptake of the 3(R)-BMIPP isomer observed in these animal studies may suggest differences in carrier-mediated myocyte uptake of the two isomers. These studies suggest that [(123)I]-3(R)-BMIPP is a candidate for clinical evaluation and may show greater myocardial uptake than the 3(S)-BMIPP isomer and may thus require reduced injected dose.

Radiolabeled ligands for imaging the muscarinic-cholinergic receptors of the heart and brain.

Interest in the potential use of cerebral SPECT and PET imaging for determination of the density and activity of muscarinic-cholinergic receptors (mAChR) has been stimulated by the changes in these receptors which occur in many neurological diseases. In addition, the important involvement of mAChR in modulating negative inotropic cardiac activity suggests that such receptor ligands may have important applications in evaluation of changes which may occur in cardiac disease. In this paper, the properties of several key muscarinic receptor ligands being developed or which have been used for clinical SPECT and PET are discussed. In addition, the ORNL development of the new iodinated IQNP ligand based on IQNB and the results of in vivo biodistribution studies in rats, in vitro competitive binding studies and ex vivo autoradiographic experiments are described. The use of radioiodinated IQNP may offer several advantages in comparison to IQNB because of its easy and high yield preparation and high brain uptake and the potential usefulness of the "partial" subtype selective IQNP isomers. We also describe the development of new IQNP-type analogues which offer the opportunity for radiolabeling with positron-emitting radioisotopes (carbon-11, fluorine-18 and bromine-76) for potential use with PET.

Positron emission tomographic investigations of central muscarinic cholinergic receptors with three isomers of [76Br]BrQNP.

We studied the potential of three radiobrominated isomers of BrQNP, (Z(-,-)-[76Br]BrQNP, E(-,-)-[76Br]BrQNP and E(-,+)-[76Br]BrQNP), as suitable radioligands for imaging of central muscarinic cholinergic receptors in the human brain. These radioligands were stereospecifically prepared by electrophilic radiobromodestannylation of the respective tributylstannyl precursors using no-carrier-added [76Br]BrNH4 and peracetic acid. Preliminary pharmacological characterizations were determined by biodistribution, autoradiography, competition, displacement and metabolite studies in rats. The (-,-)-configuration presented important specific uptakes in brain muscarinic cholinergic receptor (mAChR)-rich structures and in heart, low metabolization rates and an apparent M2 selectivity. The (-,+)-configuration revealed more rapid clearance, lower uptake, a higher metabolization rate and an apparent M1 selectivity. Reversibility of the binding was confirmed for the three radiotracers. Positron emission tomography in the living baboon brain revealed high and rapid uptake in the brain and accumulation in the mAChR-rich structures studied. At 30 min p.i., the E(-,-)-radiotracer reached a plateau in cortex, pons and thalamus with concentrations of 29%, 24% and 19% ID/l, respectively. Z(-,-)-[76Br]BrQNP also accumulated in these structures, reaching a maximal uptake (27% ID/l) in the cortex 2 h p.i. At 5 min p.i. a plateau (17% ID/l) was only observed in the cortex for the E(-, +)-[76Br]BrQNP; by contrast, the other structures showed slow washout. After 3 weeks, the (-,-)-radiotracers were studied in the same baboon pretreated with dexetimide (1 mg/kg), a well-known muscarinic antagonist. In all the mAChR structures, the highly reduced uptake observed after this preloading step indicates that these radiotracers specifically bind to muscarinic receptors. Z(-, -)-[76Br]BrQNP, which is displaced in higher amounts from M2 mAChR-enriched structures, reveals an M2 affinity. The two isomers having the (-,-)-configuration are potential probes for investigating central muscarinic receptors. The absolute configuration on the acetate chiral centre influences their muscarinic subtype selectivity and the cis-trans isomerism of the vinyl moiety affects their specific fixation.

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.

Evaluation of 1-azabicyclo[2.2.2]oct-3-yl alpha-fluoroalkyl-alpha-hydroxy-alpha-phenylacetates as potential ligands for the study of muscarinic receptor density by positron emission tomography.

Both 1-azabicyclo[2.2.2]oct-3-yl alpha-(1-fluoroeth-2-yl)-alpha-hydroxy-alpha-phenylacetate (FQNE, 5) and 1-azabicyclo[2.2.2]oct-3-yl alpha-(1-fluoropent-5-yl)-alpha-hydroxy-alpha-phenylacetate (FQNPe, 6) were prepared and evaluated as potential candidates for the determination of muscarinic cholinergic receptor (mAChR) density by positron emission tomography (PET). The results of in vitro binding assays demonstrated that although both 5 and 6 had high binding affinities for m1 and m2 mAChR subtypes, 6 displayed a higher affinity (nM, m1; KD, 0.45, m2; KD, 3.53) as compared to 5 (nM, m1; KD, 12.5, m2; KD, 62.8). It was observed that pretreatment of female Fisher rats with either 5 or 6 prior to the i.v. administration of Z-(-)(-)-[131I]-IQNP, a high-affinity muscarinic ligand, significantly blocked the uptake of radioactivity in the brain and heart measured 3 h postinjection of the radiolabeled ligand. These new fluoro QNB analogues represent important target ligands for evaluation as potential receptor imaging agents in conjunction with PET.

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.

Resolution and in vitro and initial in vivo evaluation of isomers of iodine-125-labeled 1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate: a high-affinity ligand for the muscarinic receptor.

1-Azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)- alpha-phenylacetate (IQNP, 1), is a highly selective ligand for the muscarinic acetylcholinergic receptor (mAChR). There are eight stereoisomers in the racemic mixture. The optical isomers of alpha-hydroxy-alpha-phenyl-alpha-(1-propyn-3-yl)acetic acid were resolved as the alpha-methylbenzylamine salts, and the optical isomers of 3-quinuclidinol were resolved as the tartrate salts. The E and Z isomers were prepared by varying the reaction conditions for the stannylation of the triple bond followed by purification utilizing flash column chromatography. In vitro binding assay of the four stereoisomers containing the (R)-(-)-3-quinuclidinyl ester demonstrated that each isomer of 1 bound to mAChR with high affinity. In addition, (E)-(-)-(-)-IQNP demonstrated the highest receptor subtype specificity between the m1 molecular subtype (KD, nM, 0.383 +/- 0.102) and the m2 molecular subtype (29.6 +/- 9.70). In vivo biodistribution studies demonstrated that iodine-125-labeled (E)-(-)-(+)-1 cleared rapidly from the brain and heart. In contrast, iodine-125-labeled (E)-(-)-(-)-, (Z)-(-)-(-)-, and (Z)-(-)-(+)-1 have high uptake and retention in mAChR rich areas of the brain. It was also observed that (E)-(-)-(-)-IQNP demonstrated an apparent subtype selectivity in vivo with retention in M1 (m1, m4) mAChR areas of the rain. In addition, (Z)-(-)-(-)-IQNP also demonstrated significant uptake in tissues containing the M2 (m2) mAChR subtype. These results demonstrate that the iodine-123-labeled analogues of the (E)-(-)-(-)- and (Z)-(-)-(-)-IQNP isomers are attractive candidates for single-photon emission-computed tomographic imaging of cerebral and cardiac mAChR receptor densities.

Characterization of in vivo brain muscarinic acetylcholine receptor subtype selectivity by competition studies against (R,S)-[125I]IQNB.

We have studied the in vivo rat brain muscarinic acetylcholine receptor (mAChR) m2 subtype selectivities of three quinuclidine derivatives: (R)-3-quinuclidinyl benzilate (QNB), E-(+,+)-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (E-(+,+)-IQNP), and E-(+,-)-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (E-(+,-)-IQNP), and two tricyclic ring compounds: 5-[[4-[4-(diisobutylamino)butyl]-1-phenyl]-10,11-dihydro-5H-dibenz o [b,e][1,4]diazepin-11-one [sequence: see text] (DIBD) and 11-[[4-[4-(diisobutylamino)butyl-1-phenyl]acetyl]-5,11-dihydro-6H- pyrido [2,3-b][1,4]benzodiazepin-6-one [sequence: see text] (PBID), by correlating the regional inhibition of (R,S)-[125I]IQNB with the regional composition of the m1-m4 subtypes. Subtle effects are demonstrated after reduction of the between-animal variability by normalization to corpus striatum. Substantial in vivo m2-selectivity is exhibited by QNB and DIBD, modest in vivo m2-selectivity is exhibited by E-(+,+)-IQNP, and little or no in vivo m2-selectivity is exhibited by PBID and E-(+,-)-IQNP. Surprisingly, the in vivo m2-selectivity is not correlated with the in vitro m2-selectivity. For example, QNB, which appears to be the most strongly in vivo m2-selective compound, exhibits negligible in vitro m2-selectivity. These examples indicate that a strategy which includes only preliminary in vitro screening may very well preclude the discovery of a novel compound which would prove useful in vivo.

In vivo metabolic studies of the trans-(R,R) isomer of radioiodinated IQNP: a new ligand with high affinity for the M1 muscarinic-cholinergic receptor.

E-(R,R)-IQNP is a new ligand analogue of IQNB, which has high affinity for the cholinergic-muscarinic receptor. Earlier studies have demonstrated high cerebral uptake of activity with selective localization in M1 receptor subtype areas of the brain. In this paper we describe the results of metabolic studies of E-(R,R)-IQNP directed at determining the metabolic fate of this ligand and the identification of the radioactive species observed in the brain and heart tissue. Tissue Folch extracts demonstrated that the lipid-soluble extracts from brain contained 87.0% +/- 1.65% of the activity up to 24 h. In the heart, 61.9% +/- 7.50% of the activity was extracted in the lipid-soluble extract after 30 min, decreasing to 51.4% +/- 0.65% by 4 h. In contrast, data from other tissues studied demonstrated large amounts of either aqueous soluble activity or activity which was not extracted from the tissue pellet material; analysis of lipid organic extracts revealed the following results: liver (4 h), 7.43% +/- 0.96%; serum (4 h), 3.73% +/- 0.87%; urine (24 h), 9.4%; feces (24 h), 16.5%. Thin-layer chromatographic (TLC) and high-performance liquid chromatographic (HPLC) analyses of lipid-soluble brain extracts indicated that only unmetabolized E-(R,R)-IQNP was detected (99.4% +/- 1.25%). Activity which was extracted into the organic phase from heart tissue was also determined by TLC and HPLC analysis to contain large amounts of unmetabolized ligand after 4 h (88.5% +/- 0.57%). In addition, however, low levels of two additional radioactive components were detected which increased with time. TLC analysis of the plasma lipid extracts indicated only a small amount of unmetabolized E-(R,R)-IQNP.(ABSTRACT TRUNCATED AT 250 WORDS)