Cantil: a previously unreported organ in wild-type Arabidopsis regulated by FT, ERECTA and heterotrimeric G proteins.

We describe a previously unreported macroscopic Arabidopsis organ, the cantil, named for its 'cantilever' function of holding the pedicel at a distance from the stem. Cantil development is strongest at the first nodes after the vegetative to reproductive inflorescence transition; cantil magnitude and frequency decrease acropetally. Cantils develop in wild-type Arabidopsis accessions (e.g. Col-0, Ws and Di-G) as a consequence of delayed flowering in short days; cantil formation is observed in long days when flowering is delayed by null mutation of the floral regulator FLOWERING LOCUS T. The receptor-like kinase ERECTA is a global positive regulator of cantil formation; therefore, cantils never form in the Arabidopsis strain Ler. ERECTA functions genetically upstream of heterotrimeric G proteins. Cantil expressivity is repressed by the specific heterotrimeric complex subunits GPA1, AGB1 and AGG3, which also play independent roles: GPA1 suppresses distal spurs at cantil termini, while AGB1 and AGG3 suppress ectopic epidermal rippling. These G protein mutant traits are recapitulated in long-day flowering gpa1-3 ft-10 plants, demonstrating that cantils, spurs and ectopic rippling occur as a function of delayed phase transition, rather than as a function of photoperiod per se.

Trospium Chloride Transport by Mouse Drug Carriers of the Slc22 and Slc47 Families.

BACKGROUND: The muscarinic receptor antagonist trospium chloride (TCl) is used for pharmacotherapy of the overactive bladder syndrome. TCl is a hydrophilic positively charged drug. Therefore, it has low permeability through biomembranes and requires drug transporters for distribution and excretion. In humans, the organic cation transporters OCT1 and OCT2 and the multidrug and toxin extrusion MATE1 and MATE2-K carriers showed TCl transport. However, their individual role for distribution and excretion of TCl is unclear. Knockout mouse models lacking mOct1/mOct2 or mMate1 might help to clarify their role for the overall pharmacokinetics of TCl. METHOD: In preparation of such experiments, TCl transport was analyzed in HEK293 cells stably transfected with the mouse carriers mOct1, mOct2, mMate1, and mMate2, respectively. RESULTS: Mouse mOct1, mOct2, and mMate1 showed significant TCl transport with Km values of 58.7, 78.5, and 29.3 µM, respectively. In contrast, mMate2 did not transport TCl but showed MPP+ transport with Km of 60.0 µM that was inhibited by the drugs topotecan, acyclovir, and levofloxacin. CONCLUSION: TCl transport behavior as well as expression pattern were quite similar for the mouse carriers mOct1, mOct2, and mMate1 compared to their human counterparts.

Identification of d-amino acid oxidase and propiverine interaction partners and their potential role in the propiverine-mediated nephropathy.

Propiverine, a frequently-prescribed pharmaceutical for the treatment of symptoms associated with overactive bladder syndrome, provoked massive intranuclear and cytosolic protein inclusions in rat proximal tubule epithelium, primarily consisting of the peroxisomal targeting signal 1 (PTS1) containing protein d-amino acid oxidase (DAAO). As this type of nephropathy was also observed for other drugs, the aim was to determine whether propiverine interferes with trafficking and/or import of peroxisomal proteins. To elucidate this, DAAO- and propiverine-specific interaction partners from human HEK293 and rat WKPT cell lines and rat kidney and liver homogenate were determined using co-immunoprecipitation with subsequent nano-ESI-LC-MS/MS analyses. Corroboration of the role of DAAO- and/or propiverine-specific interaction partners in the drug-induced DAAO accumulation was sought via specific immunofluorescence staining of rat kidney sections from control and propiverine-treated rats. Above analyses demonstrated the interaction of propiverine with several protein classes, foremost peroxisomal proteins (DAAO, MFE2, HAOX2) and proteins of the protein quality control system, i.e. chaperones (HSP70 and DnaJ co-chaperones), proteases and proteasomal proteins (regulatory subunits of the 26S proteasome; Rpn1/2). The immunofluorescence analysis revealed mislocalization of many PTS1-proteins (DAAO, CAT, MFE2, ACOX1, EHHADH) in rat renal sections, strongly suggesting that propiverine primarily binds to PTS1 proteins resulting in the formation of PTS1 but not PTS2 or peroxisomal membrane protein (PMP) accumulations. Moreover, chaperones involved in peroxisomal trafficking (HSC70, DnaJB1) and peroxisomal biogenesis factor proteins (PEX3, PEX5, PEX7), also presented with distinct mislocalization patterns. Concomitantly, an increased number of peroxisomes was observed, suggestive of a compensatory mechanism for the presumably suboptimally functioning peroxisomes. Overall, the data presented suggested that propiverine interacts exclusively with DAAO or with a selected number of PTS1 proteins. The consequence of this interaction is the abrogated trafficking and peroxisomal import of PTS1 proteins concomitant with their nuclear and cytosolic accumulation due to inhibited degradation and imbalanced protein homeostasis.

Trospium chloride is absorbed from two intestinal "absorption windows" with different permeability in healthy subjects.

Intestinal P-glycoprotein is regio-selectively expressed and is a high affinity, low capacity efflux carrier for the cationic, poorly permeable trospium. Organic cation transporter 1 (OCT1) provides lower affinity but higher capacity for trospium uptake. To evaluate regional intestinal permeability, absorption profiles after gastric infusion of trospium chloride (30mg/250ml=[I]2) for 6h and after swallowing 30mg immediate-release tablets in fasted and fed healthy subjects, were evaluated using an inverse Gaussian density function to model input rate and mean absorption time (MAT). Trospium chloride was slowly absorbed (MAT ∼10h) after gastric infusion involving two processes with different input rates, peaking at about 3h and 7h. Input rates and MAT were influenced by dosage form and meal. In conclusion, trospium is absorbed from two "windows" located in the jejunum and cecum/ascending colon, whose uptake capacity might result from local abundance and functional interplay of P-glycoprotein and OCT1.

In vitro and in vivo evaluation under finite conditions of a transdermal oil-in-water type emulsified formulation of propiverine hydrochloride.

A transdermal oil-in-water type emulsified formulation containing propiverine hydrochloride, used for treatment of an overactive bladder (OAB), was evaluated for in vitro skin permeation under finite conditions and in vivo transdermal absorption. Propiverine hydrochloride solubility was determined using 1,3-butyleneglycol, polyoxyethylene (2) oleylether, isostearyl alcohol, and lauryl alcohol. The solubility increased as the solubility parameter value increased. In vitro skin permeation in hairless mouse skin and in vivo transdermal absorption in rats were measured using propiverine hydrochloride dissolved in a simple solution containing these solvents. Dependent on the increase in in vitro flux, the in vivo area under the curve up to 72 h (AUC0-72) was increased. Therefore, the emulsified formulation was prepared containing these ingredients using polyoxyethylene (20) stearylether for optimization. The emulsified formulation was used to conduct in vivo single- and repeated-dose absorption studies in rats. After single-dose transdermal administration of the emulsified formulation, the AUC0-72 was equivalent to that of the simple solution. Furthermore, results using the emulsified formulation indicated an increase in AUC0-72 and significant extension of the elimination half-life, in comparison with oral administration. After repeated-dose administration, a significant minimum plasma concentration was observed compared with oral administration. These results demonstrate that the emulsified formulation is a good option for transdermal delivery of propiverine hydrochloride.

Mechanistic basis for unexpected bioavailability enhancement of polyelectrolyte complexes incorporating BCS class III drugs and carrageenans.

The objective of this study was to investigate the potential of λ-carrageenan to work as an absorption modifying excipient in combination with formulations of BCS class 3 substances. Trospium chloride was used as a model BCS class 3 substance. Polyelectrolyte complexes of trospium and λ-carrageenan were produced by layer-by-layer complexation. A λ-carrageenan-containing formulation was administered either in capsules size 9 to rats by gavage or directly into ligated intestinal loops of rats. Exceptionally strong variations were observed in the plasma concentrations of the rats that received λ-carrageenan compared to the control group, but enhanced plasma concentrations were observed only in some of the rats. In vitro permeability studies were performed across Caco2-monolayers and across excised segments of rat jejunum in a modified Ussing chamber to learn more about the mechanism of absorption enhancement. The complex did not show any effect in Caco2-cells, but led to a major enhancement of permeability across excised segments in modified Ussing chambers. Carrageenan did not lead to alterations of tight junctions. The bioavailability enhancing effect thus was most likely due to an interaction of the polyelectrolyte-drug complex with the mucus, which provided an intimate contact between the drug and the absorbing surface. A similar effect was also achievable with other types of carrageenan and was also transferable to other compounds. In conclusion, λ-carrageenan-drug complexes show interesting excipient-drug-epithelium interactions - however, for full utilization of the permeation enhancing potential, an intimate and reproducible contact between absorbing epithelia and the complex is needed.

Ion pairing with bile salts modulates intestinal permeability and contributes to food-drug interaction of BCS class III compound trospium chloride.

In the current study the involvement of ion pair formation between bile salts and trospium chloride (TC), a positively charged Biopharmaceutical Classification System (BCS) class III substance, showing a decrease in bioavailability upon coadministration with food (negative food effect) was investigated. Isothermal titration calorimetry provided evidence of a reaction between TC and bile acids. An effect of ion pair formation on the apparent partition coefficient (APC) was examined using (3)H-trospium. The addition of bovine bile and bile extract porcine led to a significant increase of the APC. In vitro permeability studies of trospium were performed across Caco-2-monolayers and excised segments of rat jejunum in a modified Ussing chamber. The addition of bile acids led to an increase of trospium permeation across Caco-2-monolayers and rat excised segments by approximately a factor of 1.5. The addition of glycochenodeoxycholate (GCDC) was less effective than taurodeoxycholate (TDOC). In the presence of an olive oil emulsion, a complete extinction of the permeation increasing effects of bile salts was observed. Thus, although there are more bile acids in the intestine in the fed state compared to the fasted state, these are not able to form ion pairs with trospium in fed state, because they are involved in the emulsification of dietary fats. In conclusion, the formation of ion pairs between trospium and bile acids can partially explain its negative food effect. Our results are presumably transferable to other organic cations showing a negative food effect.

The N-oxide metabolite contributes to bladder selectivity resulting from oral propiverine: muscarinic receptor binding and pharmacokinetics.

We characterized contribution of N-oxide metabolites [1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide (M-1) and 1-methyl-4-piperidyl benzilate N-oxide (M-2)] to the binding of muscarinic receptors in relation to the pharmacokinetics of propiverine in rats. The in vitro muscarinic receptor binding activity of M-2 was equipotent to that of propiverine, whereas M-1 was much less active. After the oral administration of propiverine (24.8-248 micromol/kg), there was relatively selective and longer-lasting binding of muscarinic receptors in the rat bladder compared with the submaxillary gland as shown by a significant increase in the apparent dissociation constant (K(d)) for specific binding of [N-methyl-(3)H]scopolamine ([(3)H]NMS). In addition, the intravesical instillation of M-2 produced a significant increase in K(d) for specific [(3)H]NMS binding in the rat bladder. Extremely high concentrations of M-1 and M-2 were detected in plasma after the oral administration of propiverine. The concentration of unbound M-2 was much higher than that of M-1 and propiverine in the rat plasma. The sum of maximal plasma unbound propiverine equivalents (C(max)) after the oral administration of propiverine at doses of 24.8, 74.3, and 248 micromol/kg was 66.0, 303, and 509 nM, respectively. The sum of corresponding area under the time-concentration curve from 0 to 12 h was 194, 2123, and 4645 nM . h, respectively. In fact, the unbound concentration of M-2 comprised more than 90% of sum of unbound propiverine equivalents in the plasma. After oral treatment with propiverine, the bladder showed the highest concentration of M-2, indicating specific distribution of this metabolite into the target organ. Thus, M-2 may contribute greatly to the relatively selective and long-lasting occupation of bladder muscarinic receptors after oral administration of propiverine.

Anticholinergic effects of cis- and trans-isomers of two metabolites of propiverine.

The muscarinic receptor antagonist propiverine used for therapy of overactive bladder undergoes first pass metabolism, leading to several active metabolites, which affect muscarinic receptors and L-type Ca(2+) channels with different potencies. M-5, the major metabolite in blood, and M-6 can be synthesized as cis- and trans-isomers. We systematically investigated the pharmacodynamic profiles of the isomers on detrusor contractile function. In murine and porcine detrusor, the effects of the derivatives were examined on contractions induced by electric field stimulation (EFS), cumulatively increasing concentrations of carbachol or high KCl concentration. EFS contractions were concentration-dependently reduced by the M-5 and M-6 isomers although to a different extent. M-5(cis) was slightly more potent than M-5(trans), but the differences did not reach statistical significance. M-6(cis) was significantly more potent than M-6(trans). Responses to carbachol were antagonized by all compounds due to rightward shifts of the concentration-response curves, but only M-5(trans) also significantly reduced the maximum response. pK (B) values obtained with Schild plot analysis indicated slightly higher potency for M-6(cis) than M-6(trans). Ca(2+) influx-dependent contractions elicited by K(+) depolarization were less impaired by low concentrations of the M-6 isomers, but strongly suppressed by 100 microM of the M-5 isomers, suggesting an additional effect of the two M-5 isomers on Ca(2+) influx. All investigated isomers of M-5 and M-6 are biologically active in reducing detrusor contraction in animal tissue. While M-5( cis,) M-6(cis), and M-6(trans) possess surmountable or partially surmountable antagonistic properties at muscarinic receptors, M-5(trans) is a strong non-competitive antagonist. However, at higher concentration ranges, all four compounds seem to have additional effects on Ca(2+) influx.

Estimating half-lives of pesticides in/on vegetation for use in multimedia fate and exposure models.

Degradation half-lives in/on vegetation are needed in environmental risk assessment of pesticides, but these data are often not available for most active ingredients. To address this, we first correlated experimental soil degradation half-life data of 41 pesticides obtained from the reviewed literature with the corresponding experimental half-lives on plant surface. Degradation half-lives in soil were found to be four times slower compared with half-lives on plant surfaces. In a second step, we explored measured plant surface half-lives directly with those in vegetation. The results were validated by comparing computed values with results obtained from an experimental set-up. The uptake and dissipation of alpha-cypermethrin (insecticide) and bromopropylate (acaricide) was studied by detecting pesticide residues in whole and peeled tomato fruits using gas chromatography. Half-lives within vegetation were found to be four times faster compared with plant surface half-lives. Using this experimental based approach, it is concluded that the estimation of degradation half-lives of pesticides in/on vegetation to be used as input data in environmental mass balance models can be directly correlated from the more abundant ready experimental degradation half-life data for soil.

Binding activities by propiverine and its N-oxide metabolites of L-type calcium channel antagonist receptors in the rat bladder and brain.

The present study was undertaken to characterize the binding activities of propiverine and its N-oxide metabolites (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide: P-4(N-->O), 1-methyl-4-piperidyl benzilate N-oxide: DPr-P-4(N-->O)) toward L-type calcium channel antagonist receptors in the rat bladder and brain. Propiverine and P-4(N-->O) inhibited specific (+)-[(3)H]PN 200-110 binding in the rat bladder in a concentration-dependent manner. Compared with that for propiverine, the K(i) value for P-4(N-->O) in the bladder was significantly greater. Scatchard analysis has revealed that propiverine increased significantly K(d) values for bladder (+)-[(3)H]PN 200-110 binding. DPr-P-4(N-->O) had little inhibitory effects on the bladder (+)-[(3)H]PN 200-110 binding. Oxybutynin and N-desethyl-oxybutynin (DEOB) also inhibited specific (+)-[(3)H]PN 200-110 binding in the rat bladder. Propiverine, oxybutynin and their metabolites inhibited specific [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS) binding in the rat bladder. The ratios of K(i) values for (+)-[(3)H]PN 200-110 to [(3)H]NMS were markedly smaller for propiverine and P-4(N-->O) than oxybutynin and DEOB. Propiverine and P-4(N-->O) inhibited specific binding of (+)-[(3)H]PN 200-110, [(3)H]diltiazem and [(3)H]verapamil in the rat cerebral cortex in a concentration-dependent manner. The K(i) values of propiverine and P-4(N-->O) for [(3)H]diltiazem were significantly smaller than those for (+)-[(3)H]PN 200-110 and [(3)H]verapamil. Further, their K(i) values for [(3)H]verapamil were significantly smaller than those for (+)-[(3)H]PN 200-110. The K(i) values of propiverine for each radioligand in the cerebral cortex were significantly (P<0.05) smaller than those of P-4(N-->O). In conclusion, the present study has shown that propiverine and P-4(N-->O) exert a significant binding activity of L-type calcium channel antagonist receptors in the bladder and these effects may be pharmacologically relevant in the treatment of overactive bladder after oral administration of propiverine.

Treatment of the overactive bladder syndrome with muscarinic receptor antagonists: a matter of metabolites?

Antagonists of muscarinic acetylcholine receptors, such as darifenacin, oxybutynin, propiverine, solifenacin, tolterodine, and trospium, are the mainstay of the treatment of the overactive bladder syndrome. Fesoterodine is a newer drug awaiting regulatory approval. We briefly review the pharmacological activity of their metabolites and discuss how active metabolites may contribute to their efficacy and tolerability in vivo. Except for trospium, and perhaps solifenacin, all of the above drugs form active metabolites, and their presence and activity need to be taken into consideration when elucidating relationships between pharmacokinetics and pharmacodynamics of these drugs. Moreover, the ratios between parent compounds and metabolites may differ depending on genotype of the metabolizing enzymes, concomitant medication, and/or drug formulation. Differential generation of active metabolites of darifenacin or tolterodine are unlikely to influence the overall clinical profile of these drugs in a major way because the active metabolites exhibit a similar pharmacological profile as the parent compound. In contrast, metabolites of oxybutynin and propiverine may behave quantitatively or even qualitatively differently from their parent compounds and this may have an impact on the overall clinical profile of these drugs. We conclude that more comprehensive studies of drug metabolites are required for an improved understanding of their clinical effects.

Propiverine and metabolites: differences in binding to muscarinic receptors and in functional models of detrusor contraction.

Propiverine is a commonly used antimuscarinic drug used as therapy for symptoms of an overactive bladder. Propiverine is extensively biotransformed into several metabolites that could contribute to its spasmolytic action. In fact, three propiverine metabolites (M-5, M-6 and M-14) have been shown to affect various detrusor functions, including contractile responses and L-type calcium-currents, in humans, pigs and mice, albeit with different potency. The aim of our study was to provide experimental evidence for the relationship between the binding of propiverine and its metabolites to human muscarinic receptor subtypes (hM(1)-hM(5)) expressed in chinese hamster ovary cells, and to examine the effects of these compounds on muscarinic receptor-mediated detrusor function. Propiverine, M-5, M-6 and M-14 bound to hM(1)-hM(5) receptors with the same order of affinity for all five subtypes: M-6 > propiverine > M-14 > M-5. In HEK-293 cells expressing hM(3), carbachol-induced release of intracellular Ca(2+) ([Ca(2+)](i)) was suppressed by propiverine and its metabolites; the respective concentration-response curves for carbachol-induced Ca(2+)-responses were shifted to the right. At higher concentrations, propiverine and M-14, but not M-5 and M-6, directly elevated [Ca(2+)](i). These results were confirmed for propiverine in human detrusor smooth muscle cells (hDSMC). Propiverine and the three metabolites decreased detrusor contractions evoked by electric field stimulation in a concentration-dependent manner, the order of potency being the same as the order of binding affinity. We conclude that, in comparison with the parent compound, loss of the aliphatic side chain in propiverine metabolites is associated with higher binding affinity to hM(1)-hM(5) receptors and higher functional potency. Change from a tertiary to a secondary amine (M-14) results in lower binding affinity and reduced potency. Oxidation of the nitrogen (M-5) further lowers binding affinity as well as functional potency.

Enantiomeric N-methyl-4-piperidyl benzilates as muscarinic receptor ligands: Radioligand binding studies and docking studies to models of the three muscarinic receptors M1, M2 and M3.

Benzilic ester derivatives with a basic moiety like N-methyl-4-piperidyl benzilates are potential drugs for the treatment of urinary incontinence, duodenal and gastric ulcers and Parkinson's disease. The effect of structural variations of chiral N-methyl-4-piperidyl benzilates was investigated using radioligand binding studies on muscarinic receptors (M1-M3). The results of the binding studies demonstrate that the absolute configuration and the aromatic substituent of benzilates have an influence on muscarinic affinity and selectivity. In this regard, (S)-configuration of benzilates and hydrophilic aromatic substituents seems to enhance muscarinic affinity. A model of the receptor ligand complex for N-methyl-4-piperidyl benzilates was obtained by molecular modelling. Both the affinity of enantiomeric benzilic esters and the subtype selectivity for muscarinic receptors are comprehensively explained by this model.

Juvenile pig detrusor: effects of propiverine and three of its metabolites.

In isolated detrusor strips, propiverine is known to be effective to decrease contractions elicited by electric field stimulation (EFS). Here we investigated whether the metabolites M-5, M-6 and M-14 of propiverine retain the pharmacological properties of the parent compound also in juvenile organisms. EFS-induced contractions of detrusor strips from juvenile pigs are more sensitive to atropine than strips from mature pigs. The atropine-resistant component of contraction is also significantly larger in juvenile pigs. Propiverine, its metabolites M-5, M-14 and also tolterodine completely reduced detrusor contraction in juvenile pigs. M-6 almost did not affect atropine-resistant contractions. We conclude that juvenile pig detrusors possess a higher atropine-resistant component of EFS-elicited contraction. Nevertheless order of potency and efficacy of propiverine and its metabolites M-5 and M-14 are similar in juvenile and mature pigs, while M-6 only reduces atropine-sensitive contractions in the juvenile organism.

Discrepancy between cell injury and benzodiazepine receptor binding after transient middle cerebral artery occlusion in rats.

We investigated postischemic alterations in benzodiazepine receptor, D1 dopamine receptor, and muscarinic acetylcholine receptor binding after transient middle cerebral artery (MCA) occlusion in rats using [3H]-flumazenil, [3H]-SCH23390, and [3H]-N-methyl-4-piperidyl benzilate ([3H]-NMPB), respectively, as radioligand. These ligand bindings were determined at 3 and 24 h and at 3 and 7 days after ischemia/reperfusion of MCA by using autoradiographic methods. Ischemic cell injury was clearly detected from 3 h after ischemia/reperfusion and progressively increased from 3-24 h after ischemia/reperfusion of MCA. The area of cell injury reached maximum at 24 h after ischemia/reperfusion of MCA. [3H]-SCH23390 binding was reduced to 47% of the contralateral side at 3 days after ischemia/reperfusion of MCA. After 7 days, [3H]-SCH23390 binding was further reduced by 20% in the striatum. [3H]-NMPB binding was slightly decreased in both the striatum and cerebral cortex at 3 days after ischemia/reperfusion of MCA, and [3H]-NMPB binding in the striatum and cerebral cortex were reduced to 42 and 62% of the contralateral side at 7 days after ischemia/reperfusion of MCA. [3H]-NMPB was also decreased at 24 h. In contrast, [3H]-flumazenil binding was not decreased in the striatum and cerebral cortex within 7 days after ischemia/reperfusion of MCA. These results suggest that [3H]-SCH23390 and [3H]-NMPB binding do not correlate with cell injury by ischemia/reperfusion, although vulnerability to ischemia/reperfusion was observed with these receptors. In addition, central benzodiazepine receptor imaging might be essentially stable to neuronal cell injury induced by transient focal cerebral ischemia in rats, in contrast to the results of PET studies.

Acetylcholinesterase inhibition increases in vivo N-(2-[18F]fluoroethyl)-4-piperidyl benzilate binding to muscarinic acetylcholine receptors.

Although the inhibition of acetylcholinesterase remains the primary treatment of Alzheimer's disease, little is known of the results of increased acetylcholine levels on muscarinic receptor occupancy or function. Using N-(2-[18F]fluoroethyl)-4-piperidyl benzilate ([18F]FEPB), a moderate affinity (Ki = 1.7 nmol/L) nonsubtype-selective muscarinic receptor antagonist, the authors examined the sensitivity of equilibrium in vivo radioligand binding in rat brain with changes in endogenous acetylcholine levels produced by treatments with acetylcholinesterase inhibitors. Phenserine administration 30 minutes before resulted in a dose-dependent into muscarinic cholinergic receptors, reaching a maximum increase of 90% in the striatum at a dose of 5 mg/kg intraperitoneally. Constant infusion of physostigmine at a dosage of 250 microg/kg/min produced an identical increase in radioligand binding. This agonist-induced increase of in vivo mAChR radioligand binding offers a new method for monitoring of the efficacy of acetylcholinesterase inhibitors or other drugs to enhance acetylcholine actions at the muscarinic receptors.

Assessment of muscarinic receptor concentrations in aging and Alzheimer disease with [11C]NMPB and PET.

Cerebral cholinergic deficits have been described in Alzheimer disease (AD) and as a result of normal aging. At the present time, there are very limited options for the quantification of cholinergic receptors with in vivo imaging techniques such as PET. In the present study, we examined the feasibility of utilizing [11C]N-methyl-4-piperidyl benzilate (NMPB), a nonselective muscarinic receptor ligand, in the study of aging and neurodegenerative processes associated with cholinergic dysfunction. Based on prior data describing the accuracy of various kinetic methods, we examined the concentration of muscarinic receptors with [11C]NMPB and PET using two- and three-compartment kinetic models. Eighteen healthy subjects and six patients diagnosed with probable AD were studied. Pixel-by-pixel two-compartment model fits showed acceptable precision in the study of normal aging, with comparable results to those obtained with a more complex and less precise three-compartment model. Normal aging was associated with a reduction in muscarinic receptor binding in neocortical regions and thalamus. In AD patients, the three-compartment model appeared capable of dissociating changes in tracer transport from changes in receptor binding, but suffered from statistical uncertainty, requiring normalization to a reference region, and therefore limiting its potential use in the study of neurodegenerative processes. After normalization, no regional changes in muscarinic receptor concentrations were observed in AD.