Substrate Report for: Methyl-4-piperidinyl-N-butyrate

UNLABELLED: Deficits of cholinergic neurotransmission contribute to various neurologic and psychiatric conditions. The neurotransmitter acetylcholine is hydrolyzed in the synaptic clefts by 2 enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). 1-[(11)C]-Methyl-4-piperidinyl-N-butyrate ((11)C-MP4B) is a radioligand for the assessment study of BuChE activity in human brain with PET. In the present study the radiation-absorbed doses of the (11)C-MP4B were estimated in humans according to the guidelines of the International Commission on Radiological Protection. Two different data acquisition protocols-dynamic organ-specific evaluation (DOSE) and whole-body scanning-were compared. Both methods are widely used for evaluation of radiation burden of (11)C-labeled PET tracers. METHODS: Fixed-bed PET on the upper neck, thorax, abdomen, or pelvic region was performed on 7 healthy subjects after injection of 707 +/- 34 MBq (mean +/- SD) of (11)C-MP4B. Brain input was derived from our previous studies on 18 healthy control subjects and 10 patients with Alzheimer's disease. Regions of interest were drawn on transverse images of all visible organs. Radiation dose estimates were calculated from organ residence times using the MIRDOSE3 software. Urine samples were collected after imaging to estimate tracer extraction. To compare the estimates for absorbed doses between the whole-body scan approach and the DOSE method, we simulated whole-body data acquisition methods used in (11)C dosimetry studies with our fixed-bed data. RESULTS: The organs with the highest radiation-absorbed doses were the liver, urinary bladder, kidneys (renal cortex), upper large intestine, trabecular bone, salivary glands, and heart wall. Up to 60% of the injected dose was excreted via the urinary pathway, and the clearance was relatively rapid, as 30% of the radioactivity was excreted within 60 min after injection. With a 2-h voiding interval the effective dose was 4.2 microSv/MBq. CONCLUSION: (11)C-MP4B causes less radiation burden than previously studied (11)C-labeled PET tracers. No intolerably high absorbed doses were observed in critical organs. With 740 MBq of injected radioactivity, the radiation burden is equivalent to 3.11 mSv. This would allow multiple PET examinations per year to be performed on the same subject. The DOSE method and the simulated whole-body imaging approach produced similar results.

1-(11)C-Methyl-4-piperidinyl n-butyrate ((11)C-MP4B) is a new radiopharmaceutical for the in vivo assessment of butyrylcholinesterase (BuChE) activity using PET. To quantify in vivo activity of BuChE with a kinetic model, investigators must determine the time course of radioactivity associated with unchanged (11)C-MP4B. We aimed at clarifying the metabolic fate and whole-body distribution of intravenously administered (11)C-MP4B in man.
METHODS: High-performance liquid chromatography and thin-layer chromatography assays were performed to determine the amounts of intact (11)C-MP4B and its radioactive hydrolysis product in blood withdrawn during PET. In addition, we evaluated the distribution and kinetics of (11)C-MP4B uptake in human brain and main organs.
RESULTS: The analysis of plasma samples of 28 human subjects (10 patients with Alzheimer's disease [AD] and 18 healthy controls) showed that the level of unmetabolized (11)C-MP4B decreases rapidly from 28% +/- 14% (mean +/- SD) at 0.5 min to 7% +/- 6% at 15 min after injection. Large individual variation was observed in the rate of plasma (11)C-MP4B hydrolysis, but no significant differences were found in the degradation of (11)C-MP4B either between male and female or between healthy subjects and patients. The whole-body distribution of (11)C-MP4B showed the highest activities in the urinary bladder, renal pelvis, stomach, salivary glands, liver, kidneys, spleen, vertebral column, and brain. In patients with AD, (11)C-MP4B activity in the brain was highest in cerebellum, followed by striatum, pons, and thalamus. Lower (11)C-MP4B activity was seen in cortical areas. CONCLUSION: Our results indicate that (11)C-MP4B is excreted rapidly through the renal system. Careful analysis of plasma metabolites is required to determine the accurate arterial input function for quantitative PET measurement. Biodistribution of (11)C-MP4B in the brains of patients with AD appears to be in accordance with the distribution of BuChE seen in postmortem studies of human brain, except for the observed higher activity in striatum than in cortex. Further studies of the cerebral distribution and regional kinetic analysis of (11)C-MP4B are in progress.

There is currently great interest in developing radiolabeled substrates for acetylcholinesterase and butyrylcholinesterase that would be useful in the in vivo imaging of patients with Alzheimer's disease. Using a simple in vitro spectrophotometric assay for determination of enzymatic cleavage rates, the structure-activity relationship for a short series of 1-methyl-4-piperidinyl esters was investigated. Relative enzymatic hydrolysis rates for the well-characterized 1-methyl-4-piperidinyl acetate, propionate, and i-butyrate esters were in agreement with literature values. The 4 and 5 carbon esters of 1-methyl-4-piperidinol were specific for butyrylcholinesterase and cleaved in the rank order n-valerate > n-butyrate >> 2-methylbutyrate, iso-valerate. These spectrophotometric results were also in agreement with in vitro hydrolysis rates in mouse blood and with in vivo regional retention of radioactivity in mouse brain of 11C-labeled analogs. Brain uptake and apparent enzymatic rate constants for 1-[11C]methyl-4-piperidinyl n-butyrate and n-valerate were calculated from in vivo measurements in M. nemistrina using positron emission tomography. Based on higher brain uptake of radioactivity and superior pharmacokinetics, 1-[11C]methyl-4-piperidinyl n-butyrate was identified as a new radiopharmaceutical for the in vivo measurement of butyrylcholinesterase activity.

Cholinesterase activity associated with neuritic plaques (NPs) and neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) brains exhibit altered histochemical properties, such as requiring lower pH (6.8) for optimal cholinesterase staining compared to the pH (8.0) for best visualization of cholinesterases in neurons. Furthermore, visualization of NPs and NFTs can be prevented by agents like the peptidase inhibitor/metalloantibiotic bacitracin. The anomalous behavior of cholinesterases associated with pathological lesions needs to be elucidated because of the putative links between these enzymes and the disease process in AD. In this study, cholinesterases were extracted from AD and normal brain tissue to determine whether the differences observed in histochemical analyses in the two sources were reflected in kinetic properties measured in solubilized enzymes. Isolated brain enzymes from both these sources exhibited comparable kinetic parameters with respect to pH dependence, substrate affinity and inhibitor sensitivity and were not significantly affected by other agents that blocked cholinesterase histochemical visualization, such as the structurally diverse metal-chelating antibiotics bacitracin, doxycycline, minocycline and rifampicin. Although the cholinesterases from AD brain tissue examined here represented a total pool of these enzymes from AD brain, rather than enzymes specifically from NPs and NFTs, their kinetic behavior being comparable to cholinesterases isolated from normal brain tissues implies that these enzymes do not undergo disease-related modification in their primary structures. This suggests that the atypical histochemical behavior of cholinesterases in NPs and NFTs may result from interaction of cholinesterases with other molecules within these lesions, mediated by transition metal ions known to be present in AD pathology lesions.

UNLABELLED: Deficits of cholinergic neurotransmission contribute to various neurologic and psychiatric conditions. The neurotransmitter acetylcholine is hydrolyzed in the synaptic clefts by 2 enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). 1-[(11)C]-Methyl-4-piperidinyl-N-butyrate ((11)C-MP4B) is a radioligand for the assessment study of BuChE activity in human brain with PET. In the present study the radiation-absorbed doses of the (11)C-MP4B were estimated in humans according to the guidelines of the International Commission on Radiological Protection. Two different data acquisition protocols-dynamic organ-specific evaluation (DOSE) and whole-body scanning-were compared. Both methods are widely used for evaluation of radiation burden of (11)C-labeled PET tracers. METHODS: Fixed-bed PET on the upper neck, thorax, abdomen, or pelvic region was performed on 7 healthy subjects after injection of 707 +/- 34 MBq (mean +/- SD) of (11)C-MP4B. Brain input was derived from our previous studies on 18 healthy control subjects and 10 patients with Alzheimer's disease. Regions of interest were drawn on transverse images of all visible organs. Radiation dose estimates were calculated from organ residence times using the MIRDOSE3 software. Urine samples were collected after imaging to estimate tracer extraction. To compare the estimates for absorbed doses between the whole-body scan approach and the DOSE method, we simulated whole-body data acquisition methods used in (11)C dosimetry studies with our fixed-bed data. RESULTS: The organs with the highest radiation-absorbed doses were the liver, urinary bladder, kidneys (renal cortex), upper large intestine, trabecular bone, salivary glands, and heart wall. Up to 60% of the injected dose was excreted via the urinary pathway, and the clearance was relatively rapid, as 30% of the radioactivity was excreted within 60 min after injection. With a 2-h voiding interval the effective dose was 4.2 microSv/MBq. CONCLUSION: (11)C-MP4B causes less radiation burden than previously studied (11)C-labeled PET tracers. No intolerably high absorbed doses were observed in critical organs. With 740 MBq of injected radioactivity, the radiation burden is equivalent to 3.11 mSv. This would allow multiple PET examinations per year to be performed on the same subject. The DOSE method and the simulated whole-body imaging approach produced similar results.

OBJECTIVE: We tested the premise that cholinesterase inhibitor therapy should target butyrylcholinesterase (BuChE) in Alzheimer's disease (AD), not acetylcholinesterase (AChE) alone, because both enzymes hydrolyze acetylcholine, and BuChE is increased in AD cerebral cortex.
METHODS: To examine this issue in vivo, we quantified human cerebral cortical BuChE activity using tracer kinetic estimates (k(3)) of 1-[(11)C]methyl-4-piperidinyl n-butyrate ([(11)C]BMP) hydrolysis determined by positron emission tomography. Validation of the putative positron emission tomography method included regional distribution, positive correlation with age, and attenuation by the nonselective cholinesterase inhibitor physostigmine, but no attenuation by the AChE-selective inhibitor donepezil. Positron emission tomography scans in AD patients (n = 15) and control subjects (n = 12) measured both BuChE (using [(11)C]BMP) and AChE activity (using N-[(11)C] methylpiperidin-4-yl propionate, an established method).
RESULTS: As expected, AChE activity in AD cerebral cortex was decreased to 75 +/- 13% of normal (p = 0.00001). Contrary to prediction, accompanying BuChE activity also was decreased to 82 +/- 14% of normal (p = 0.001). INTERPRETATION: Failure to observe increased [(11)C]BMP hydrolysis in vivo makes it less likely that incremental BuChE contributes importantly to acetylcholine hydrolysis in AD. The findings do not support the premise that inhibitor therapy should target BuChE so as to prevent increased levels of BuChE from hydrolyzing acetylcholine in AD cerebral cortex.

In Alzheimer's disease, cerebral cortical butyrylcholinesterase (BChE) activity is reported to be elevated. Our aim was to develop a novel (18)F-labeled tracer for quantifying cerebral BChE activity by positron emission tomography. With in vitro screening of N-[(14)C]ethylpiperidin-3- and 4-ylmethyl esters, N-[(14)C]ethylpiperidin-4-ylmethyl butyrate was selected as a lead for (18)F-labeling, affording N-[(18)F]fluoroethylpiperidin-4-ylmethyl butyrate. The (18)F-labeled butyrate showed the required properties for in vivo BChE measurement, that is, the lipophilic nature of the authentic ester, high specificity to BChE, a moderate hydrolysis rate, and the hydrophilic nature of the metabolite.

1-(11)C-Methyl-4-piperidinyl n-butyrate ((11)C-MP4B) is a new radiopharmaceutical for the in vivo assessment of butyrylcholinesterase (BuChE) activity using PET. To quantify in vivo activity of BuChE with a kinetic model, investigators must determine the time course of radioactivity associated with unchanged (11)C-MP4B. We aimed at clarifying the metabolic fate and whole-body distribution of intravenously administered (11)C-MP4B in man.
METHODS: High-performance liquid chromatography and thin-layer chromatography assays were performed to determine the amounts of intact (11)C-MP4B and its radioactive hydrolysis product in blood withdrawn during PET. In addition, we evaluated the distribution and kinetics of (11)C-MP4B uptake in human brain and main organs.
RESULTS: The analysis of plasma samples of 28 human subjects (10 patients with Alzheimer's disease [AD] and 18 healthy controls) showed that the level of unmetabolized (11)C-MP4B decreases rapidly from 28% +/- 14% (mean +/- SD) at 0.5 min to 7% +/- 6% at 15 min after injection. Large individual variation was observed in the rate of plasma (11)C-MP4B hydrolysis, but no significant differences were found in the degradation of (11)C-MP4B either between male and female or between healthy subjects and patients. The whole-body distribution of (11)C-MP4B showed the highest activities in the urinary bladder, renal pelvis, stomach, salivary glands, liver, kidneys, spleen, vertebral column, and brain. In patients with AD, (11)C-MP4B activity in the brain was highest in cerebellum, followed by striatum, pons, and thalamus. Lower (11)C-MP4B activity was seen in cortical areas. CONCLUSION: Our results indicate that (11)C-MP4B is excreted rapidly through the renal system. Careful analysis of plasma metabolites is required to determine the accurate arterial input function for quantitative PET measurement. Biodistribution of (11)C-MP4B in the brains of patients with AD appears to be in accordance with the distribution of BuChE seen in postmortem studies of human brain, except for the observed higher activity in striatum than in cortex. Further studies of the cerebral distribution and regional kinetic analysis of (11)C-MP4B are in progress.

There is currently great interest in developing radiolabeled substrates for acetylcholinesterase and butyrylcholinesterase that would be useful in the in vivo imaging of patients with Alzheimer's disease. Using a simple in vitro spectrophotometric assay for determination of enzymatic cleavage rates, the structure-activity relationship for a short series of 1-methyl-4-piperidinyl esters was investigated. Relative enzymatic hydrolysis rates for the well-characterized 1-methyl-4-piperidinyl acetate, propionate, and i-butyrate esters were in agreement with literature values. The 4 and 5 carbon esters of 1-methyl-4-piperidinol were specific for butyrylcholinesterase and cleaved in the rank order n-valerate > n-butyrate >> 2-methylbutyrate, iso-valerate. These spectrophotometric results were also in agreement with in vitro hydrolysis rates in mouse blood and with in vivo regional retention of radioactivity in mouse brain of 11C-labeled analogs. Brain uptake and apparent enzymatic rate constants for 1-[11C]methyl-4-piperidinyl n-butyrate and n-valerate were calculated from in vivo measurements in M. nemistrina using positron emission tomography. Based on higher brain uptake of radioactivity and superior pharmacokinetics, 1-[11C]methyl-4-piperidinyl n-butyrate was identified as a new radiopharmaceutical for the in vivo measurement of butyrylcholinesterase activity.

Alzheimer disease (AD) is accompanied by a marked loss of acetylcholinesterase (AChE) activity associated with cortical cholinergic axons and cholinoceptive neurons. Simultaneous with this loss, cholinesterase (ChE) activity emerges in AD cortex in the form of AChE and butyrylcholinesterase activity associated with plaques, tangles, and amyloid angiopathy. Our observations have shown that the ChEs associated with the pathological lesions of AD (ADChEs) possess different enzymatic properties and quite possibly are of a different source as compared with the ChEs associated with normal neurons and axons. The ADChEs most likely have noncholinergic functions involved in the pathogenesis of AD. The postulated functions include acting as proteases/peptidases, participating directly in the amyloidogenic processing of the amyloid precursor protein, and causing aberrant growth of neuronal processes. The therapeutic and diagnostic implications of ADChEs are discussed.