Background

[PubMed]

The current state of molecular genetics of Alzheimer’s disease (AD) focuses on four genes: the β-amyloid precursor protein (β-APP) gene, the presenilin 1 gene, the presenilin 2 gene, and the apolipoprotein E (APOE) gene (2). The ε4 allele of the APOE gene is associated with an increased risk for late-onset AD (>50 years of age). Few of the imaging studies to date have been based on these genetic abnormalities. The characteristic neuropathology is neurofibrillary tangles and amyloid plaques. The result of the phenotypic changes is caused by an alteration in the processing of β-APP favoring the production of the potentially toxic Aβ42 protein. Binding of radioligands to Aβ42 protein in vivo has been reported (3). There has also been a report on the use of 2-[¹⁸F]fluoro-2-deoxyglucose ([¹⁸F]FDG) in a subpopulation of AD patients with the APOE gene (4), although this is an indirect measure of the phenotype. The authors found changes in FDG distribution before clinical manifestations.

Muscarinic cholinergic M₂ subtype-selective ligands were developed based on the observation that this subtype is lost in the cerebral cortex in AD (5-7). Postmortem quantization of muscarinic subtypes indicated a selective loss of the M₂ subtype in cortical regions, whereas the M₁ subtype was preserved. Thus, an M₂-selective ligand labeled with a positron-emitting radionuclide would allow determination of M₂ subtype density in the living human brain. Several investigators have pursued muscarinic receptor imaging with positron emission tomography (2) and single-photon emission computed tomography (SPECT) (8, 9). Most tracers for cholinergic receptors have not demonstrated subtype selectivity. Alternatively, ligands that are subtype selective in vitro typically do not cross the blood-brain barrier (BBB). In addition to muscarinic receptors, ligands for the nicotinic receptors (10, 11) and acetylcholinesterase (12) have been investigated. Acetylcholinesterase (13-15) has been a target for radioligand development because its levels also change in AD. In addition, the vesicular acetylcholine (ACh) transporter has been studied recently in patients with AD using [¹²³I]iodobenzovesamicol (12). The probe for acetylcholinesterase (N-[¹¹C]methylpiperdin-4-yl propionate, [¹¹C]PMP) has been used in AD patients and found to show a decrease that is more uniform and broadly distributed than the focal defects found with [¹⁸F]FDG (12). 3-((3-(3-fluoropropyl)thio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (FP-TZTP), a muscarinic agonist based on a series of non-fluorinated analogs first proposed by Sauerberg et al. (16), was radiolabeled with the positron-emitting radionuclide ¹⁸F ([¹⁸F]FP-TZTP) (17). [¹⁸F]FP-TZTP was found to be a promising imaging agent for the M2 system in the brain.

Synthesis

[PubMed]

Nucleophilic fluorination of (3-(3-((3-(methanesulfonyoxy)-propyl)thio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine with K[¹⁸F]F/Kryptofix2.2.2 to form [¹⁸F]FP-TZTP (18). A one-step radiosynthesis of [¹⁸F]FP-TZTP was developed and can also be conducted with an automated synthesis unit. The manual synthesis produced [¹⁸F]FP-TZTP in a radiochemical yield of 23.4% ± 4.3% (EOS, n = 69) with a specific activity of 117 ± 54 MBq/μmol, 4377 ± 2011 mCi/μmol (EOB, n = 100). The automated synthesis unit provided [¹⁸F]FP-TZTP in a radiochemical yield of 18.8% ± 2.4% (EOS, n = 25) with a specific activity of 111 ± 70 MBq/μmol, 4112 ± 2572 mCi/μmol (EOB, n = 25) (18).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

FP-TZTP showed a K_i of 7.4 nM for M₁ and 2.2 nM for M₂. It did not bind to M₃ receptors or other biogenic amine receptors (19).

Rat hepatocytes were incubated with [¹⁸F]FP-TZTP with (carrier-added) or without (no-carrier-added) the addition of 20 μg of unlabeled FP-TZTP. The time course of metabolites from the sequential analysis of the no-carrier-added experiment revealed that the parent compound was nearly consumed within 2 h, concomitant with the formation of seven radiolabeled metabolites. The carrier-added experiment showed slower metabolism. The 30-min, carrier-added sample was selected for identification of the metabolites by liquid chromatography tandem mass spectrometry (LC/MS/MS). Structures were confirmed for all compounds by comparison of retention time and MS/MS fragmentation with the synthesized standards. N-Oxidation provided the major radioactive metabolite. Other major metabolites were formed from sulfur oxidation, demethylation of the tertiary amine, and dehydrogenation of the tetrahydropyridine ring, respectively. In addition to the major metabolites of FP-TZTP identified above, two more polar radioactivity peaks were found in the radiochromatogram. The peak eluting at the solvent front was identified as [¹⁸F]fluoride. The remaining peak was unidentified based on the MS/MS data. Compared with rat hepatocytes, human hepatocytes produced a similar metabolite profile. The N-oxide was also the major metabolite identified by LC/MS and radiochromatography. However, N-oxidation metabolites were not obtained with the same dominance as observed in rat hepatocytes. Demethylation and S-oxidation metabolites were also produced by human hepatocytes. The ring oxidation metabolite and its hydroxylated analog were also found in human hepatocytes (20).

Animal Studies

Human Studies

[PubMed]

[¹⁸F]FP-TZTP dosimetry in humans was determined from its biodistribution in monkeys by i.v. of 93-130 MBq (2.5-3.5 mCi) [¹⁸F]FP-TZTP (30). There was early accumulation of activity in liver and kidney, with subsequent appearance of activity in gallbladder and urine, reflecting pathways for tracer excretion. The effective dose equivalent was 0.023 mSv/MBq (85 mrem/mCi). The critical organ was urinary bladder (0.073 mGy/MBq, 269 mrad/mCi), followed by pancreas (0.047 mGy/MBq, 173 mrad/mCi), gallbladder (0.040 mGy/MBq, 147 mrad/mCi), and brain (0.023 mGy/MBq, 86 mrad/mCi).

[¹⁸F]FP-TZTP PET was studied in healthy volunteers (31). In plasma, the parent compound represented 68 ± 8%, 41 ± 9%, and 14 ± 4% of radioactivity at 20, 40, and 120 min, respectively. The plasma-free fraction was 5.9 ± 1.2%. A model with one tissue compartment produced an excellent fit for the full 120 min of data, so that the additional parameters of a two-compartment model were unidentifiable. K1 values in gray matter regions were high, 0.36 to 0.56 ml/min/ml, and showed excellent correlation with cerebral blood flow. V values, representing total tissue binding, were similar in cortical regions, basal ganglia, and thalamus but were significantly higher (p < 0.01) in amygdala. Unlike the results in the monkey, binding in the cerebellum was similar to that in the cerebral cortex.

In the first clinical studies, an age-related increase in M₂ receptor binding potential was observed using [¹⁸F]FP-TZTP and PET in normal subjects (32). There was a significant increase not only in the average volume of distribution but also for variance in the older subjects. This infers that other aspects of the systems biology were coming into play, and therefore the data should be modeled by a more complex set of equations. The increase in variance for the volume of distribution was caused by using age discrimination only. This suggested the possibility that the age-related changes might be associated with a specific genotype, such as the APOE-ε4 allele. In the following study, Cohen et al. (33) found that the gray matter distribution volume for [¹⁸F]FP-TZTP was significantly higher in the APOE-ε4-positive normal subjects than in the APOE-ε4-negative normal subjects, whereas there were no differences in global cerebral blood flow. Given that [¹⁸F]FP-TZTP measures the muscarinic system rather than only receptor density because of its agonist properties, changes in receptor binding affinity attributable to changes in G protein binding and competition with ACh at the muscarinic receptor can be monitored. The increased volume of distribution in elderly normal subjects with APOE-ε4 positivity may be because of a decreased concentration of ACh in the synapse, which would lead to higher binding of [¹⁸F]FP-TZTP. This type of competition was shown to be possible in the studies in monkeys using physostigmine (21). As a result, the use of [¹⁸F]FP-TZTP can be considered an in vivo measurement of muscarinic systems biology, rather than of receptor density alone.

Supplemental Information

[Disclaimers]

Synthesis Protocol A

Synthesis Protocol B

Preclinical Pharmacology

NIH Support

Intramural Research Program

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