Background

[PubMed]

2-(2-(5-[¹¹C]Methoxypyridin-3-yl)ethynyl)pyridine ([¹¹C]M-PEPy) is a radioligand developed for positron emission tomography (PET) imaging of metabotropic glutamate (mGlu) receptor subtype 5 (mGluR5 or mGluR₅) in the central nervous system (CNS) (1, 2). ¹¹C is a positron emitter with a physical half-life of 20.3 min.

Glutamate is a major excitatory neurotransmitter at CNS synapses. Many neuroanatomical CNS projection pathways contain glutamatergic neurons (3). Glutamate produces its excitatory effects by acting on cell-surface ionotropic glutamate or mGluRs (4). The mGluRs are G-protein–coupled receptors, and the eight mGluR subtypes are further subdivided into groups I, II, and III. The group I receptors include mGluR1 and mGluR5, and they are found predominantly in postsynaptic locations. The mGluR5s are found with high to moderate density in the frontal cortex, caudate, putamen, nucleus accumbens, olfactory tubercle, hippocampus, and dorsal horn of the spinal cord, whereas the density in the cerebellum is low. These receptors couple with phospholipase C and up- or downregulate neuronal excitability. They have been implicated in a variety of diseases in the CNS, including anxiety, depression, schizophrenia, Parkinson’s disease, and drug addiction or withdrawal. These receptors are also involved in the modulation of various pain states, which makes them attractive targets for therapeutic drug development.

PET and single-photon emission computed tomography imaging of radioligands that target mGluR5s can be used to visualize and study the CNS mGluR5s in normal and pathological states. Some mGluR5 antagonists have been successfully labeled, but their in vivo visualization has been hampered by high lipophilicity, unfavorable brain uptake kinetics, or high metabolism (5, 6). 2-Methyl-6-(2-phenylethynylpyridine (MPEP) and its analogs, M-MPEP and M-PEPy, have been identified as potent and highly selective noncompetitive antagonists for mGluR5 (1, 7-9). M-MPEP was reported to have approximately six-fold higher in vitro affinity (50% inhibition concentration = 3.4 nM) compared to 5-[(2-methyl-1,3-thizaol-4-yl)-ethynyl]pyridine (MTEP), an mGluR5 antagonist with moderately high affinity (2, 9). Yu et al. (1) synthesized [¹¹C]M-PEPy and demonstrated the feasibility of using it as a PET ligand for in vivo imaging.

Synthesis

[PubMed]

Cosford et al. (9) reported the synthesis of the phenolic precursor 5-(2-(pyridin-2-yl)ethynyl)pyridin-3-ol of M-PEPy. Briefly, sodium methoxide was reacted with 3,5-dibromopyridine in dimethylformamide at 60ºC for 18 h to produce 3-bromo-5-methoxypyridine. M-PEPy was prepared by Sonogoshira cross-coupling of 2-ethynylpyridine with 3-bromo-5-methoxypyridine. M-PEPy was converted to the phenolic precursor by reaction with methylene bromide in the presence of aluminum bromide at 0–25ºC for 1 h. The yield was 76%. Yu et al. (1) reported the radiosynthesis of [¹¹C]M-PEPy by radiolabeling this phenolic precursor with [¹¹C]methyl iodide ([¹¹C]CH₃I) under basic conditions. [¹¹C]CH₃I was prepared from [¹¹C]carbon dioxide with the use of the ¹⁴N(p, α)¹¹C reaction. The best yield condition was determined to be with the use of potassium hydroxide (KOH) as the base. In this radiolabeling reaction, 4–10 mg of solid KOH powder was added to 0.5–1.0 mg of the precursor dissolved in acetonitrile before [¹¹C]CH₃I was added to the reaction mixture. The mixture was heated at 90ºC for 6 min. The radioligand was purified with high-performance liquid chromatography (HPLC). The radiochemical purity was >98.1 ± 1.6% (n = 9), and the specific activity was 31 ± 4.4 GBq/μmol (840 ± 120 mCi/μmol). The radiochemical yield was 1.47 ± 0.70 GBq (39.8 ± 18.8 mCi) at the end of synthesis (EOS). The time of synthesis was ~45 min.

Severance et al. (2) reported the radiosynthesis of [¹¹C]M-PEPy with the precursor desmethyl-M-PEPy with the use of [¹¹C]methyl triflate, which was synthesized from [¹¹C]methyl iodide. Desmethyl-M-PEPy was dissolved in acetone and mixed with aqueous sodium hydroxide. [¹¹C]Methyl triflate was transported into the mixture at room temperature over a period of 5 min, and then mixture was heated at 60ºC for 2 min. The final product was purified with semipreparative HPLC; the radiochemical purity was >99%. The total time of synthesis was 30 min from the end of bombardment with an average yield of 30 ± 5.5% (n = 10) based on [¹¹C]CO₂ at EOS. The average specific activity of [¹¹C]M-PEPy was 70.3 ± 33.3 GBq/μmol (1.9 ± 0.9 Ci/μmol) (n = 9).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Severance et al. (2) studied the in vitro binding of [¹¹C]M-MPEPy with the use of 20-μm sections of human prefrontal cortex, striatum, hippocampus, occipital cortex, and cerebellum, as well as rat brain sections. The sections were incubated with [¹¹C]M-MPEP for 40 min. The sections were then washed and studied with a phosphor imager. Nonspecific binding was evaluated by coincubation with unlabeled M-MPEPy. High specific binding was observed in human brain sections of hippocampus, striatum, ventral striatum, frontal cortex, and occipital cortex. Similar high specific binding was also observed in the hippocampus and cortex of the rat brain sections. Minimal specific binding was found in the both human and rat cerebellum. The hippocampus/cerebellum ratio was ~8 in the human sections and ~37 in the rat sections.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

NIH/NIBIB grant EB01850, PHS MH062185.

References

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Yu M., Tueckmantel W., Wang X., Zhu A., Kozikowski A.P., Brownell A.L. Methoxyphenylethynyl, methoxypyridylethynyl and phenylethynyl derivatives of pyridine: synthesis, radiolabeling and evaluation of new PET ligands for metabotropic glutamate subtype 5 receptors. Nucl Med Biol. 2005;32(6):631–40. [PubMed: 16026710]

2.

Severance A.J., Parsey R.V., Kumar J.S., Underwood M.D., Arango V., Majo V.J., Prabhakaran J., Simpson N.R., Van Heertum R.L., Mann J.J. In vitro and in vivo evaluation of [11C]MPEPy as a potential PET ligand for mGlu5 receptors. Nucl Med Biol. 2006;33(8):1021–7. [PubMed: 17127176]

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Patel S., Ndubizu O., Hamill T., Chaudhary A., Burns H.D., Hargreaves R., Gibson R.E. Screening cascade and development of potential Positron Emission Tomography radiotracers for mGluR5: in vitro and in vivo characterization. Mol Imaging Biol. 2005;7(4):314–23. [PubMed: 16080024]

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Patel S., Krause S.M., Hamill T., Chaudhary A., Burns D.H., Gibson R.A. In vitro characterization of [3H]MethoxyPyEP, an mGluR5 selective radioligand. Life Sci. 2003;73(3):371–9. [PubMed: 12757844]

9.

Cosford N.D., Roppe J., Tehrani L., Schweiger E.J., Seiders T.J., Chaudary A., Rao S., Varney M.A. [3H]-methoxymethyl-MTEP and [3H]-methoxy-PEPy: potent and selective radioligands for the metabotropic glutamate subtype 5 (mGlu5) receptor. Bioorg Med Chem Lett. 2003;13(3):351–4. [PubMed: 12565928]

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Shigemoto R., Nomura S., Ohishi H., Sugihara H., Nakanishi S., Mizuno N. Immunohistochemical localization of a metabotropic glutamate receptor, mGluR5, in the rat brain. Neurosci Lett. 1993;163(1):53–7. [PubMed: 8295733]