Background

[PubMed]

Glutamate is a major excitatory neurotransmitter at neuronal synapses in the central nervous system (CNS) (1, 2). Glutamate produces excitatory effects by acting on cell-surface ionotropic glutamate or metabotropic glutamate receptors (mGluRs). The mGluRs are GTP-binding protein (G-protein)–coupled receptors that play important roles in regulating the activity of many synapses in the CNS, and many neuronal projection pathways contain mGluRs. There are eight mGluR subtypes, which 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. mGluR1 is found in moderate to high density in the cerebellum, caudate, putamen, thalamus, cingulate cortex, and hippocampus, with low density in the pons. mGluR5 is usually found in moderate to high density in the frontal cortex, caudate, putamen, nucleus accumbens, olfactory tubercle, and hippocampus, with low density in the cerebellum. mGluR1 and mGluR5 are positively coupled to phospholipase C in the regulation of neuronal excitability (3). Dysfunction of mGluR1 and mGluR5 is implicated in a variety of diseases in the CNS, including anxiety, depression, schizophrenia, Parkinson’s disease, and drug addiction or withdrawal (2, 4).

Positron emission tomography (PET) radioligands targeting mGluR5 can visualize and analyze mGluR5 expression in normal physiological and pathological conditions (4-8). However, only a few mGluR1 ligands have been studied. N-(4-(6-(Isopropylamino)pyrimidin-4-yl)-1,3-thiazol-2-yl)-4-methoxy-N-methylbenzamide (ITMM) was shown to be a selective mGluR1 antagonist with nanomolar affinity (K_i = 12.6 nM), with little inhibition of mGluR5. Fujinaga et al. (9) labeled ITMM with ¹¹C to prepare N-(4-(6-(isopropylamino)pyrimidin-4-yl)-1,3-thiazol-2-yl)-4-[¹¹C]methoxy-N-methylbenzamide ([¹¹C]ITMM) for use with in vivo PET imaging of mGluR1 distribution in rat brains. Toyohara et al. (10) evaluated [¹¹C]ITMM in mouse and human brains. [¹¹C]ITMM was shown to be a suitable PET radioligand for mapping mGluR1 in the human brain.

Synthesis

[PubMed]

Fujinaga et al. (9) synthesized [¹¹C]ITMM by methylation of the hydroxyl precursor with [¹¹C]MeI (prepared with [¹¹C]CO₂) for 5 min at 70°C. Subsequent separation with high-performance liquid chromatography (HPLC) produced a radiochemical purity >99%. Average radiochemical yield was 22 ± 5% (n = 21) based on [¹¹C]CO₂. The specific activity of [¹¹C]ITMM was 95–140 GBq/µmol (2.56–3.78 Ci/µmol) at the end of synthesis (EOS). The total synthesis time was 25 ± 3 min from the end of bombardment (EOB). For higher specific activity, [¹¹C]ITMM was prepared via methylation of the hydroxyl precursor with [¹¹C]MeI (prepared with [¹¹C]CH₄) for 5 min at 70°C. Average radiochemical yield was 6 ± 1% (n = 3) based on [¹¹C]CH₄. The specific activity of [¹¹C]ITMM was 4,370–7,840 GBq/µmol (118–212 Ci/µmol) at EOS. The total synthesis time was 29 ± 1 min.

Toyohara et al. (10) prepared [¹¹C]ITMM by methylation of the hydroxyl precursor with [¹¹C]methyl triflate under NaOH as a base using an automated synthesizer for 1 min at room temperature. Subsequent separation with HPLC produced a radiochemical purity >98%. Average radiochemical yield was 66.0 ± 27.4% (n = 4) based on [¹¹C]methyl triflate. The specific activity of [¹¹C]ITMM was 97.9–351.1 GBq/µmol (2.64–9.49 Ci/µmol) at EOS. The total synthesis time was <25 min from the EOB.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

In vitro binding affinity of ITMM was determined with rat brain homogenates using [¹⁸F]fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide ([¹⁸F]FITM) as the mGluR1 radioligand (9). The K_i values were 12.6 ± 1.2 nM and 5.4 ± 1.2 nM for ITMM and FITM, respectively. Both agents showed IC₅₀ values >10,000 nM for mGluR5. ITMM (LogD 2.57) was found to be more lipophilic than FITM (LogD 1.45).

In vitro [¹¹C]ITMM autoradiographic imaging studies were performed on rat brain sections (9). [¹¹C]ITMM bound heterogeneously to the brain sections, with the highest accumulation of radioactivity in the mGluR1-rich cerebellum, followed by the thalamus and striatum, with the lowest radioactivity in the pons. JNJ-16259685 (1,000 nM), another mGluR1 antagonist, blocked radioactivity signals by >70% in the cerebellum, thalamus, and striatum.

Animal Studies

Human Studies

[PubMed]

Toyohara et al. (10) performed dynamic PET imaging scans for 90 min with a normal male subject (27 years old) after injection of 565 MBq (15.3 mCi) [¹¹C]ITMM (8 nmol). Radioactivity in the cerebellum reached a plateau at 40 min. The volume of distribution (V_T) values were 2.7, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, and 0.5 for the cerebellum, thalamus, temporal cortex, parietal cortex, frontal cortex, occipital cortex, caudate, and putamen, respectively. [¹¹C]ITMM remained 84.7% intact in the blood at 10 min and declined to 60.5% intact at 60 min. Plasma radioactivity decreased rapidly and exhibited a three-phase clearance pattern with half-lives of 1, 4, and 29 min.

References

1.

Pin J.P., Duvoisin R. The metabotropic glutamate receptors: structure and functions. Neuropharmacology. 1995;34(1):1–26. [PubMed: 7623957]

2.

Ferraguti F., Crepaldi L., Nicoletti F. Metabotropic glutamate 1 receptor: current concepts and perspectives. Pharmacol Rev. 2008;60(4):536–81. [PubMed: 19112153]

3.

Abe T., Sugihara H., Nawa H., Shigemoto R., Mizuno N., Nakanishi S. Molecular characterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/Ca2+ signal transduction. J Biol Chem. 1992;267(19):13361–8. [PubMed: 1320017]

4.

Gasparini F., Lingenhohl K., Stoehr N., Flor P.J., Heinrich M., Vranesic I., Biollaz M., Allgeier H., Heckendorn R., Urwyler S., Varney M.A., Johnson E.C., Hess S.D., Rao S.P., Sacaan A.I., Santori E.M., Velicelebi G., Kuhn R. 2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist. Neuropharmacology. 1999;38(10):1493–503. [PubMed: 10530811]

5.

Ametamey S.M., Kessler L.J., Honer M., Wyss M.T., Buck A., Hintermann S., Auberson Y.P., Gasparini F., Schubiger P.A. Radiosynthesis and preclinical evaluation of 11C-ABP688 as a probe for imaging the metabotropic glutamate receptor subtype 5. J Nucl Med. 2006;47(4):698–705. [PubMed: 16595505]

6.

Ametamey S.M., Treyer V., Streffer J., Wyss M.T., Schmidt M., Blagoev M., Hintermann S., Auberson Y., Gasparini F., Fischer U.C., Buck A. Human PET studies of metabotropic glutamate receptor subtype 5 with 11C-ABP688. J Nucl Med. 2007;48(2):247–52. [PubMed: 17268022]

7.

Baumann C.A., Mu L., Johannsen S., Honer M., Schubiger P.A., Ametamey S.M. Structure-activity relationships of fluorinated (E)-3-((6-methylpyridin-2-yl)ethynyl)cyclohex-2-enone-O-methyloxime (ABP688) derivatives and the discovery of a high affinity analogue as a potential candidate for imaging metabotropic glutamate recepors subtype 5 (mGluR5) with positron emission tomography (PET). J Med Chem. 2010;53(10):4009–17. [PubMed: 20411954]

8.

Honer M., Stoffel A., Kessler L.J., Schubiger P.A., Ametamey S.M. Radiolabeling and in vitro and in vivo evaluation of [(18)F]-FE-DABP688 as a PET radioligand for the metabotropic glutamate receptor subtype 5. Nucl Med Biol. 2007;34(8):973–80. [PubMed: 17998101]

9.

Fujinaga M., Yamasaki T., Yui J., Hatori A., Xie L., Kawamura K., Asagawa C., Kumata K., Yoshida Y., Ogawa M., Nengaki N., Fukumura T., Zhang M.R. Synthesis and evaluation of novel radioligands for positron emission tomography imaging of metabotropic glutamate receptor subtype 1 (mGluR1) in rodent brain. J Med Chem. 2012;55(5):2342–52. [PubMed: 22316010]

10.

Toyohara J., Sakata M., Fujinaga M., Yamasaki T., Oda K., Ishii K., Zhang M.R., Moriguchi Jeckel C.M., Ishiwata K. Preclinical and the first clinical studies on [(11)C]ITMM for mapping metabotropic glutamate receptor subtype 1 by positron emission tomography. Nucl Med Biol. 2013;40(2):214–20. [PubMed: 23265669]