Preclinical and the first clinical studies on [11C]ITMM for mapping metabotropic glutamate receptor subtype 1 by positron emission tomography

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Abstract

Introduction

Preclinical studies and first positron emission tomography (PET) imaging studies were performed using N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-4-[11C]methoxy-N-methylbenzamide ([11C]ITMM) to map metabotropic glutamate receptor type 1 (mGluR1) in the human brain.

Methods

[11C]ITMM was synthesized by O-methylation of the desmethyl precursor with [11C]methyl triflate in the presence of NaOH at room temperature. In vitro selectivity and brain distributions of [11C]ITMM in mice were characterized. Radiation absorbed-dose by [11C]ITMM in humans was calculated from mouse distribution data. Acute toxicity of ITMM at 4.72 mg/kg body weight (> 74,000-fold clinical equivalent dose of [11C]ITMM) was evaluated. Mutagenicity of ITMM was studied by the Ames test. Clinical PET imaging of mGluR1 with [11C] ITMM was performed in a healthy volunteer.

Results

ITMM had low activity for a 28-standard receptor binding profile. Regional brain uptake of [11C]ITMM in mice was heterogeneous and consistent with known mGluR1 distributions. The radiation absorbed-dose by [11C]ITMM in humans was sufficiently low for clinical use, and no acute toxicity or mutagenicity of ITMM occurred. A 90-min dynamic PET scan with [11C]ITMM in a healthy volunteer showed a gradual increase of radioactivity in the cerebellum. Total distribution volume of [11C]ITMM was highest in the cerebellum, followed by thalamus, cerebral cortex, and striatum; regional differences in brain radioactivity corresponded to the mGluR1 distribution in the brain. Peripherally, [11C]ITMM was stable in humans: 60% of the plasma radioactivity remained in the unchanged form for 60 min.

Conclusions

[11C] ITMM is a suitable radioligand for imaging mGluR1 in the human brain providing acceptable dosimetry and pharmacological safety at the dose required for PET.

Introduction

Glutamate is an excitatory neurotransmitter in the central nervous system (CNS) and is involved in many pathological conditions of the CNS. Glutamate receptors are divided into metabotropic (mGluRs) and ionotropic types based on their biological functions and molecular structures [1]. The mGluRs are classified into three groups, including eight subtypes, according to sequence homology, G protein coupling mechanisms, and pharmacological activity [2], [3]. The mGluR subtype 1 (mGluR1) is one of the group I receptors localized postsynaptically in the cerebellar Purkinje cells, striatonigral and striatopallidal projection neurons, and striatal interneurons [4], [5]. The distribution of mGluR1 in the rodent brain has been identified by in vitro autoradiography and immunohistochemistry [6], [7]. These studies demonstrated a wide distribution of mGluR1 in the rat brain with high levels of expression in the cerebellum, moderate or low levels in the thalamus, striatum, and cerebral cortex, and a very low level in the brainstem. Group I mGluRs (mGluR1 and mGluR5) play a role in regulating ion channels and synaptic transmission, and synaptic plasticity, which underlies memory and learning [8], [9], [10]. It has been reported that mGluR1 may be a drug target for the treatment of diseases, such as stroke, epilepsy, pain, cerebellar ataxia, Parkinson's disease, anxiety, and mood disorders [11], [12], [13], [14], [15]. Therefore, in vivo imaging of mGluR1 may provide crucial information about its functions in the living human brain under healthy and pathological conditions, and facilitate the development of CNS drugs targeting mGluR1. Several groups have developed radiotracers for selective imaging of mGluR1 in the human brain by positron emission tomography (PET) [16], [17], [18], [19], [20], [21], [22], [23]. Despite these efforts, there have been no clinical studies using radioligands for mGluR1 in the human brain. Conversely, three mGluR5 PET ligands, i.e., [11C]APB688, [18F]SP203, and [18F]FPEP, have entered clinical trials [24]. Recently, Fujinaga et al. developed N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-4-[11C]methoxy-N-methylbenzamide ([11C]ITMM) as a novel PET ligand for mGluR1 in the rodent brain [25]. An in vitro binding study showed high affinity and selectivity (mGluR1 Ki = 12.6 nM, mGluR5/mGluR1 > 306) of ITMM to mGluR1. [11C]ITMM distributions in the rat brain measured by in vitro autoradiography and small animal PET were consistent with the regional distribution of mGluR1. In contrast, only a very low and uniform distribution of radioactivity was found in the mGluR1 knockout mouse brain. Moreover, brain uptake of [11C]ITMM was selectively blocked by pretreatment with the mGluR1-selective ligand JNJ16259685 [26] and carrier loading. These findings prompted us to undertake a preclinical study of [11C]ITMM.

In the present study, we further characterized in vitro selectivity of ITMM and brain distributions of [11C]ITMM in mice. We also calculated the radiation dosimetry of [11C]ITMM for humans based on a distribution study in mice and examined the acute toxicity and mutagenicity of ITMM in a preclinical study. Finally, we present the first report of clinical PET imaging of mGluR1 with [11C]ITMM in a healthy subject.

Section snippets

General

ITMM and its precursor, 4-hydroxy-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide (O-desmethyl precursor), were prepared by the method described previously [25]. All chemical reagents were obtained from commercial sources. Male ddY mice were obtained from Japan SLC Inc. (Shizuoka, Japan).

The animal studies were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology. The clinical study of [11C]ITMM was also approved by the

Radiosynthesis

[11C]ITMM was synthesized by O-methylation of the O-desmethyl precursor with [11C]methyl triflate under the NaOH (1.0 equivalent) as a base (Fig. 1). The use of excess NaOH (7.4 equivalent) also led to sufficient radiochemical yields (93.7%, decay corrected). The total synthesis time was within 30 min from the end of bombardment. The decay-corrected radiochemical yields of [11C]ITMM based on [11C]methyl triflate were 66.0% ± 27.4% (range, 33.8–93.7) (n = 4). The radiochemical purity of [11C]ITMM was

Discussion

In previous in vitro membrane binding and in vivo small animal PET studies, Fujinaga et al. demonstrated that [11C]ITMM has potential for mapping mGluR1 in the CNS as a PET ligand [25]. In the present study, we further characterized the in vitro selectivity of ITMM and regional brain distributions of [11C]ITMM in mice by the ex vivo tissue dissection method. Then, we investigated the dosimetry of [11C]ITMM and the acute toxicity and mutagenicity of ITMM as a preclinical study. Finally, we

Acknowledgments

This work was supported by Grant-in-Aid for Scientific Research (B) No. 24390298 from the Japan Society for the Promotion of Science (to Kiichi Ishiwata). We thank Mr. Kunpei Hayashi and Ms. Hatsumi Endo for technical assistance.

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