In vivo imaging of vesicular monoamine transporter 2 in pancreas using an ¹⁸F epoxide derivative of tetrabenazine

Abstract

Objectives

Development of imaging agents for pancreatic beta cell mass may provide tools for studying insulin-secreting beta cells and their relationship with diabetes mellitus. In this paper, a new imaging agent, [¹⁸F](+)-2-oxiranyl-3-isobutyl-9-(3-fluoropropoxy)-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinoline [¹⁸F](+)4, which displays properties targeting vesicular monoamine transporter 2 (VMAT2) binding sites of beta cells in the pancreas, was evaluated as a positron emission tomography (PET) agent for estimating beta cell mass in vivo. The hydrolyzable epoxide group of (+)4 may provide a mechanism for shifting biodistribution from liver to kidney, thus reducing the background signal.

Methods

Both ¹⁸F- and ¹⁹F-labeled (+) and (−) isomers of 4 were synthesized and evaluated. Organ distribution was carried out in normal rats. Uptake of [¹⁸F](+)4 in pancreas of normal rats was measured and correlated with blocking studies using competing drugs, (+)dihydrotetrabenazine [(+)-DTBZ] or 9-fluoropropyl-(+)dihydro tetrabenazine [FP-(+)-DTBZ, (+)2].

Results

In vitro binding study of VMAT2 using rat brain striatum showed a K_i value of 0.08 and 0.15 nM for the (+)4 and (±)4, respectively. The in vivo biodistribution of [¹⁸F](+)4 in rats showed the highest uptake in the pancreas (2.68 %ID/g at 60 min postinjection). In vivo competition experiments with cold FP-(+)-DTBZ, (+)2, (3.5 mg/kg, 5 min iv pretreatment) led to a significant reduction of pancreas uptake (85% blockade at 60 min). The inactive isomer [¹⁸F](−)4 showed significantly lower pancreas uptake (0.22 %ID/g at 30 min postinjection). Animal PET imaging studies of [¹⁸F](+)4 in normal rats demonstrated an avid pancreatic uptake in rats.

Conclusion

The preliminary results suggest that the epoxide, [¹⁸F](+)4, is highly selective in binding to VMAT2 and it has an excellent uptake in the pancreas of rats. The liver uptake was significantly reduced through the use of the epoxide group. Therefore, it may be potentially useful for imaging beta cell mass in the pancreas.

Introduction

The pancreas is a dual-function organ (active as endocrine and exocrine organ). As such, parasympathetic and sympathetic neurons terminate in the pancreas and these neurons tightly control the functions [1], [2]. The endocrine tissue accounts for only a small percentage of the pancreatic mass (about 1% to 2%); it is found scattered in the islets of Langerhans and consisted predominantly of beta cells. Beta cells produce insulin in response to metabolic demands, and loss of beta cells results in insulin deficiency and diabetes. In type 1 diabetes, common in juveniles, an autoimmune process dramatically disables beta cells. In type 2 diabetes, beta cell mass decreases as part of a slower, more insidious process that also involves peripheral insulin resistance and increased demand for insulin. Since there is a significant beta cell mass reserve, symptoms related to unstable glucose homeostasis are not obvious until beta cell mass has been reduced by more than 50–60% [3]. Unfortunately, most studies measuring beta cell mass have relied on postmortem examination of the pancreas because until recently it has been impossible to prospectively measure beta cell mass in vivo.

Although there is no cure for diabetes, several promising therapies for modifying the disease course are in clinical trials. These approaches mainly focus on preserving or replacing beta cell mass and include pancreas and/or islet cell transplantation [4], [5], [6], [7], [8] or islet cell regeneration from stem cells. The ability to monitor beta cell mass in vivo would greatly facilitate development of disease-modifying therapies for diabetes mellitus [4], [5], [6], [7], [8], [9]. Studies using labeled beta cell-specific antibodies and antibody fragments as in vivo imaging agents have shown some promise but are not suitable for routine clinical use due to a relatively low cellular specificity (low pancreas accumulation vs. high liver and kidney accumulations) [10]. Additional beta cell mass ligands have been reported, but except for VMAT2 imaging agents, none have been successfully utilized to image diabetes in humans [1], [5], [6], [7], [8], [10], [11], [12], [13], [14], [15], [16], [17].

PET imaging of VMAT2 binding sites in the basal ganglia area of the brain using [¹¹C](+)-dihydrotetrabenazine ([¹¹C](+)-DTBZ) (Fig. 1) has been successfully applied in the diagnosis of Parkinson's disease for the past decade [18], [19], [20]. Recently, high level of VMAT2 gene expression in the pancreas was detected [21], [22], [23]. The feasibility of using [¹¹C](+)-DTBZ, a VMAT2 ligand, for PET imaging of the pancreas in monkeys and humans has also been reported [3], [21], [24], [25]. However, ¹¹C has a very short half-life (t_1/2=20 min). Analogs of DTBZ labeled with ¹⁸F, which has a longer half-life (t_1/2=110 min), would be more practical for a widespread application. Previously, we had successfully tested a novel DTBZ derivative, an optically pure fluoropropoxyl-derivative [FP-(+)-DTB, (+)2] (Fig. 1) in animals [26], [27]. It displayed excellent binding affinities (K_i=0.11 nM) for VMAT2. With the use of [¹⁸F](+)2, PET imaging of rat pancreas has been successfully reported [26], [27], [28].

Despite the success of using [¹⁸F](+)2 for PET imaging, it is not ideal for mapping beta cell mass in the pancreas. One of the major obstacles for pancreas imaging is the anatomical proximity of pancreas to liver, which contributes to a high background noise. The liver is a highly active organ, which traps a large number of lipophilic compounds. It subsequently secretes the sequestered lipophilic compounds through the bile or metabolizes the compounds through oxidative mechanisms leading to more water-soluble metabolites. Thus, they are readily excreted through kidney. The tetrabenazine (TBZ) derivatives, such as (±)-TBZ, (+)-DTBZ and (+)2, are neutral and inherently very lipophilic; therefore, normally liver excretion is the predominant route of excretion for these molecules. To further explore the feasibility of imaging beta cell mass, we have made an effort to shift the in vivo excretion pattern from the liver to the kidney, because the kidney is located distant from the target organ, the pancreas. We have designed a new “metabolizable” epoxide derivative of TBZ, which contains an epoxide ring at the C2 position of the TBZ core structure (Fig. 1). By adding the epoxide group we can preserve the binding affinity to VMAT2 sites. It is hypothesized that the epoxide ring will be less stable in vivo. When the epoxide ring is opened, or “hydrolyzed”, in vivo, the resulting hydroxyl derivative will likely be more water soluble. As such, the in vivo kinetics of the hydrolyzed epoxide tracer will show a lower liver accumulation and shift the metabolic path of the tracer towards kidney excretion. By lowering the liver uptake and retention we hope to improve the signal-to-noise ratio and provide an enhanced contrast between the pancreas and the background (higher pancreas-to-liver ratio). The choice of selecting epoxide as an in vivo labile tracer, which may be “hydrolyzed” in vivo, is based on a large number of literature reports suggesting that mammalian epoxide hydrolases commonly present in all types of cells may likely hydrolyze the epoxide ring in vivo [29], [30], [31]. We report herein the synthesis and initial evaluation of the pancreas localization of [¹⁸F](+)-2-oxiranyl-3-isobutyl-9-(3-fluoropropoxy)-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinoline, [¹⁸F](+)4, in normal rats.