(R)-[11C]Emopamil as a novel tracer for imaging enhanced P-glycoprotein function

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Abstract

Introduction

2-Isopropyl-5-[methyl-(2-phenylethyl)amino]-2-phenylpentanenitrile (emopamil; EMP) is a calcium channel blocker of the phenylalkylamine class, with weak substrate properties for P-glycoprotein (P-gp). A weak substrate for P-gp would be suitable for measuring enhanced P-gp function. This study was performed to synthesise (R)- and (S)-[11C]EMP and characterise their properties as P-gp tracers.

Methods

We synthesised (R)- and (S)-[11C]EMP and compared their biodistribution, peripheral metabolism, and effects of the P-gp inhibitor cyclosporine A (CsA, 50 mg/kg). We compared the brain pharmacokinetics of (R)-[11C]EMP and (R)-[11C]verapamil [(R)-[11C]VER] at baseline and CsA pretreatment with small animal positron emission tomography (PET).

Results

(R)- and (S)-[11C]EMP were synthesised from (R)- and (S)-noremopamil, respectively, by methylation with [11C]methyl triflate in the presence of NaOH at room temperature. (R)- and (S)-[11C]EMP yields were ~ 30%, with specific activity > 74 GBq/μmol and radiochemical purity > 99%. (R)-[11C]EMP showed significantly greater uptake in the mouse brain than (S)-[11C]EMP. Both showed homogeneous non-stereoselective regional brain distributions. (R)- and (S)-[11C]EMP were rapidly metabolised to hydrophilic metabolites. Unchanged plasma (S)-[11C]EMP level was significantly lower than that of (R)-[11C]EMP 15 minutes post-injection, whilst > 88% of radioactivity in the brain was intact at 15 minutes post-injection and was non-stereoselective. CsA pretreatment increased brain activity ~ 3-fold in mice, but was non-stereoselective. The baseline area-under-the-curve (AUC) of brain radioactivity (0–60 minutes) of (R)-[11C]EMP was 2-fold higher than that of (R)-[11C]VER, but their AUCs after CsA pretreatment were comparable.

Conclusions

(R)-[11C]EMP is a novel tracer for imaging P-gp function with higher baseline uptake than (R)-[11C]VER.

Introduction

The blood–brain barrier (BBB) plays an important role in both protecting the brain from xenobiotics and maintenance of homeostasis in the internal environment of the central nervous system (CNS) [1]. The BBB has multiple transporters involved in brain-to-blood efflux of both endogenous molecules and xenobiotics in the brain. Amongst these transporters at the BBB, P-glycoprotein (P-gp) plays an important role in restricting the entry into the brain of xenobiotics from the circulating blood. Therefore, altered P-gp function at the BBB has been proposed as a possible aetiology of CNS pathophysiology. For example, enhanced P-gp function may be responsible for drug resistance in several diseases, such as epilepsy [2], [3], depression [4] and human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) [5]. In contrast, decreased P-gp function may decrease clearance of β-amyloid from interstitial fluid in the brain to the plasma, which would result in a predisposition for β-amyloid deposition [6], [7], [8], [9], [10].

To date, a number of strong substrates for P-gp, such as [11C]verapamil ([11C]VER) [11], [12], [13] and [11C]desmethyl loperamide [14], [15], have been developed for imaging P-gp function with positron emission tomography (PET). These strong substrates can measure decreased function of P-gp as increased uptake of tissue radioactivity. However, this method would be unlikely to be able to measure enhanced P-gp function, because baseline tissue radioactivity is already below the limit of detection. Therefore, weak substrates would be suitable for measuring enhanced P-gp function. Along with this strategy, [11C]phenytoin [16] and 6,7-dimethoxy-2-[3-(5-[11C]methoxy-1,2,3,4-tetrahydro-naphthalen-1-yl)-propyl]-1,2,3,4-tetrahydro-isoquinoline [17] were developed and evaluated as P-gp tracers. Another strategy to overcome the low baseline signal is to develop a radiotracer with a higher baseline brain distribution by increasing passive diffusion using a more lipophilic tracer. However, increased lipophilicity could increase P-gp affinity and leads to higher non-specific binding, too.

Emopamil (EMP) is a calcium channel blocker of the phenylalkylamine class. The chemical structure of EMP closely resembles that of VER. Interestingly, EMP partially reversed the multi-drug resistance in human KB cell lines [18]. This reversing potency of EMP was approximately half the level of VER [18]. Furthermore, brain uptake index (BUI) of (S)-EMP was markedly high (110%) and comparable to that of the highly diffusible inert substance iodoantipyrine (120%) [19], whilst the BUI for VER is below 50%. These findings prompted us to develop [11C]EMP as a good candidate for visualising enhanced P-gp function using PET. EMP includes a chiral quaternary carbon centre, and previous research has indicated that the optical isomers differ significantly in their biological effects [19], [20], [21]. Therefore, we first synthesised (R)- and (S)-[11C]EMP and compared their biodistribution, peripheral metabolism and blocking effect on P-gp in mice. Then, we compared the brain pharmacokinetics of (R)-[11C]EMP and (R)-[11C]VER with small animal PET in rats.

Section snippets

General

(R)-Norverapamil and (R)-verapamil hydrochloride were purchased from ABX GmbH (Radeberg, Germany). Cyclosporine A (CsA, Sandimmune® Injection, 50 mg/mL) was purchased from Novartis Pharma (Tokyo, Japan). Eight-week-old male ddY mice and 7-week-old male Wistar rats were purchased from Japan SLC (Hamamatsu, Japan). The animals were allowed to acclimate to the laboratory environment for at least 1 week prior to use. The Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology

Tissue distribution in mice

The tissue distributions of radioactivity after injection of (R)- and (S)-[11C]EMP are summarised in Table 1, Table 2, respectively. The biodistributions of both enantiomers were not markedly different except in the liver, brain and blood. The lung showed the highest initial uptake followed by the kidney, heart and pancreas. The level of radioactivity in the brain increased for the first 5 minutes and then decreased gradually thereafter. (R)-[11C]EMP showed significantly higher radioactivity in

Discussion

In the present study, we synthesised (R)- and (S)-[11C]EMP and compared their biodistribution, peripheral metabolism, and effects of the P-gp inhibitor CsA in mice. The TACs of liver were largely different between (R)- and (S)-enantiomer, might indicate the differences of the hepatic metabolism. There were no differences of TACs between (R)- and (S)-enantiomer in the other organs. Furthermore, regional brain distribution and effects of CsA treatment were not largely different between (R)- and (S

Acknowledgments

We thank Mr. Kunpei Hayashi and Mr. Masanari Sakai (SHI Accelerator Service) for their technical support with the cyclotron operation and radiosynthesis. We also thank Dr. Seijiro Hosokawa (Waseda University) for his valuable advice. This work was supported in part by a Grant-in Aid for Scientific Research (B) 25293271 from Japan Society for the Promotion of Science (JSPS) and a Grant-in-Aid for the Global COE Programme “Practical Chemical Wisdom” from the Ministry of Education, Culture,

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