Evaluation of [¹¹C]laniquidar as a tracer of P-glycoprotein: radiosynthesis and biodistribution in rats

Abstract

At present, P-glycoprotein (P-gp) function can be studied using positron emission tomography (PET) together with a labelled P-gp substrate such as (R)-[¹¹C]verapamil. Such a tracer is, however, less suitable for investigating P-gp (over)expression. Laniquidar is a third-generation P-gp inhibitor, which has been used in clinic trials for modulating multidrug resistance transporters. The purpose of the present study was to develop the radiosynthesis of [¹¹C]laniquidar and to assess its suitability as a tracer of P-gp expression.

The radiosynthesis of [¹¹C]laniquidar was performed by methylation of the carboxylic acid precursor with [¹¹C]CH₃I. The product was purified by HPLC and reformulated over a tC₁₈ Seppak, yielding a sterile solution of [¹¹C]laniquidar in saline. For evaluating [¹¹C]laniquidar, rats were injected with 20 MBq [¹¹C]laniquidar via a tail vein and sacrificed at 5, 15, 30 and 60 min after injection. Several tissues and distinct brain regions were dissected and counted for radioactivity. In addition, uptake of [¹¹C]laniquidar in rats pretreated with cyclosporine A and valspodar (PSC 833) was determined at 30 min after injection. Finally, the metabolic profile of [¹¹C]laniquidar in plasma was determined.

[¹¹C]Laniquidar could be synthesized in moderate yields with high specific activity. Uptake in brain was low, but significantly increased after administration of cyclosporine A. Valspodar did not have any effect on cerebral uptake of [¹¹C]laniquidar. In vivo rate of metabolism was relatively low. Further kinetic studies are needed to investigate the antagonistic behaviour of [¹¹C]laniquidar at tracer level.

Introduction

The blood–brain barrier (BBB) is the main barrier between blood and brain. Its purpose is to maintain homeostasis in the central nervous system (CNS). Furthermore, it protects the CNS from endo- and exogenous toxins that are circulating in blood [1], [2].

Only small lipophilic compounds can enter the brain by passive diffusion via the cell membrane. In addition, very small hydrophilic molecules can penetrate the brain via tight junctions between the cells [3]. All other molecules have to pass the BBB via active transport systems, such as active influx and efflux transporters. Several transporters have been identified. The most important, according to our current knowledge, and best characterized efflux transporter is P-glycoprotein (P-gp) [4].

Changes of P-gp expression in the BBB may play a role in several brain disorders and therefore it is of interest to measure its functionality with positron emission tomography (PET) [5], [6]. In recent years, several PET tracers have been developed and characterised in vitro and in vivo for imaging P-gp. Amongst others, these are [^94mTc] complexes, [⁶⁴Cu] complexes, [⁶⁸Ga] complexes, [¹¹C]loperamide, [¹¹C]desmethyl loperamide and [¹¹C]verapamil [7], [8], [9], [10], [11], [12], [13], [14].

[¹¹C]Verapamil is the best validated P-gp PET tracer and has been already used in humans [15], [16], [17]. Initially, the racemic mixture was used, but more recently enatiomerically pure (R)-[¹¹C]verapamil was developed enabling quantification [18] and used in PET studies [19], [20], [21], [22], [23].

There are, however, several limitations associated with the use of (R)-[¹¹C]verapamil, especially its extensive peripheral metabolism, in which radioactive metabolites are formed that can penetrate the BBB and also are substrates for P-gp. In addition, polar radiolabelled metabolites are formed, which might result in a non-P-gp-mediated signal [24]. Moreover, effects of medication (antiepileptic drugs) on peripheral metabolism of (R)-[¹¹C]verapamil are found in epilepsy patients [21]. From these results, it was concluded that an average arterial plasma input function derived from healthy volunteers cannot be used for the analysis of patient studies.

Overexpression of multidrug transporters (MDR-1) at the BBB reduces drug penetration into cerebral tissue. This has been shown to be relevant in patients with epilepsy and brain tumours [6], [25]. In case of overexpression of P-gp, cerebral uptake of (R)-[¹¹C]verapamil will also be reduced, resulting in poor counting statistics and, consequently, in reduced precision of PET measurements. To measure overexpression of P-gp, a better strategy would be to use a specific inhibitor instead of a substrate of P-gp, such as laniquidar. Laniquidar is a third-generation inhibitor, which has been used in Phase I and II clinical trials [26], [27] as well as in extensive animal studies [28]. Laniquidar is a noncompetitive inhibitor, actively binds with high affinity to the P-gp transporter (IC₅₀ value of 0.51 μM) and is less active as a substrate [29]. Therefore it is likely that it will not actively be transported out of the brain, but will remain in cerebral tissue. As P-gp contains several binding sites, its overexpression should lead to increased uptake of [¹¹C]laniquidar. Other potential advantages of [¹¹C]laniquidar are its high selectivity and lack of interactions with cytochrome P450 isoenzymes and therefore insensitive to liver metabolism, which in theory should lead to relatively low levels of labelled metabolites of [¹¹C]laniquidar [30].

The purpose of the present study was to develop a synthesis for [¹¹C]laniquidar and to evaluate [¹¹C]laniquidar as a potential P-gp tracer both in control rats and in rats pretreated with P-gp modulators cyclosporine-A and valspodar (PSC 833).