The peripheral benzodiazepine receptor ligand PK11195 binds with high affinity to the acute phase reactant α1-acid glycoprotein: implications for the use of the ligand as a CNS inflammatory marker¹

Abstract

The peripheral benzodiazepine receptor ligand PK11195 has been used as an in vivo marker of neuroinflammation in positron emission tomography studies in man. One of the methodological issues surrounding the use of the ligand in these studies is the highly variable kinetic behavior of [¹¹C]PK11195 in plasma. We therefore undertook a study to measure the binding of [³H]PK11195 to whole human blood and found a low level of binding to blood cells but extensive binding to plasma proteins. Binding assays using [³H]PK11195 and purified human plasma proteins demonstrated a strong binding to α1-acid glycoprotein (AGP) and a much weaker interaction with albumin. Immunodepletion of AGP from plasma resulted in the loss of plasma [³H]PK11195 binding demonstrating: (i) the specificity of the interaction and (ii) that AGP is the major plasma protein to which PK11195 binds with high affinity. PK11195 was able to displace fluorescein-dexamethasone from AGP with IC₅₀ of <1.2 μM, consistent with a high affinity interaction. These findings are important for understanding the behavior of the ligand in positron emission tomography studies for three reasons. Firstly, AGP is an acute phase protein and its levels will vary during infection and pathological inflammatory diseases such as multiple sclerosis. This could significantly alter the free plasma concentrations of the ligand and contribute to its variable kinetic behavior. Secondly, AGP and AGP-bound ligand may contribute to the access of [¹¹C]PK11195 to the brain parenchyma in diseases with blood brain barrier breakdown. Finally, local synthesis of AGP at the site of brain injury may contribute the pattern of [¹¹C]PK11195 binding observed in neuroinflammatory diseases.

Introduction

PK11195 (1-[2-chlorophenyl]-N-methyl-N-[1-methyl-propyl]-3-isoquinoline carboxamide) is a high affinity ligand of the peripheral benzodiazepine receptor (PBR). Labeled with carbon-11, PK11195 has been used in positron emission tomography studies as an in vivo marker of neuroinflammation and injury in a range of diseases including multiple sclerosis (MS) [1], Alzheimer’s disease (AD) [2], Rasmussen’s encephalitis [3], ischemic stroke [4] and herpes simplex encephalitis [5]. It is believed that the increased binding of [¹¹C]PK11195 seen in these studies is primarily due to the presence of activated microglia, the brain’s resident phagocytic cell, and that as such it may provide an in vivo marker of disease activity in the brain [1].

PBRs are present at low levels in normal central nervous tissue, located primarily on astrocytes and microglia [6]. A number of studies have found that in animal models of brain injury there is time-dependent increase in the binding of PK11195 at the site of damage [7], [8], [9], [10], [11], [12]. The increase in ligand binding is believed to be due to an upregulation in the expression of the PBR, although in a number of studies this has not been substantiated by immunohistochemical data [7], [10]. The question of which glial cell type in CNS disease is primarily responsible for the increased ligand binding is not fully resolved and the answer is most likely model dependent. In a rat experimental allergic encephalitis model of multiple sclerosis it has been demonstrated that activated microglia and macrophages are responsible for a significant part of the observed increase in PK11195 binding [1], [11]. However Kuhlmann and Guilarte [9] demonstrated that both activated astrocytes and microglia were capable of expressing high levels of PBR following neurotoxic injury induced by trimethyltin.

Despite the increasing use of [¹¹C]PK11195 as a PET ligand in studies of human brain disease, there remain a number of methodological issues surrounding the interpretation of results from these studies [1], [2]. Firstly, the very low signal from [¹¹C]PK11195 in the normal brain results in a poorly defined pattern of binding. This in turn makes it difficult to define normal reference tissue in diseases that are disseminated throughout the brain, such as AD and MS. Secondly, standard methods of extracting reference data from the plasma compartment are not possible due to the highly variable kinetic behavior of [¹¹C]PK11195 in plasma. This unstable kinetic behavior has been attributed to variable plasma protein binding [1].

We therefore undertook a study to investigate the distribution of the ligand in blood and to identify its binding to plasma proteins. Our results demonstrate that PK11195 binds with high affinity to the acute phase reactant α1-acid glycoprotein and the relevance of this interaction with regards to the use of the ligand in PET studies is discussed.