The effect of neuroleptic drugs on DPPC/sphingomyelin/cholesterol membranes

Highlights

• Neuroleptics fluidize and disorder the lipid bilayers composed of DPPC, sphingomyelin, and cholesterol.

• Drug protonated species produce stronger effects on the lipid bilayer.

• The driving force for neuroleptics insertion into the bilayer is entropic in nature.

• High drug concentrations revert fluidization or abolishes bilayer thermal transition.

• Some neuroleptics induce the formation of drug rich domains in the lipid bilayer.

• Neuroleptic-protein receptor binding occur in a fluidized and disordered membrane.

Abstract

The hydrophobic nature of neuroleptic drugs renders that these molecules interact not only with protein receptors, but also with the lipids constituting the membrane bilayer. We present a systematic study of the effect of seven neuroleptic drugs on a biomembrane model composed of DPPC, sphingomyelin, and cholesterol. Differential scanning calorimetry (DSC) measurements were used to monitor the gel-fluid phase transition of the lipid bilayer at three pH values and also as a function of drug concentration. The implementation of a new methodology to mix lipids homogeneously allowed us to assemble bilayers completely free of organic solvents. The seven neuroleptics were: trifluoperazine, haloperidol decanoate, clozapine, quetiapine, olanzapine, aripiprazole, and amisulpride. The DSC results show that the insertion of the drug into the bilayer produces a fluidization and a disordering of the bilayer. The bilayer perturbation is qualitatively the same for all the studied drugs, but quantitatively different. The driving force for the neuroleptic drug to place itself in the lipid bilayer is entropic in nature, signaling to the importance of the size and geometry of the drugs. The drug protonated species produce stronger effects than their non-protonated forms. At high concentrations two of the neuroleptics revert the fluidization effect and another completely abolishes the gel-fluid transition. The DSC data and the associated discussion contribute to the understanding of the interactions between neuroleptic drugs and lipid membranes.

Introduction

Since the accidental discovery of the antipsychotic action of chlorpromazine in the 1950s (Madras, 2013), neuropsychiatric disorders are often treated using a variety of neuroleptic drugs. They are mainly employed in disorders associated with psychotic symptoms, particularly in schizophrenia. One important goal in neuropharmacology is to link a given therapeutic effect with a particular neuroleptic action mechanism. In this context, it has been reported that the activity of neuroleptic drugs is connected to their affinity to dopamine receptors such as the D2 protein (Kapur et al., 2000; Lieberman et al., 1998; Nyberg and Farde, 2000; Seeman, 1992) and to the serotonin 5-HT_2A receptors (Gerlach, 1991; Meltzer et al., 1989). However, this action mechanism cannot explain all the clinical effects associated with neuroleptic therapy (Dziedzicka-Wasylewska, 2004; Kapur et al., 2000; Nyberg and Farde, 2000), the challenge being then to understand why in many cases the mechanism based on a specific binding approach cannot explain both the desired and undesired pharmacological effects. Given that both the neuroleptic drugs and the receptor proteins are in the lipid membrane bilayer forming an integral biological system, to fully understand the action mechanism it is necessary to study the influence of neuroleptics on the lipid bilayer.

Lipidomic analyses reveal that about 50 % of the brain's dry weight is lipid (Lim and Wenk, 2009). In general, lipid composition in brain cells varies substantially depending on several factors such as homeostasis and submembrane compartments (Ingólfsson et al., 2017). The lipid function in neuronal transmission has been the subject of intense research after the lipid rafts hypothesis was put forward (Simons and Ikonen, 1997). Lipid rafts define transient membrane domains enriched with cholesterol and sphingolipids that function as protein-anchoring platforms (Farooqui et al., 2009; Simons and Ikonen, 1997; Korade and Kenworthy, 2008; Róg and Vattulainen, 2014; Sezgin et al., 2017). The domain formation takes place at several scales of length and time, from nanometer to micrometer, and from microseconds to seconds (Sevcsik and Schütz, 2016). It has been postulated that such lipid domains play a fundamental role in synaptic transmission, action potential regulation, signaling, anesthesia, and neuropsychiatric disorders (Egawa et al., 2016; Wallace, 2010; Yang et al., 2017). Clearly, a biophysical assessment of bilayers composed of mixtures of biologically relevant lipids is of importance. In these bilayers, there is a permanent electrostatic repulsion between the polar heads of the lipids, which are interacting favorably with water molecules. This repulsion is compensated by the Van der Waals forces existing between the hydrophobic tails of the lipid, producing the structure of the membrane which effectively excludes water from its lipid region. When a molecule inserts into the bilayer the Van der Waals interactions are weakened and thereby the organization of the membrane is disturbed, this phenomenon arising from the interplay between enthalpy and entropy. One of the most convenient methods to experimentally monitor the membrane perturbation caused by the presence of a drug is differential scanning calorimetry (DSC), where the thermodynamic parameters characterizing the gel to fluid phase transition (Lewis et al., 2007) of the lipid bilayer can be determined with precision at many different environmental conditions. It must be mentioned that another technique that provides essentially the same information regarding the membrane fluidity/rigidity properties is electron paramagnetic resonance spectroscopy, that have been used in a variety of systems (Theodoropoulou and Marsh, 1999; Budai et al., 2003; Zhaoa et al., 2007; Pentak et al., 2008).

In this work, we performed a thorough calorimetric investigation to study the effect of neuroleptic drugs on membranes composed of DPPC, sphingomyelin, and cholesterol (hereon denoted by PSC). The combinations of these lipids induce raft-like domains, which have been proposed as key for functioning membrane organization, including functions of the neuronal plasma membrane (Hendrich et al., 2007; Ingólfsson et al., 2017; Korade and Kenworthy, 2008; Wallace, 2010). Based on a recent report (Oropeza-Guzman and Ruiz-Suárez, 2018), we developed a new methodology to assemble the PSC vesicles in water or buffer without using organic solvents. Using DSC, we monitored the gel to fluid transition of a PSC bilayer and PSC membranes doped with the seven neuroleptic drugs whose chemical structure is depicted in Fig. 1. We obtained the calorimetric response of all drugs at three pH values and as a function of drug concentration for three neuroleptics. The data are discussed in terms of the fluidization or rigidization of the lipid bilayer, as a result of the neuroleptic drug presence.