Kinetics and Thermodynamics of Chlorpromazine Interaction with Lipid Bilayers: Effect of Charge and Cholesterol Patrícia T. Martins, † Adrian Velazquez-Campoy, ‡,§ Winchil L. C. Vaz, † Renato M. S. Cardoso, † Joana Vale ́ rio, ∥ and Maria Joa ̃ o Moreno* ,† † Chemistry Department FCTUC, Largo D. Dinis, Rua Larga, 3004-535 Coimbra, Portugal ‡ Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, 50018 Zaragoza, Spain, Unidad Asociada BIFI-IQFR, CSIC, Zaragoza, Spain § Fundació n ARAID, Diputació n General de Aragó n, Spain ∥ Instituto de Tecnologia Química e Bioló gica − UNL, Av. da Repú blica-EAN, 2780-157 Oeiras, Portugal * S Supporting Information ABSTRACT: Passive transport across cell membranes is the major route for the permeation of xenobiotics through tight endothelia such as the blood−brain barrier. The rate of passive permeation through lipid bilayers for a given drug is therefore a critical step in the prediction of its pharmacodynamics. We describe a detailed study on the kinetics and thermodynamics for the interaction of chlorpromazine (CPZ), an antipsychotic drug used in the treatment of schizophrenia, with neutral and negatively charged lipid bilayers. Isothermal titration calorimetry was used to study the partition and translocation of CPZ in lipid membranes composed of pure POPC, POPC:POPS (9:1), and POPC:Chol:POPS (6:3:1). The membrane charge due to the presence of POPS as well as the additional charge resulting from the introduction of CPZ in the membrane were taken into account, allowing the calculation of the intrinsic partition coefficients (K P ) and the enthalpy change (ΔH) associated with the process. The enthalpy change upon partition to all lipid bilayers studied is negative, but a significant entropy contribution was also observed for partition to the neutral membrane. Because of the positive charge of CPZ, the presence of negatively charged lipids in the bilayer increases both the observed amount of CPZ that partitions to the membrane (K P obs ) and the magnitude of ΔH. However, when the electrostatic effects are discounted, the intrinsic partition coefficient was smaller, indicating that the hydrophobic contribution was less significant for the negatively charged membrane. The presence of cholesterol strongly decreases the affinity of CPZ for the bilayer in terms of both the amount of CPZ that associates with the membrane and the interaction enthalpy. A quantitative characterization of the rate of CPZ translocation through membranes composed of pure POPC and POPC:POPS (9:1) was also performed using an innovative methodology developed in this work based on the kinetics of the heat evolved due to the interaction of CPZ with the membranes. ■ INTRODUCTION Passive transport across cell membranes is the major route for the permeation of xenobiotics through tight epithelia, such as the vascular endothelium that constitutes the blood brain barrier. The rate of passive permeation through lipid bilayers, a critical step in the prediction of pharmacodynamics, is usually evaluated from the drug hydrophobicity with little consid- eration for the rate of insertion/desorption or translocation through the lipid bilayer. However, in most cases, the rate of the interaction (rather then the equilibrium partition) is the most relevant parameter, 1−3 and therefore it is very important to have kinetic details. Chlorpromazine, a phenothiazine derived antipsychotic agent, is recommended in psychiatric disorders where neuro- leptic sedative treatment is needed. It is amphiphilic and self- aggregates at a critical concentration, forming micelle-like structures, which undergo temperature- and concentration- dependent phase transitions. 4 Critical micelle concentrations (CMC) reported for chlorpromazine by different authors, using different techniques and experimental conditions, are scattered over a range of 2 orders of magnitude (from 10 −5 to 10 −3 M). It should be noted that chlorpromazine has a tertiary amine (pK a = 9.35 5 ), and therefore intermolecular interactions depend strongly on the solution pH and ionic strength. 6 At conditions similar to those used in this study (22 °C, 10 mM phosphate buffer at pH 7.3, with 140 mM NaCl), the CMC was found to be 2 × 10 −4 M. 6 Aggregation of CPZ at concentrations well below its CMC is well supported by literature, 7,8 and the structure of a dimer has actually been proposed. 4 To guarantee that CPZ is predominantly in the monomeric form, its concentration in the aqueous solution at pH ≈ 7 must be maintained below 3 × 10 −5 M. 9 Received: October 21, 2011 Published: January 31, 2012 Article pubs.acs.org/JACS © 2012 American Chemical Society 4184 dx.doi.org/10.1021/ja209917q | J. Am. Chem. Soc. 2012, 134, 4184−4195