Immobilized Artificial Membrane Research Articles

Stereoselective binding of chiral ligands to single nucleotide polymorphisms of the human organic cation transporter-1 determined using cellular membrane affinity chromatography

Membranes from stably transfected cell lines that express two point mutations of the human organic cation transporter-1 (hOCT1), R488 M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form two cellular membrane affinity chromatography (CMAC) columns, CMAC(hOCT1 G465R) and CMAC(hOCT1 R488M). Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [ 3H] MMP + (1-methyl-4-phenylpyridinium) as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol, and isoproterenol. The chromatographic data obtained were used to refine a previously developed pharmacophore for hOCT1.

The synthesis and characterization of cellular membrane affinity chromatography columns for the study of human multidrug resistant proteins MRP1, MRP2 and human breast cancer resistant protein BCRP using membranes obtained from Spodoptera frugiperda (Sf9) insect cells

CMAC (cellular membrane affinity chromatography columns) have been developed for the study of the human multidrug transporters MRP1, MRP2 and the breast cancer resistance protein (BCRP). The columns were constructed using the immobilized artificial membrane (IAM) stationary phase and cellular membrane fragments obtained from Spodoptera frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp cDNA, using a baculovirus expression system. The resulting CMAC(Sf9(MRP1)), CMAC(Sf9(MRP2)) and CMAC(Sf9(BCRP)) columns and a control column produced using membrane fragments from non-transfected Sf9 cells, CMAC(Sf9), were characterized using frontal affinity chromatography using [(3)H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9(MRP1)) column, etoposide and furosemide on the CMAC(Sf9(MRP2)) column and etoposide and fumitremorgin C on the CMAC(Sf9(BCPR)) column. The binding affinities (K(i) values) obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. (S)-verapamil displaced [(3)H]-etoposide on the CMAC(Sf9(MRP1)) column to a greater extent than (R)-verapamil and the relative IC(50) values of the enantiomers were calculated using the changes in the retention times of the marker. The observed enantioselectivity and calculated IC(50) values were consistent with previously reported data. The results indicated that the CMAC(Sf9(MRP1)), CMAC(Sf9(MRP2)) and CMAC(Sf9(BCRP)) columns can be used for the study of binding to the MRP1, MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This is the first example of the use of membranes from a non-mammalian cell line in an affinity chromatographic system.

Assessment of the Enzymatic Activity and Inhibition using HPFA with a Microreactor, Trypsin, Absorbed on Immobilized Artificial Membrane

The purpose of this study was to develop a new method to assess the activity and inhibition of the immobilized enzyme trypsin based on the frontal analysis of enzymatic reaction products. This novel technique was performed by on-line monitoring of the absorption at 253 nm of N-benzoyl-L-arginine (BA) from the hydrolysis of N-benzoyl-L-arginine ethyl ester (BAEE). A microreactor was constructed by immobilizing trypsin on an immobilized artificial membrane (IAM)-packed column. Trypsin was non-covalently and dynamically immobilized on-line in the hydrophobic interface of an IAM liquid chromatographic stationary phase. The trypsin-IAM stationary phase was bioactive. Due to the enzymatic reaction, the substrate of BAEE was completely hydrolyzed when the BAEE concentration was below 0.5 mmol/L and partly hydrolyzed when the BAEE concentration ranged from 0.75 to 1.0 mmol/L. By the addition of soybean trypsin inhibitor (STI), phenylmethane sulfonyl fluoride (PMSF), and benzamidine into the substrate solution, the results obtained from the frontal analysis showed that the activity of trypsin on IAM was strongly inhibited not only by STI but by both benzamidine and PMSF.

Drug–Membrane Interaction on Immobilized Liposome Chromatography Compared to Immobilized Artificial Membrane (IAM), Liposome/Water, and Octan‐1‐ol/Water Systems

AbstractThe objective of this study was to investigate drug–membrane interaction by immobilized liposome chromatography (ILC; expressed as lipophilicity index log Ks) and the comparison with lipophilicity indices obtained by liposome/H2O, octan‐1‐ol/H2O, and immobilized artificial membrane (IAM) systems. A set of structurally diverse monofunctional compounds and drugs (nonsteroidal anti‐inflammatory drugs and β‐blockers) were selected in this study. This set of solutes consists of basic or acidic functionalities which are positively or negatively charged at physiological pH 7.4. No correlation was found between log Ks from ILC and lipophilicity indices from any of the other membrane model systems for the whole set of compounds. For structurally related compounds, significant correlations could be established between log Ks from ILC and lipophilicity indices from IAM chromatography and octan‐1‐ol/H2O. However, ILC and liposome/H2O systems only yield parallel partitioning information for structurally related large molecules. For hydrophilic compounds, the balance between electrostatic and hydrophobic interactions dominating drug partitioning is different in these two systems.

Prediction of immobilized artificial membrane‐liquid chromatography retention of some drugs from their molecular structure descriptors and LFER parameters

In this work multiple linear regression (MLR) was carried out for the prediction of immobilized artificial membrane (IAM) retention factors of 40 basic and neutral drugs in two mobile phase compositions. We developed some MLR models by using linear free energy relationships (LFER) parameters and also theoretically derived molecular descriptor. Root mean square error of MLR model in prediction of log k(wPBS)(IAM) and k(wMOPS)(IAM) are 0.332 and 0.351, respectively, while these values are 0.371 and 0.500 for LFER models. Inspections to these values indicate that the statistical parameters of MLR models are better than LFER models. The credibility of MLR models was evaluated by using leave-many-out cross-validation and y-scrambling procedures. The results of these tests indicate the applicability of theoretically derived molecular descriptors and LFER parameters prediction of IAM retention of drugs.

Enantioselective retention of β-blocking agents on human serum albumin and α 1-acid glycoprotein HPLC columns: Relationships with different scales of lipophilicity

The enantioselective retention of thirteen β-blockers on HPLC stationary phases supporting human serum albumin (HSA) or α 1-acid glycoprotein (AGP) was investigated. Eight β-blockers were enantiomerically resolved on the AGP column whereas only four β-blockers were resolved on the HSA column. Moreover, interactions between β-blockers and AGP were much stronger than those with HSA. Retention values on both HSA and AGP for less retained enantiomers related well with various lipophilicity parameters, with the best relationships found with log ⁡ k w IAM values obtained on HPLC stationary phases supporting phospholipids, i.e. the so-called Immobilized Artificial Membrane (IAM). Differently from n-octanol lipophilicity values, these values encode both lipophilic. Electrostatic intermolecular recognition forces which may be involved in the interaction between ionized analytes, such as β-blockers, and proteins. However, their effectiveness to describe non-specific interactions with serum–proteins for other classes of drugs needs further investigations. Analyses performed on AGP with eluent containing dimethyloctylamine (DMOA) as the displacer demonstrated that enantioselective sites bind to both (−)-forms and (+)-forms, but the binding to (−)-forms is stronger. The enantiomer competition to bind to a same site may be relevant from a pharmacokinetic point of view when racemic mixtures are administered. Finally, in contrast to previously reported data in the literature, we found that AGP can bind enantioselectively not only the more lipophilic congeners but also the less lipophilic ones.

Prediction of volume of distribution values in human using immobilized artificial membrane partitioning coefficients, the fraction of compound ionized and plasma protein binding data

We developed an improved Lombardeo's method (J. Med. Chem., 2004) for prediction of VD ss in human. With Elog D substituted by log k IAM, together with f i (7.4) (the fraction of compound ionized at pH 7.4) and log f u (logarithmic fraction of compound unbound in plasma), the predictive equation of f ut (the fraction of the compound unbound in tissues) for the 121 compounds was built, predictive VD ss was further obtained from the Øie–Tozer equation. Our model could be applicable to structurally diverse compounds, including acids, bases, neutrals, and ampholytes. Interior and exterior validation results indicated the model had a robust predictive ability. Compared to the Lombardeo's and Hollósy's (J. Med. Chem., 2006) methods, our model can be generally applicable with better predictive accuracy.

A molecular chromatographic approach to analyze the cell diffusion of arginase inhibitors

Our group demonstrated that arginase inhibition reduces endothelial dysfunction in spontaneously hypertensive rats [C. Demougeot, A. Prigent-Tessier, C. Marie, A. Berthelot, J. Hypertens. 23 (2005) 971; C. Demougeot, A. Prigent-Tessier, T. Bagnost, C. Andre, Y. Guillaume, M. Bouhaddi, C. Marie, A. Berthelot, Life Sci. 80 (2007) 1128] which opens perspectives in the development of drugs against hypertension. In previous papers [T. Bagnost, Y.C. Guillaume, M. Thomassin, J.F. Robert, A. Berthelot, A. Xicluna, C. Andre, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 856 (2007) 113; T. Bagnost, Y.C. Guillaume, M. Thomassin, A. Berthelot, C. Demougeot, C. Andre, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 873 (2008) 37], we developed a biochromatographic column for studying the binding of an arginase inhibitor with this enzyme and the effect of magnesium on this binding. In this paper, the interaction of arginase inhibitors with an immobilized artificial membrane (IAM) has been studied using a biochromatographic approach. This IAM provided a biophysical model system to study the inhibitor passive transport across cells. It was demonstrated that more the inhibitor cross the cell membrane by passive diffusion more it is potent. As well, an analysis of the thermodynamics of the interaction of the arginase inhibitors with the IAM showed that van der Waals, hydrogen and ionic bonds were the main forces between the arginase inhibitors and the polar head groups of the IAM surface.

Prediction of drug bioavailability in humans using immobilized artificial membrane phosphatidylcholine column chromatography and in vitro hepatic metabolic clearance

This study reports a rapid screening method for the prediction of oral drug bioavailability in humans based on combined immobilized artificial membrane (IAM) chromatographic capacity factor (k(IAM)) and in vitro stability in hepatic microsomes. The fraction of drug absorbed (F(a)) in humans was predicted for a set of 15 structurally diverse commercial drugs based on k(IAM) values using a mobile phase consisting of acetonitrile: Dulbecco's phosphate-buffered saline. The hepatic intrinsic clearance (CL'(int)) was calculated from in vitro disappearance half-life, and the oral bioavailability was predicted using in vitro hepatic clearance (CL(h)) and F(a). Significant correlations were observed for the relationships between predicted hepatic extraction ratios (ER(h)) and actual presystemic metabolism (r = 0.854) and between predicted and observed oral bioavailabilities (r = 0.805; p < 0.01). The IAM capacity factor together with the hepatic microsomal disappearance half-life may be useful in identifying compounds with high oral absorption potential in early drug discovery processes.

High Throughput Screening of Physicochemical Properties and In Vitro ADME Profiling in Drug Discovery

Current advances of new technologies with robotic automated assays combined with highly selective and sensitive LC-MS enable high-speed screening of lead series libraries in many in vitro assays. In this review, we summarize state of the art high throughput assays for screening of key physicochemical properties such as solubility, lipophilicity, pKa, drug-plasma protein binding and brain tissue binding as well as in vitro ADME profiling. We discuss two primary approaches for high throughput screening of solubility, i.e. an automated 96-well plate assay integrated with LC-MS and a rapid multi-wavelength UV plate reader. We address the advantages of newly developed miniaturized techniques for high throughput pKa screening by capillary electrophoresis combined with mass spectrometry (CE-MS) with automated data analysis flow. Several new lipophilicity approaches other than octanol-water partitioning are critically reviewed, including rapid liquid chromatographic retention based approach, immobilized artificial membrane (IAM) partitioning and liposome, and potential microemulsion electrokinetic chromatography (MEEKC) for accurate screening of LogP. We highlight the sample pooling (namely cassette dosing, all-in-one, cocktail) as an efficient approach for high throughput screening of physicochemical properties and in vitro ADME profiling with emphasis on the benefit of on-line quality control. This cassette dosing approach has been widely adapted in drug discovery for rapid screening of in vivo pharmacokinetic parameters with significantly increased capacity and dramatically reduced animal usage.

Lipophilicity of some GABAergic phenols and related compounds determined by HPLC and partition coefficients in different systems

Some phenolic compounds, like propofol and thymol, have been shown to act on the GABAA receptor. Taking into account the hydrophobicity of these compounds, their interaction with the membrane surrounding the receptor and consequent non-specific effect on receptor modulation cannot be neglected. In the present work, we determined and correlated several lipophilic parameters for both GABAergic agents and three other related phenolic compounds (eugenol, carvacrol and chlorothymol), including logPo/w, retention data in high performance liquid chromatography (HPLC) by using C18 and immobilized artificial membrane (IAM) columns at different temperatures, and partition coefficients determined in phospholipid liposomes. The correlation results demonstrated the high capacity of the compounds assayed to interact with phospholipid membrane phases, which can be predicted by simple model systems as logPo/w or HPLC. The values obtained by HPLC using a fast screening IAM column were the quantitatively closest to the partition coefficients determined in liposome systems, due to the capacity of this column to permit the establishment of molecular interactions like those found in phospholipid membranes. Finally, the fact that all the compounds studied are able to interact with membranes would suggest the participation of some alteration of the GABAA receptor lipid environment as part of the receptor modulation exerted by phenolic compounds.

Partition Coefficients of Polyphenols for Phosphatidylcholine Investigated by HPLC with an Immobilized Artificial Membrane Column

The biological activity of polyphenols in vitro reflects their affinity for cell membranes and the amount of cellular incorporation. The interaction of polyphenols with phosphatidylcholine was investigated by HPLC with an immobilized artificial membrane (IAM) column. The IAM partition coefficients (K(IAM)) of the polyphenols, calculated by retention times, correlated well with the amounts of polyphenols incorporation into the liposomes.

Lipophilicity of Substituted Aurones and Related Compounds Measured on Immobilized Artificial Membrane (IAM) and Conventional C8 (MOS) Columns

A high performance liquid chromatographic method utilizing an immobilized artificial membrane (IAM HPLC) column has been developed to separate the members of a library containing 38 aurone and thioaurone type structures and to characterise their lipophilicity. The experimental lipophilicity data (k′IAM) have been compared with the previously determined ones C8(MOS) column (k′MOS) and with their calculated lipophilicity parameters (CLOGP). In general, good correlations between the measured and calculated lipophilicities have been found both for the IAM and MOS column. The IAM column showed higher efficiency in separation of isomeric aurones or thioaurones than the MOS one and, consequently, it showed higher potential in differentiation of their lipophilicities. Our findings proved the usefulness of the HPLC method in fast characterisation of the lipophilicity of drug candidates closely related in structure.

Development of an immobilized GPR17 receptor stationary phase for binding determination using frontal affinity chromatography coupled to mass spectrometry

A liquid chromatographic stationary phase containing immobilized membranes from cells expressing the P2Y-like receptor GPR17 is described. Cellular membranes from 1321N1 cells transiently transfected with GPR17 vector [GPR17(+)] and from the same cell line transfected with the corresponding empty vector [GPR17(−)] were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6-mm-i.d. glass columns to create GPR17(+)–IAM and GPR17(−)–IAM stationary phases. Frontal chromatography experiments on both GPR17(+)–IAM and GPR17(−)–IAM demonstrated the presence of a specific interaction with GPR17 only in the former that was maximized by increasing the membrane/IAM ratio. GPR17(+)–IAM was used in frontal affinity chromatography experiments to calculate the dissociation constants ( K d) of three ligands—the antagonist cangrelor (formerly AR-C69931MX, a P2Y 12/P2Y 13 antagonist), MRS2179 (a P2Y 1 receptor antagonist), and the agonist UDP—all of which have been reported to also interact with GPR17. Immobilized GPR17 retained its ability to specifically bind the three analytes, as demonstrated by the agreement of the calculated K d values with previously reported data. Preliminary ranking experiments suggest the application of GPR17(+)–IAM in ranking affinity studies for the selection of new potential candidates.

Retention Behavior of Neutral and Positively and Negatively Charged Solutes on an Immobilized‐Artificial‐Membrane (IAM) Stationary Phase

AbstractThe retention behavior of neutral, positively charged, and negatively charged solutes on the IAM.PC.DD2 stationary phase was investigated and compared. A set of monofunctional compounds and complex drugs (steroids, nonsteroidal anti‐inflammatory drugs, and β‐blockers) were selected for this study, i.e., neutral solutes and solutes with acidic or basic functionalities which are positively charged or negatively charged at pH 7.0. The correlation between the retention factor log kw at pH 7.0 on the IAM.PC.DD2 stationary phase and the partition coefficient log Poct or the distribution coefficient log D7.0 showed that the retention mechanism depends on the charge state and structural characteristics of the compounds. The neutrals were least retained on the IAM.PC.DD2 stationary phase, and positively charged solutes were more retained than negatively charged ones. This implies that the retention of the charged solutes is controlled not only by lipophilicity but also by the electrostatic interaction with the phospholipid, with which positively charged solutes interact more strongly than negatively charged ones.

Prediction of retention indices of drugs based on immobilized artificial membrane chromatography using Projection Pursuit Regression and Local Lazy Regression

The relationship between the logarithm of retention indices (log k(IAM)) of 55 diverse drugs in immobilized artificial membrane (IAM) chromatography and molecular structure descriptors was established by linear and non-linear modeling methods--Projection Pursuit Regression (PPR) and lazy learning method-Local Lazy Regression (LLR). The descriptors calculated from the molecular structures by the software CODESSA and a widely accepted property parameter ClogP were used to represent the characteristics of the compounds. The best multi-linear regression (BMLR) method in the CODESSA was used to select the most important molecular descriptors from a large set of descriptors and to develop the linear and non-linear quantitative structure retention relationship (QSRR) models. By comparing these different methods, the LLR model gave the best predictive results for the drugs studied in the present work with the square of correlation coefficient (R(2)) of 0.9540, 0.9305; root mean square error (RMSE) of 0.2418, 0.3949; for the training set and test set, respectively. It was proved that the LLR method was a promising method for QSRR modeling with good predictive capability for the retention indices of drugs in immobilized artificial membrane chromatography, and could be used in other similar chromatography research fields.

QSAR of Antiproliferative Activity of N‐Substituted 2‐Amino‐5‐(2,4‐dihydroxyphenyl)‐1,3,4‐thiadiazoles in Various Human Cancer Cells

AbstractAntiproliferative activities in vitro against some human cancer cell lines of N‐substituted 2‐amino‐5‐(2,4‐dihydroxyphenyl)‐1,3,4‐thiadiazoles were correlated with their structure‐dependent properties. Phase affinity: lipophilicity and phospholipophilicity were determined by Reversed‐Phase Overpressured Layer Chromatography (RP‐8 OPLC) and Immobilized Artificial Membrane (IAM) High‐Performance Liquid Chromatography (HPLC), respectively. Electronic and steric descriptors were calculated by computational methods. The results indicate that phospholipophilicity and electronic properties expressed by partial charge of atoms of 2‐amino‐1,3,4‐thiadiazole moiety are the main factors that influence the antiproliferative effects of studied compounds. The Quantitative Structure–Activity Relationship (QSAR) equations obtained by the Multiple Linear Regression (MLR) analysis predict activity of compounds of high and average effects precisely and can be used in screening of new potential antiproliferative analogues of 2‐amino‐5‐(2,4‐dihydroxyphenyl)‐1,3,4‐thiadiazole set. At the same time, these dependences as well as Quantitative Activity–Activity Relationship (QAAR) equations point to some differences in the individual human cancer cell lines studied.

Prediction of oral absorption in humans by experimental immobilized artificial membrane chromatography indices and physicochemical descriptors

The purpose of the present study was to examine the human oral absorption (HOA) predictability of the experimentally determined immobilized artificial membrane (IAM) chromatography capacity factor ( log k ′ IAM ) in conjunction with physicochemical descriptors. Transcellular permeation was modeled based on determination of log k ′ IAM considering pH partition hypothesis, and the independent variables were polar surface area (PSA) and molecular weight (MW). The correlation between log k ′ IAM determined at different pH and n-octanol/water partition coefficient (log P) and contribution of polarity (PSA) and size (MW) in the transcellular permeation model were the extension to the previous work. A data set of 37 compounds with partition coefficient values taken from the literature was employed to show importance of ionic interaction in oral absorption prediction. The highest log k ′ IAM value among screened pH 4.5, 5.5, 6.5 and 7.4 ( log k ′ IAM 4.5 − 7.4 ) in conjunction with PSA predicted HOA with coefficient of determination (CD) of 0.9001 compare to log k ′ IAM 4.5 − 7.4 alone with CD of 0.8454 after excluding bretylium from the set of 28 structurally diverse drugs for known reason. PSA helped to avoid over estimation of HOA for amiloride, famotidine and furosemide. The model was tested for its applicability in drug development program and found to predict oral absorption using physically meaningful and structurally related properties making them relatively straightforward for a medicinal chemist to interpret.

Statins (HMG-coenzyme A reductase inhibitors)–biomimetic membrane binding mechanism investigated by molecular chromatography

Many studies have demonstrated that the statin beneficial effects on cardiovascular diseases like coronary are linked to their hypocholesterolemic properties. These lipid-lowering drugs are the first-line pharmacologic therapy for hypercholesterolemia. In this paper, the interaction of a series of statin molecules STCOOH (pravastatin (prava), mevastatin (meva), simvastatin (simva) and fluvastatin (fluva)) with a phosphatidylcholine monolayer immobilized on to porous silica particles has been studied using a biochromatographic approach (molecular chromatography). The immobilized artificial membrane (IAM) provided a biophysical model system to study the binding of the statin molecules to a lipid membrane. For all the test statin molecules, linear retention plots were observed at all temperatures. An analysis of the thermodynamics (i.e., enthalpy (Δ H°), entropy (Δ S°*)) of the interaction of the statin molecules with the immobilized monolayer was also carried out. The Δ H° and Δ S°* values were negative due to van der Waals interactions and hydrogen bonding between the statin molecules with the polar head groups of the phospholipid monolayer (polar retention effect). The statin elution order was: Prava ⋘ Meva < Atorva ≪ Simva < Fluva. This result associated with IC50 data of each statin molecule confirmed that pravastatin, which exhibited the lowest association with the lipid monolayer, was taken up by a membrane transporter. In addition, the logarithm of the statin retention factor with the lipid membrane extrapolated to a total aqueous bulk solvent at pH 7.0 ( log k ′ w − IAM ) was measured and compared to the octanol–water partition coefficient (log P). The observed significant correlation showed an affinity enhanced with the increase in the molecule lipophilicity and confirmed that the hydrophobic forces played an important role in the statin molecule–biomembrane association mechanism. As well, the affinity of STCOOH to IAM is high and changes slightly with the bulk solvent pH, because the number of protons linked to binding is low. This confirmed the importance to take into account the electrostatic interaction in this association mechanism. At pHs lower than ≈7.0, the binding process is accompanied by protons release and at higher pHs protons are taken up. A change in the phospholipid monolayer phosphate group p K a has been proposed to contribute to the positive number of protons exchanged at pHs higher than ≈7.0. This demonstrated that this phosphate residue could function as a general base/proton shuttle by facilitating the deprotonation of STCOOH, i.e., this hyper-reactive residue in the IAM surface could play a catalytic function.

Magnesium cation effect on passive diffusion of statin molecules: Molecular chromatography approach

Recently, immobilized artificial membranes (IAMs) have been introduced as HPLC column packing materials. IAMs consist of phosphatidylcholine residues, the most common phospholipids in natural membranes, covalently bound to silica propylamine and consequently mimic fluid phospholipid bilayer. Thus, the immobilized artificial membrane provided a biophysical model system to study the passive diffusion of the statin molecules through the cellular membrane. Statins or 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA R) inhibitors are widely used for reducing the circulating atherogenic lipid fractions and decreasing cardiovascular morbidity and mortality. This paper describes magnesium cation (Mg 2+) effect on five statin molecules (pravastatin, mevastatin, atorvastatin, simvastatin and fluvastatin)–IAM surface association using a molecular chromatography approach. An analysis of the thermodynamics (i.e. enthalpy (Δ H°), entropy (Δ S°*)) of the interaction of the statin molecules with the immobilized monolayer was also carried out. The Δ H° and Δ S°* values were negative due to van der Waals interactions and hydrogen bonding between the statin molecules with the polar head groups of phospholipid monolayer (polar retention effect). However, the increase of statin–IAM association, with the Mg 2+ concentration increase, was associated with an increase of these thermodynamic data. This explains that this interaction was also governed by hydrophobic and electrostatic bonds which became preponderant. The statin elution order was: Pravastatin ⋘< Mevastatin ≪ Atorvastatin ⋘ Simvastatin < Fluvastatin. This result confirmed that pravastatin, which exhibited the lowest association with the lipid monolayer, was taken up by a membrane transporter. It appeared equally that Mg 2+ supplementation (Mg 2+ concentration range 0.0–2.6 mmol L −1, including its biological concentration range, i.e. 0.75–0.90 mmol L −1) could increase the statin passive diffusion into hepatocytes and their pharmacological actions on cholesterol biosynthesis.