Immobilized Artificial Membrane Research Articles

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.

Electrostatic interactions and ionization effect in immobilized artificial membrane retention: A comparative study with octanol–water partitioning

The present study is a continuation of our efforts to investigate the effect of electrostatic interactions and ionization on immobilized artificial membrane (IAM) retention. The previous set of neutral and basic drugs was extended to include acids and ampholytes and analogous buffer conditions in the mobile phase were used, namely morpholinepropanesulfonic acid and phosphate buffer saline, adjusted at pH 7.4. The important contribution of electrostatic forces in IAM retention of positively charged species was further justified by the results of the present study, while analogous electrostatic interactions for ionized acidic drugs were not found to affect significantly the affinity for the IAM stationary phase. The critical role of shielding or exposure of the charged centers on the IAM surface, as a result of the effect of the aqueous component of the mobile phase, was evaluated by the use of water instead of buffer for a number of drugs. Measurements at pH 5.0 demonstrated the effect of ionization in IAM retention despite the partial compensation by electrostatic interactions in the case of protonated basic drugs. Silanophilic interactions were also found to play a potential role as secondary interactions in IAM retention. IAM chromatographic indices were compared to octanol–water distribution coefficients and the corresponding relationships established. Finally, solvation analysis was applied in the aim to gain insight in the balance of forces between IAM retention and octanol–water partitioning. The results showed that apart from electrostatic interactions, there is no significant differentiation between the two systems.

Characterization of the acidity of residual silanol groups in immobilized artificial membranes

Residual silanol acidity and activity of one immobilized artificial membrane (IAM) column have been measured from the retention of LiNO 3 in the column with a methanol/buffer (1 mM in Na +) (60:40, v/v) mobile phase buffered to different pH values. Just one type of silanol with p s s K a = 7.61 (near the pH limit recommended by the manufacturer) was found, although these silanols show large activity. The results obtained have been compared with those obtained previously for Resolve C18, Resolve Silica, Symmetry C18, Symmetry Silica, XTerra MS C18, underivatized XTerra, Lichrospher 100 RP-18, Purospher RP-18e, Luna C18 (2) and Chromolith Perfomance RP-18e, showing that the IAM column is similar to Luna C18 and Symmetry C18 in terms of silica quality, as measured by Li + retention. A warning about the use of IAM columns for emulation of biological systems at physiological pH 7 is given because the ionized silanols may contribute to the retention of some drugs at this pH.

Is phospholipid-saturated alkyl column a convenient replacement for immobilized-artificial-membrane?

Three phosphatidylcholine (PC)-saturated C 8/C 18 stationary phases prepared using biologically representative membrane lipids (purchasable L-α-PC and pure 1,2-dipalmitoyl- sn-glycero-3-phosphatidylcholine) have been developed to compare with IAM (immobilized-artificial-membrane) and C 8/C 18 columns. These PC-coated stationary phases were found to be stable and reproducible for retention experiments. The retention characteristics of nucleobases on these coated phases deviate significantly from those on the IAM counterpart, but surprisingly similar to those of the underlying C 8/C 18 columns. An inter-phase model has been proposed and explored for interpretation of the results.

Development and characterization of immobilized human organic anion transporter-based liquid chromatographic stationary phase: hOAT1 and hOAT2

This paper reports the development of liquid chromatographic columns containing immobilized organic anion transporters (hOAT1 and hOAT2). Cellular membrane fragments from MDCK cells expressing hOAT1 and S2 cells expressing hOAT2 were immobilized on the surface of the immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The resulting stationary phases were characterized by frontal affinity chromatography, using the marker ligand [ 3H]-adefovir for the hOAT1 and [ 14C]- p-aminohippurate for the hOAT2 in the presence of multiple displacers. The determined binding affinities ( K d) for eight OAT1 ligands and eight OAT2 ligands were correlated with literature values and a statistically significant correlation was obtained for both the hOAT1 and hOAT2 columns: r 2 = 0.688 ( p < 0.05) and r 2 = 0.9967 ( p < 0.0001), respectively. The results indicate that the OAT1 and OAT2 have been successfully immobilized with retention of their binding activity. The use of these columns to identify ligands to the respective transporters will be presented.

Monohydroxyflavones: Distribution Coefficients and Affinities for Reverse-Phase (C18) and Immobilized Artificial Membrane (IAM) Adsorbents

Distribution coefficients (D) were measured for flavone, monohydroxyflavones, and monomethoxyflavones equilibrated between 1-octanol and aqueous buffer (50 mM MOPS, pH=7.4). The values of LogD were used to determine substitution constants referred to as π values. Hydroxyl groups at the 3 or 5 position of flavone had positive π values (increased hydrophobicity) while hydroxyl groups at other positions had negative π values (increased hydrophilicity). For each monohydroxyflavone, chromatographic capacity factors (k’) were determined for both reverse phase (C-18) and immobilized artificial membrane (IAM) columns. For the IAM column, relatively large k’ values were observed for both 3-hydroxyflavone and 5-hydroxyflavone indicating that hydroxyl groups at positions 3 and 5 of flavones promote high affinities for phospholipid structures. These results should aid in refinement of quantitative structure activity relationships (QSAR's) that are useful for drug development based on flavonoids as lead compounds.

Investigation of the lipophilic behaviour of some thiazolidinediones: Relationships with PPAR-γ activity

Various lipophilicity aspects of five well-known PPAR-γ ligands, belonging to the thiazolidinedione (TZD) class, ciglitazone (CSZ), troglitazone (TGZ), netoglitazone (NGZ) and the ampholytic pioglitazone (PGZ) and rosiglitazone (RGZ), have been explored. The compounds were found to be highly lipophilic as assessed by direct octanol–water partitioning experiments and further confirmed by reversed phase HPLC measurements under different conditions. Immobilised artificial membrane (IAM) chromatographic indices were also determined as an alternative expression of lipophilicity. They were found to show less diversity forming two clusters. Experimental log D/log P values were compared to those predicted by three widely used calculation systems. For the two ampholytic TZDs, the lipophilicity and retention/pH profiles were established over a broad pH range and compared to the corresponding calculated profiles. Lipophilicity indices derived under the different conditions were further compared to biological activity, concerning in vitro transactivation (pEC 50) and binding affinity (p K i) data, taken from literature. The most active TZD (RGZ) in both transactivation and binding assay proved to be the less lipophilic analogue. An equation relating pEC 50 data to experimental log D 7.4 or reversed-phase log k w values could be established, while p K i data did not lead to satisfactory correlation.

Comparison between immobilized artificial membrane (IAM) HPLC data and lipophilicity in n-octanol for quinolone antibacterial agents

The membrane phospholipid affinity of ten quinolone antibacterial agents, including both acidic and zwitterionic compounds, was measured by HPLC on two different immobilized artificial membrane (IAM) stationary phases, namely IAM.PC.MG and IAM.PC.DD2; it is expressed as the logarithm of the retention factor measured with (or extrapolated to) 100% aqueous eluent at pH 7.0, log k w IAM . Quinolones are a class of highly potent, orally active, broad-spectrum antibacterial agents. For these compounds, lipophilicity values in n-octanol found in the literature, either calculated or measured, are not consistent with each other and are too low to be compatible with their pharmacokinetic properties. The log k w IAM values obtained in this study showed no relation with any of the lipophilicity values in the literature (clog P(a), clog P(b), MLP, log D 7.4). In contrast, they were collinear with a new lipophilicity scale we had previously obtained by an original ion-pair reversed-phase HPLC method set up to estimate the lipophilicity of the neutral forms, log P N . Moreover, when comparing the retention of quinolones on IAM to the retention of structurally unrelated neutral compounds, we observed that they interact with phospholipids with the same affinity as neutral isolipophilic compounds. The use of an eluent at pH 5.5, instead of pH 7.0, increased the retention on IAM not only for acidic, but also for zwitterionic congeners, indicating that phospholipid affinity is enhanced in the experimental conditions that depress the ionization of the acidic function, even when the ionization of the amino function increases simultaneously. To gain an insight into the mechanism of quinolones/serum-protein interactions, we investigated about possible relationships between quinolones affinity data for serum proteins and IAM data. Quinolone affinity for both HSA and AGP was already demonstrated poorly related to n-octanol lipophilicity values, probably due to the occurrence of electrostatic interactions. Only poor relationships were found between IAM and HSA affinity data, whereas quite good relationships were found with AGP affinity data. However, IAM.PC.DD2 data correlated better than those on IAM.PC.MG with quinolone affinity for both serum-proteins, mainly due to the fact that IAM.PC.MG phase is scarcely discriminative for the compounds with the highest retention values. The results suggest that IAM retention can produce a lipophilicity scale that, unlike solvent/water partition coefficients, is consistent with the pharmacokinetic behaviour of zwitterionic quinolones.

Complex retention behavior of pyrimidines on biomembrane-mimic immobilized-artificial-membrane phase

The influence of the chemical substitutions on the interfacial interactions of pyrimidines with the phospholipid-mimic immobilized-artificial-membrane (IAM) chromatographic stationary phase was evaluated. Monocyclic pyrimidine nucleic acid bases (nucleobases) were revealed behaving differently from their bicyclic purine counterparts substantially. The computed electrostatic potential surfaces for both the IAM phase and the interacting nucleobases are intuitive in deconvoluting the retention patterns of pyrimidines molecularly. A structure-retention model has also been derived using quantitative 3D-QSAR methodology pertinent to the IAM-retention of pyrimidines for the potential use in molecular design. IAM phase is found particularly suitable in assessing the retention of pyrimidines with bulky or elongated exocyclic substituents in the biological context than the alkyl-based chromatographic counterparts.

Immobilized P2X 2 purinergic receptor stationary phase for chromatographic determination of pharmacological properties and drug screening

The purinergic receptor signaling system plays an important role in communication between cells in the nervous system and opens new opportunities for screening of potential drugs. Our objective was to explore the pharmacological properties and establish a new methodology for ligand screening for the P2X 2 receptor, which has been developed by the combinatorial library approach Systematic Evolution of Ligands by Exponential enrichment (SELEX). To this end, membranes of 1321N1 cells stably transfected with rat P2X 2 receptors were resuspended in 2% cholate detergent and subsequently coupled onto an immobilized artificial membrane (IAM). The IAM–cholate–P2X 2 mixture was then dialyzed, centrifuged and packed into a FPLC column. Equilibrium binding to the receptor and competition between ATP and the purinergic antagonists suramin and 2′3′- O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) were analyzed by a chromatographic assay using 32P alpha ATP as a radioligand. Our data indicate that suramin does not compete with ATP for the ligand binding site and TNP-ATP is a competitive antagonist, confirming previous studies [C.A. Trujillo, A.A. Nery, A.H. Martins, P. Majumder, F.A. Gonzalez, H. Ulrich, Biochemistry 45 (2006) 224–233]. In addition, we demonstrate that this assay can be used in in vitro selection procedures for RNA aptamers binding to P2X 2 receptors. The results demonstrate that the receptor can be immobilized in a stable format and reused over an extended period of time, facilitating the exploration of ligand–receptor interactions and screening of combinatorial pools for possible ligands.

Interactions of Polyphenols with Immobilized Artificial Membrane and Human Serum Albumin Determined by High Performance Liquid Chromatography

Within recent years, a promising new field of chromatography has opened up with immobilization of phospholipids and human serum albumin in columns and their use in prediction of drug candidates' pharmacokinetics. It is important to investigate early in the drug discovery both the oral absorption of new drug and the extent to which it binds to plasma proteins. In the last few decades plant polyphenols became very popular phytochemicals due to their presumed role in the prevention of various degenerative diseases, such as various types of cancer and cardiovascular diseases [1]. Until recently, relatively little information was available on their absorption and subsequent distribution and excretion in humans. The aim of our work was to investigate interactions of 30 polyphenols with immobilized artificial membrane (IAM) and human serum albumin (HSA) by high performance liquid chromatography in order to predict their oral absorption and plasma protein binding. The interaction of polyphenols with IAM was evaluated by using IAM.PC.DD 2 column. The solvent system (methanol-buffer) was used as a mobile phase with a varying content of organic modifier. The interaction of polyphenols with phospholipids was expressed as the logarithm of capacity factor, which was in the range from 1.345 to 3.581. The HSA binding values were derived from the gradient retention times using Chiral HSA column with buffer and 2-propanol as a mobile phase. The binding of polyphenols to HSA was in the range from 81.8% to 99.8%. Furthermore, the correlation between experimental and pharmacokinetic parameters predicted by different computer programs was considered in order to establish relationship between chromatographic behavior and predicted parameters. According to our research, chromatographic parameters proved to be useful for evaluation of lipophilicity, oral absorption and plasma protein binding properties of investigated polyphenols.

Quantitative structure–retention relationship studies with immobilized artificial membrane chromatography: II: Partial least squares regression

We aimed to establish quantitative structure–retention relationship (QSRR) with immobilized artificial membrane (IAM) chromatography using easily understood and obtained physicochemical molecular descriptors and to elucidate which descriptors are critical to affect the interaction process between solutes and immobilized phospholipid membranes. The retention indices (log k IAM) of 55 structurally diverse drugs were determined on an immobilized artificial membrane column (IAM.PC.DD2) directly or obtained by extrapolation method for highly hydrophobic compounds. Ten simple physicochemical property descriptors (clog P, rings, rotatory bond, hydro-bond counting, etc.) of these drugs were collected and used to establish QSRR and predict the retention data by partial least squares regression (PLSR). Five descriptors, clog P, rotatory bond (RotB), rings, molecular weight (MW) and total surface area (TSA), were reserved by using the Variable Importance for Projection (VIP) values as criterion to build the final PLSR model. An external test set was employed to verify the QSRR based on the training set with the five variables, and QSRR by PLSR exhibited a satisfying predictive ability with R p = 0.902 and RMSE p = 0.400. Comparison of coefficients of centered and scaled variables by PLSR demonstrated that, for the descriptors studied, clog P and TSA have the most significant positive effect but the rotatable bond has significant negative effect on drug IAM chromatographic retention.

The Use of Immobilised Artificial Membrane (IAM) Chromatography for Determination of Lipophilicity

The Use of Immobilised Artificial Membrane (IAM) Chromatography for Determination of Lipophilicity

Estimation of Volume of Distribution in Humans from High Throughput HPLC-Based Measurements of Human Serum Albumin Binding and Immobilized Artificial Membrane Partitioning

The volume of distribution (VD) in humans of 179 known drug molecules (acids, bases, and neutrals) has been modeled using two biomimetic-binding measurements. The phospholipid binding (log K (IAM)) and the plasma protein binding (log K (HSA)) have been calculated from gradient HPLC retention times on immobilized artificial membrane (IAM) and on human serum albumin (HSA) columns, respectively. The log VD values showed good correlation with the compounds' relative binding to IAM and HSA as follows: log VD=0.44 log K (IAM)-0.22 log K (HSA)-0.66; n=179, r2=0.76, s=0.33, and F=272. It was also observed that positively charged molecules bind relatively more to IAM, while negatively charged ones bind more to HSA, in line with the empirical observation that bases tend to have a larger volume of distribution than acids. These results suggest that with the help of these two simple high throughput HPLC-based biomimetic binding measurements an important in vivo drug disposition property can be estimated for use in early drug discovery.

Relationship between immobilized artificial membrane chromatographic retention and human oral absorption of structurally diverse drugs

Capacity factors are determined for a set of drugs for which human oral absorption (HOA) data are available, using immobilized artificial membrane (IAM) chromatography. The compound set represented acidic, basic, neutral and amphoteric drugs from various structure classes and having low to high human oral absorption. Effect of mobile phase pH on retention was investigated to determine the optimal condition for better correlation with HOA. The retention (capacity factor, k ′ IAM ) of each drug was measured by reverse phase HPLC using an IAM.PC.DD2 (1 cm × 3 mm i.d., 12 μm) column with an eluent of acetonitrile—0.01 M phosphate buffer at pH 4.5–7.4. The pH dependent k ′ IAM was in accordance with pH partition theory. Using non-linear regression analysis the obtained log k ′ IAM values were compared with published data on HOA in order to establish correlation. The better correlation with HOA was observed when the highest log k ′ IAM value selected among pH 4.5–7.4 ( R 2 = 0.8566) for each drug rather than obtained at more traditional pH 7.4 ( R 2 = 0.7403). Finally, it was confirmed by Cook's D outlier test that there was no influential observation in the model that affect the relationship between IAM capacity factor and HOA. The assay conditions were optimized and validated to make it suitable for routine analysis and for compound characterization in early discovery where permeability may be an issue.

Computational prediction of solubilizers’ effect on partitioning

A computational model for the prediction of solubilizers’ effect on drug partitioning has been developed. Membrane/water partitioning was evaluated by means of immobilized artificial membrane (IAM) chromatography. Four solubilizers were used to alter the partitioning in the IAM column. Two types of molecular descriptors were calculated: 2D descriptors using the MOE software and 3D descriptors using the Volsurf software. Structure–property relationships between each of the two types of descriptors and partitioning were established using partial least squares, projection to latent structures (PLS) statistics. Statistically significant relationships between the molecular descriptors and the IAM data were identified. Based on the 2D descriptors structure–property relationships R 2 Y = 0. 99 and Q 2 = 0.82–0.83 were obtained for some of the solubilizers. The most important descriptor was related to log P. For the Volsurf 3D descriptors models with R 2 Y = 0.53–0.64 and Q 2 = 0.40–0.54 were obtained using five descriptors. The present study showed that it is possible to predict partitioning of substances in an artificial phospholipid membrane, with or without the use of solubilizers.

Quantitative structure-retention relationship studies using immobilized artificial membrane chromatography I: Amended linear solvation energy relationships with the introduction of a molecular electronic factor

Linear solvation energy relationships (LSERs) amended by the introduction of a molecular electronic factor were employed to establish quantitative structure-retention relationships using immobilized artificial membrane (IAM) chromatography, in particular ionizable solutes. The chromatographic indices, log k IAM, were determined by HPLC on an IAM.PC.DD2 column for 53 structurally diverse compounds, including neutral, acidic and basic compounds. Unlike neutral compounds, the IAM chromatographic retention of ionizable compounds was affected by their molecular charge state. When the mean net charge per molecule ( δ) was introduced into the amended LSER as the sixth variable, the LSER regression coefficient was significantly improved for the test set including ionizable solutes. The δ coefficients of acidic and basic compounds were quite different indicating that the molecular electronic factor had a markedly different impact on the retention of acidic and basic compounds on IAM column. Ionization of acidic compounds containing a carboxylic group tended to impair their retention on IAM, while the ionization of basic compounds did not have such a marked effect. In addition, the extra-interaction with the polar head of phospholipids might cause a certain change in the retention of basic compounds. A comparison of calculated and experimental retention indices suggested that the semi-empirical LSER amended by the addition of a molecular electronic factor was able to reproduce adequately the experimental retention factors of the structurally diverse solutes investigated.

Chromatographic Estimation of Drug Disposition Properties by Means of Immobilized Artificial Membranes (IAM) and C18 Columns

Chromatographic retention measurement in immobilized artificial membranes (IAMs) is considered a fast and reliable method to predict biological properties (drug distribution) because of the IAM structure, which consists of phospholipid analogues bonded covalently to silica particles. A new parameter (d) is proposed to estimate the similarity between IAM columns, conventional HPLC columns, and drug distribution systems, and thus the performance of chromatographic systems to predict drug distribution. An IAM.PC.DD2 column has been used for this study, together with two XTerra columns (MSC18 and RP18), at several acetonitrile-water mobile phases. According to the d parameter, good correlations should be obtained between chromatographic systems (both IAM and C18) and octanol-water partition coefficient (log P), and thus both types of columns could be used to obtain log P values. The IAM.PC.DD2 system shows a close similarity to human skin partition, tadpole narcosis, and blood-brain permeability processes, showing that it can be useful as a model for these biological processes. Controversially, it is shown that human skin permeation is more similar to C18 partition than to IAM partition. Other biological processes such as blood-brain distribution and tissue-blood partition show a poor similarity to IAM and C18 systems, demonstrating that estimation of these drug distribution processes by chromatographic measurements may not be adequate.

Development of an immobilized mutant organic cation transporter column for on‐line screening using chromatographic techniques

Purpose To develop and validate and immobilize in a liquid chromatographic column format two point mutations of the human Organic Cation Transporters (R488M and G465R). Methods The membranes used in the study were obtained from a transfected MDCK cell line that expressed hOCT1-R488M or G465R and hOCT1-transfected MDCK cells [hOCT(+)]. The cells were homogenized, solubilized, and subsequently mixed with 160 mg of immobilized artificial membrane (IAM) stationary phase and the resulting mixture was dialyzed for 2 days. The resulting stationary phase mixture was packed into a HR 5/2 glass column to yield a 150 mm x 5 mm (ID) chromatographic bed. The column was placed in a HPLC system containing an on-line scintillation detector. A series of nine displacer ligands with [3H]-MPP as the marker ligand were run through the R488M and the G465R column to obtain elution profiles showing a front and plateau regions. Results The R488M point mutation resulted in a change of the binding properties of the catechols and failed to exhibit the enantioselectivity observed for verapamil on the hOCT1 transporter, while the G465R exhibited similar binding properties as the hOCT1 however resulted in a non-functional transporter. Conclusions The results suggest that the R488M mutation is not located at the transport site but in the vicinity of this site, while the G465R is believed to be in the transport site.

Molecular lipophilicity determination of a huperzine series by HPLC: Comparison of C18 and IAM stationary phases

Two hydrophobic parameters (log k w−C18 and log k w−IAM, respectively) of a huperzine A series were extrapolated by high performance liquid chromatography (HPLC) using both C18 and immobilised artificial membrane (IAM) columns. A mathematical correlation between C18 and IAM hydrophobic parameters was completed, suggesting a similar behaviour on both columns. This behaviour was principally led by hydrophobic forces. The theoretical lipophilicity (log P) of each compound was computed using Pallas ® software and compared to experimental values, showing a similar lipophilic behaviour. Finally, the huperzine log k w−IAM and log k w−C18 values were correlated with the relative bound percentage of huperzine in human serum albumin, confirming that hydrophobic forces are predominant in the huperzine–HSA binding mechanism.