Immobilized artificial membranes (IAMs) are chromatographic surfaces prepared by covalently immobilizing cell membrane phospholipids. IAM surfaces mimic fluid cell membranes. Solute capacity factors (k'IAM) measured on IAM columns correlate very well with solute equilibrium partition coefficients (Km') measured in fluid liposome systems. For 23 structurally unrelated compounds, log-(k'IAM) correlates with log(Km') with a linear correlation coefficient r = 0.907. This indicates that solute partitioning between the IAM bonded phase and the aqueous mobile phase is similar to the solute partitioning between liposomes and the aqueous phase. Although both IAM chromatography and liposome partitioning can be used as in vitro methods to predict solute partitioning into cell membranes, IAM chromatography is experimentally convenient compared to liposome systems. To study the effect of lipid structure on drug binding to IAMs, IAMs were prepared from three different phosphatidylcholine ligands: (i) a diacylated phosphatidylcholine ligand, (ii) a single chain ether phosphatidylcholine ligand, and (iii) a single chain phosphatidylcholine ligand that lacks a glycerol backbone. Solute retention data were identical for all of these IAMs, and consequently, predictions of solute binding to fluid membranes were also identical. This indicates that the structure of the phosphatidylcholine ligand that is immobilized is not critical for the binding of solutes. Since the structure is not important, the binding of solutes to membranes is a bulk phase property, i.e., it is the interface created by the ligands that determines the solute binding properties, not the ligands themselves. Solute partitioning using octanol/water systems does not correlate with k'IAM unless a homologous series of hydrophobic solutes is being evaluated.

Drug-membrane interactions have recently been studied by immobilized artificial membrane (IAM) chromatography (Pidgeon, C.; et al. J. Med. Chem. 1995, 38, 590-595. Ong, S.; et al. Anal. Chem. 1995, 67, 755-762), and the molecular recognition properties of IAM surfaces toward drug binding/partitioning appear to be remarkably close to the molecular recognition properties of fluid membranes. The structural requirements of chromatography surfaces to emulate biological partitioning are unknown. To begin to elucidate the surface structural requirements needed to predict drug partitioning into membranes, three bonded phases were prepared. The chromatography bonded phases were prepared by immobilizing (i) a single-chain analog containing the phosphocholine (PC) headgroup (IAM.PC.DD), (ii) a long-chain alcohol containing polar OH groups protruding from the surface (12-OH-silica), and (iii) a long-chain fatty acid containing OCH3 groups protruding from the surface (12-MO-silica). The 12-OH-silica surface can be considered as an immobilized "octanol" phase with OH groups protruding from the surface and is therefore a solid phase model of octanol/water partitioning systems. As expected, improved capability of predicting solute-membrane interactions as found for the chromatographic surface containing the PC polar head-group because the PC headgroup is also found in natural cell membranes. For instance, the IAM.PC.DD column predicted drug partitioning into dimyristoylphosphatidylcholine liposomes (r = 0.864) better than 12-OH-silica (r = 0.812), and 12-MO-silica (r = 0.817). IAM. PC.DD columns also predicted intestinal drug absorption (r = 0.788) better than 12-OH-silica (r = 0.590) and 12-MO-silica (r = 0.681); reversed phase octadecylsilica (ODS) columns could not predict intestinal absorption (r = 0.10). Collectively, these results suggest that chromatographic surfaces containing interfacial polar groups, i.e., PC, OH, and OCH3, model drug-membrane interactions, but surfaces lacking interfacial polar functional groups (e.g., ODS surface) are poor models. Most interestingly, drug partitioning into octanol/water systems does not correlate with drug binding to the immobilized octanol phase. However, drug partitioning into immobilized octanol correlates with drug partitioning into liposomes (r = 0.812).

Mixed ligand immobilized artificial membranes (IAMs) are surfaces that contain at least two immobilized membrane phospholipids which are designated as either the primary phospholipid or the secondary phospholipid. The primary immobilized phospholipid refers to the immobilized phospholipid that has the highest surface density. For this work, the primary immobilized phospholipid was a single-chain ether phosphatidylcholine (PC) analog. Four mixed-ligand IAMs were prepared by use of immobilized PC as the primary immobilized phospholipid. The secondary immobilized phospholipid ligand was either phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, or phosphatidic acid. All of these secondary phospholipids are bonded at approximately 6-10 mol % relative to the molar amount of immobilized PC. Each secondary phospholipid contains functional groups in the polar head group region that require protecting groups during the immobilization process. The four-step synthetic strategy to prepare mixed-ligand IAMs involves (i) immobilization of the PC analog at high density to silica propylamine (SPA), (ii) immobilization of the second phospholipid (PL) analog at low density, (iii) end capping residual amines with a long-chain anhydride followed by end capping with a short-chain anhydride, and (iv) deprotection of the polar head group protecting groups. The surface density of the mixed PLs bonded to the silica support was approximately 130 mumol of PLs/g of SPA. High-performance liquid chromatography using these mixed lipid IAMs can be exploited to rapidly predict the membrane binding properties of drugs.