Membrane, artificial phospholipids

The evaluation of the apparent ionization constants (i) can indicate in partition experiments the extent to which a charged form of the drug partitions into the octanol or liposome bilayer domains, (ii) can indicate in solubility measurements, the presence of aggregates in saturated solutions and whether the aggregates are ionized or neutral and the extent to which salts of dmgs form, and (iii) can indicate in permeability measurements, whether the aqueous boundary layer adjacent to the membrane barrier, Umits the transport of drugs across artificial phospholipid membranes [parallel artificial membrane permeation assay (PAMPA)] or across monolayers of cultured cells [Caco-2, Madin-Darby canine kidney (MDCK), etc.]. 

In both Navanax neurons (65) and an artificial phospholipid bilayer membrane (66). salicylic acid (1-30 mM) increased K" " permeability but decreased Cl- permeability resulting in a net Increase in membrane conductance. To account for the selective effect of salicylic acid (and other benzoic acids) on the two permeabilities, it was proposed that the anions of the organic acids adsorb to membranes to produce either a negative surface potential (66) or an increase in the anionic field strength of the membrane (47, 48). 

This book is written for the practicing pharmaceutical scientist involved in absorption-distribution-metabolism-excretion (ADME) measurements who needs to communicate with medicinal chemists persuasively, so that newly synthesized molecules will be more drug-like. ADME is all about a day in the life of a drug molecule (absorption, distribution, metabolism, and excretion). Specifically, this book attempts to describe the state of the art in measurement of ionization constants (p Ka), oil-water partition coefficients (log PI log D), solubility, and permeability (artificial phospholipid membrane barriers). Permeability is covered in considerable detail, based on a newly developed methodology known as parallel artificial membrane permeability assay (PAMPA). 

This review describes recent improvements in the measurement of the passive transport of molecules across artificial phospholipid membranes anchored inside... 

One decade has passed since the parallel artificial membrane permeation assay (PAM PA) was first introduced in 1998 [47]. Since then, PAM PA rapidly gained wide popularity in drug discovery [3, 48-51]. Today, PAMPA is the most widely used physicochemical membrane permeation model. The term PAMPA is nowusedas the general name for a plate-based (HTS enabled), biter-supported (filter immobilized) artificial membrane. Typically, phospholipids dissolved in an organic solvent are impregnated into the filter to construct a PAMPA membrane. 

The question is How much can one infer from Dr. Thomas diffusion experiments on the diffusion mechanism in large (bulk) artificial bilayer membranes about mechanisms on bilayer membranes of phospholipids with proteins inclusion Some part of the formulation may break down because we pass from bulk to surface only, from macro- to microdescription. 

Dr. Thomas drew our attention to the fact that, although biological membranes are thin in comparison to his preparations, their diffusion constants may be long. I have measured permeability coefficients (transmembrane "diffusion ) for a number of solutes for artificial phospholipid bilayers (liposomes). The values follow and are to be compared for calculated permeabilities for a solute diffusion across a comparable thickness of water. 

During recent decades, the use of artificial phospholipid membranes as a model for biological membranes has become the subject of intensive research. As discussed above, biological membranes are composed of complex mixtures of lipids, sterols, and proteins. Defined artificial membranes may therefore serve as simple models of membranes that have many striking similarities with biological membranes. A comparison of some important physicochemical properties of biological and artificial membranes is given in Table 1.8 [2]. 

Tab. 5.1 Relative blocking potency of local anesthetics on natural membranes and various physicochemical effects of local anesthetics on artificial phospholipid membranes. (Reprinted from Tab. 1 of ref.

Vaz WLC, Derzko ZI, Jacobson K. Photobleaching measurements of the lateral diffusion of lipids and proteins in artificial phospholipid bilayer membranes. Cell Surface Rev. 1982 8 83-135. 

A EXPERIMENTAL FIGURE 17-8 Vesicle buds can be visualized during in vitro budding reactions. When purified COPII coat components are incubated with isolated ER vesicles or artificial phospholipid vesicles (liposomes), polymerization of the coat proteins on the vesicle surface induces emergence of highly curved buds. In this electron micrograph of an in vitro budding reaction, note the distinct membrane coat, visible as a dark protein layer, present on the vesicle buds. [From K. Matsuoka etal., 1988, Ce//93(2) 263.[... 

Hence, the local binding affinity can be determined. Using this approach a systematic study varying the charge density of the membrane allowed for a locally resolved analysis of the protein-membrane binding affinity. The results showed that binding of ASYN to artificial phospholipid membranes is initiated by the N-terminus (Fig. 11) [126]. 

To evade the interferences due to metabolism or intracellular compartmentalization and sequestration, isolated membrane preparations in the form of vesicles have proved useful, and can be obtained either directly from isolated natural membranes [32,36] or, going one step further, by extracting transport-related proteins and reconstituting them into artificial phospholipid vesicles (liposomes) [34,98]. The preparation of the natural membrane vesicles is aided by the natural tendency of membrane fragments to form closed vesicles spontaneously under suitable conditions. Reconstituted vesicles are more difficult to obtain, for even rather pure preparations of transport components show little tendency to spontaneously integrate themselves in an artificial lipid membrane. Nonetheless, some successful attempts have been described in the literature of such an incorporation. 

P.J. Henderson, J.D.McGivan, and J.B. Chappell, Biocftem./., 111,521 (1969). The Action of Certain Antibiotics on Mitochondrial, Erythrocyte and Artificial Phospholipid Membranes The Role of Induced Proton Permeability. 

Artificial phospholipid vesicles (liposomes) are used to transport vaccines, drugs, enzymes or other substances to target cells or organs. They also make excellent model systems for studying biological ion transport across cell membranes. The vesicles, which are several hundred nanometres in diameter, do not suffer from interference from residual natural ion-channel peptides or ionophores, unlike purified natural cells. For example, the synthetic heptapeptide 5.23 forms pores that promote chloride efflux in vesicle models. Similarly, the ion-pair receptor 2.108 can ferry NaCl from vesicles as an ion-pair ionophore (see Chapter 2, Section 2.6.2), while the hydraphile 5.24 has been shown to transport Na using Na NMR spectroscopy through the bilayer walls of a vesicle model system. 

According to Pollard et al.[i2], the Afi forms cation-selective channels which are capable of transporting calcium and some other monovalent cations when incorporated in artificial phospholipid bilayer membranes. Whether such an effect occurs in intact cells requires further study. These theories assume that A is a substance not normally produced by cells but only in brain tissue of certain aged mammals and in patients with A)9-type amyloidosis. This assumption would be in contradiction to recent reports which show that in cell cultures, under normal metabolic conditions, soluble Ap is produced. Establishing a functional role for Ap remains a challenge. 

There is also inside-outside (transverse) asymmetry of the phospholipids. The choline-containing phospholipids (phosphatidylcholine and sphingomyelin) are located mainly in the outer molecular layer the aminophospholipids (phosphatidylserine and phos-phatidylethanolamine) are preferentially located in the inner leaflet. Obviously, if this asymmetry is to exist at all, there must be limited transverse mobility (flip-flop) of the membrane phospholipids. In fact, phospholipids in synthetic bilayers exhibit an extraordinarily slow rate of flip-flop the half-life of the asymmetry can be measured in several weeks. However, when certain membrane proteins such as the erythrocyte protein gly-cophorin are inserted artificially into synthetic bilayers, the frequency of phospholipid flip-flop may increase as much as 100-fold. 

Artificial membrane systems can be prepared by appropriate techniques. These systems generally consist of mixtures of one or more phospholipids of natural or synthetic origin that can be treated (eg, by using mild sonication) to form spherical vesicles in which the lipids form a bilayer. Such vesicles, surrounded by a lipid bilayer, are termed liposomes. 

The popular applications of the adsorption potential measurements are those dealing with the surface potential changes at the water/air and water/hydrocarbon interface when a monolayer film is formed by an adsorbed substance. " " " Phospholipid monolayers, for instance, formed at such interfaces have been extensively used to study the surface properties of the monolayers. These are expected to represent, to some extent, the surface properties of bilayers and biological as well as various artificial membranes. An interest in a number of applications of ordered thin organic films (e.g., Langmuir and Blodgett layers) dominated research on the insoluble monolayer during the past decade. 

A very promising method, immobilized artificial membrane (IAM) chromatography, was developed by Pidgeon and co-workers [299-304,307], where silica resin was modified by covalent attachment of phospholipid-like groups to the surface. The retention parameters mimic the partitioning of drugs into phospholipid bilayers. The topic has been widely reviewed [47,298,307,309-311]. 

In the bulk of this chapter we will focus on the rapidly emerging new in vitro technology based on the use of immobilized artificial membranes, constructed of phospholipid bilayers supported on lipophilic filters. One objective is to complete the coverage of the components of the transport model explored in Chapter 2, by considering the method for determining the top curve (horizontal fine) in the plots... 

The survey of over 50 artificial lipid membrane models (pION) in this chapter reveals a new and very promising in vitro GIT model, based on the use of levels of lecithin membrane components higher than those previously reported, the use of negatively charged phospholipid membrane components, pH gradients, and artificial sink conditions. Also, a novel direction is suggested in the search for an ideal in vitro BBB model, based on the salient differences between the properties of the GIT and the BBB. 

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