| 摘要 |
The invention described here improves the PAMPA (parallel artificial membrane permeability assay) high-throughput method used in lead compound selection and optimization in pharmaceutical and biotechnological research and development, and for identifying active compounds with the right plant distribution properties in agrochemical research and development. The invention described here is a robust method and apparatus for measurement of two physical properties, permeability and membrane retention of compounds, which overcomes several of the shortcomings in the prior art. This invention includes reagents specifically designed for enhancing the sensitivity of the assay, to allow a UV detection system to be used for concentration measurements, to accurately estimate and compensate for the effects of membrane retention and unstirred water layer, and to increase the speed of the assay. In this invention, concentrated phospholipid membrane barriers are used, consisting of 10-74% wt/vol commercially-available soybean lecithin extract dissolved in dodecane. Sample concentrations in both the donor and the acceptor compartments of the permeation cells are quickly measured by direct UV spectrophotometry. To reduce the excessive membrane retention of compounds by the concentrated phospholipid membrane barriers, an artificial sink state is created in the acceptor compartment of the permeation cells, by using surfactants, cyclodextrins, or water-soluble lipophilic polymers, which have low absorption of UV energy. A secondary sink state is created with ionizable molecules when the permeation cell contains a pH gradient between the donor and the acceptor solutions. The resultant "double-sink" condition forms a basis for successful modeling of the passive-diffusion transport of molecules in the human gastrointestinal tract (GIT). Also, this condition accelerates the transport of certain molecules across membranes, shortening the measurement time, and increasing the assay throughput. A new permeability equation is used, that takes into account (a) pH gradients between the two sides of the membrane barrier and (b) the retention of the molecules by the membrane, which the pH-gradient sink condition is unable to eliminate entirely. When combined with the prior art PAMPA practice of placing solubilizers, such as surfactants, bile salts, and water-soluble lipophilic polymers, into the donor compartment to overcome problems of low aqueous solubility of sample molecules, the said GIT model becomes modified to serve as a blood-brain barrier (BBB) absorption model. This invention can also improve performance in prior art PAMPA membrane barriers, and in several other types of membranes, such as, (i) nonporous synthetic membrane material (e.g., silicone rubber), (ii) dialysis membranes with a particular molecular weight cut-off, and (iii) monolayers of cultured cells (e.g., Caco-2, MDCK, or HT29) deposited onto the surface of porous microfilter supports.
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