Permeability 2013 11 08 Contents Permeability Overview Permeability

Permeability 김소연 2013. 11. 08

Contents Permeability Overview Permeability Fundamentals Permeability Effect Permeability Structure Modification Strategies Problem

Peameability Overview � � Permeability is the velocity of molecule passage through a membrane barrier. Permeability is a determinant of intestinal absorption and oral bioavailability. Optimizing passive diffusion is productive because it is the predominant mechanism for absorption of most commercial drugs. Permeability is increased by removing ionizable groups, increasing Log P, and decreasing size and polarity.

Permeability Fundamentals � Different membranes can have different permeabilities for a compound.

Passive Diffusion Luminal Cytoplasm Abluminal lipid bilayer Membrane � � � Passive diffusion is driven by a concentration gradient (High->Low Concentration) It has been estimated that 95% of commercial drugs are predominantly absorbed in the GI tract by passive diffusion It is important to remember that permeability is much higher for more liphophilic molecules than for polar molecule p. H and p. Ka play important roles in passive diffusions.

Passive Diffusion

Passive Diffusion � The passive diffusion of acids is much higher at low p. H. � The passive diffusion of bases is low at low p. H.

Passive Diffusion � � For an acid passive diffusion is enhanced in the direction of the higher p. H because of the ionization equilibrium. On the other hand, base passive diffusion is enhanced in the direction of the lower p. H.

Endocytosis � � Compounds may be engulfed by the membrane, pass through the cell within the vesicle, and be released on the other side. Minor interest for small molecule drug discovery. Active uptake � � Compound binds to a transmembrane protein and moves through the membrane. Active transport requires the expenditure of energy, commonly two ATPs for each molecule transported.

Palacellular � � GI tract or other organs are sometimes termed leaky because of the somewhat loose junctions between the cells that allow molecules to slip between. Paracellular permeability in the GI tract is available primarily to compounds that have a molecular weight less than 180 Da and are polar. Efflux � � � Efflux, the active transport of compounds from inside the cell or membrane back into the lumenal space. Pgp(p-glycoprotein) BCRP (breast cancer resistance protein)

Combined Permeability � � GI track – Passive diffusion – Absorption BBB – Active transport, efflux

Factors that affect permeability � Concentration gradient � p. H polarity � Expression of transporter � Transporter Km � Affinity of the compound for various transporters � The size of the pores between membrane barrier

Permeability Effect Living System Bioavailability In vitro discovery Cell based Activity assay

Bioavailability � � Compounds with low permeability typically have low bioavailability In this case, passive diffusion is limited for the highly charged acidic compound (p. Ka= 4. 5).

Cell based Activity assay � Both Good enzyme activity and permeability were required in order to produce good bioactivity in the cell based assay.

Cell based Activity assay

Modification Strategies Permeability Structure Modification Strategies Ionizable group to non-ionizable group Add lipophilicity Isosteric replacement of polar groups Esterify carboxylic acid Reduce hydrogen bonding and polarity Reduce size Add nonpolar side chain Prodrug

Modification Strategies � Ionizable group to non-ionizable group

Modification Strategies � Add lipophilicity

Modification Strategies � Isosteric replacement of polar groups

Modification Strategies � Esterify carboxylic acid

Modification Strategies � Reduce hydrogen bonding and polarity

Modification Strategies � Reduce size

Modification Strategies � Add nonpolar side chain

Modification Strategies � Prodrug

Problem � � � � 1. What is the predominant permeability mechanism for absorption of most commercial drugs? 2. What are the structural properties of compounds that undergo paracellular permeation? 3. How will passive diffusion permeability change as p. H increases from 4. 5 to 8 for: (a) basic compound, (b) acidic compound? 4. List important permeability barriers for drug discovery. 5. Which of the following structural modifications likely will improve permeability? : (a) change an amine to a methyl, (b) add a hydroxyl group, (c) remove a propyl group, (d) change a carboxylic acid to an ethyl ester, (e) change a carboxylic acid to a tetrazole. 6. For the following lead compared, what structural modifications could you make that might improve permeability?

Problem � � 7. Permeability is important for which of the following? : (a) absorption in intestine, (b) CYP metabolism, (c) BBB penetration, (d) dissolution in the intestinal lumen, (e) in vitro cell-based assay, (f) to reach intracellular targets in vivo. 8. Following are groups that could be added to a lead compound that is MW 300 and has Clog. P 2. 0. Rank them from lowest to highest predicted permeability of the product: (a) – CH 3, (b) –OH, (c) –OCH 3, (d) –COOH. 9. Following are groups that could be added to a lead compound that is MW 450 and has Clog. P 4. 5. Rank them from lowest to highest predicted permeability of the product: (a) – C 6 H 5, (b) –CH 3, (c) –C 3 H 7. 10. Following are groups that could be added to a lead compound that is MW 250 and has Clog. P 0. 0. Rank them from lowest to highest permeability of the product: (a) –CH 3, (b) –C 6 H 11, (c) –C 3 H 7

Contents Permeability Method Overview In Silico Permeability Method In Vitro Permeability Method In Depth Peameability Method

Permeability Method Overview � � � In silico methods are available for calculating in vivo and in vitro permeability, such as intestinal absorption, Caco-2, and parallel artificial membrane permeability assay (PAMPA). High-throughput permeability methods utilize high-performance liquid chromatography (immobilized artificial membrane), artificial membranes (PAMPA), and cell layers(Caco-2). In-depth study of permeability uses portal vein cannulation and in situ perfusion.

In Silico Permeability Method

In Vitro Permeability Method � IAM � � HPLC Test compounds partition between the aqueous mobile phase and the phospholipid phase. Compounds are rank ordered by k, which indicates a higher lipophilicity or phospholipids affinity. The parameters of this affinity correlate with permeation.

In Vitro Permeability Method � Cell layer Permeability assay Caco-2

In Vitro Permeability Method

In Vitro Permeability Method � Artificial Membrane Permeability assay • PAMPA reduces the cost and increases the throughput of permeability assays. • Instead of a barrier made of living cells, the PAMPA barrier is made of phospholipids solubilized in along-chain hydrocarbon.

In Vitro Permeability Method � Comparisn of Caco-2 and PAMPA Method • Comparison of Caco-2 and PAMPA permeability values from the same compound can provide insight on permeability mechanisms.

In Depth Permeability Methods