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Permeability: transporting drugs through (lipid) membranes

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  1. Permeability: transporting drugs through (lipid) membranes Paula Garcia 1st Physical Chemistry Symposium, November 30, 2005

  2. Factors Determining Oral Bioavailability Physicochemical factors: Dissolution (solid to solution) Aqueous Solubility Membrane Permeability Biochemical factors Efflux (or counter-transport) Metabolic (in)stability: microflora intestines liver

  3. Passive Diffusion Active Transport Paracellular Efflux Endocytosis Cellular Barrier PAMPA Structure Motifs MW Polarity Caco-2 MDCK Passive diffusion: major absorption pathway Permeation Mechanisms Di, L., Kerns, E., Fan, O.J., Carter, G.T., Eur. J. Med. Chem. 2003, 38, 223.

  4. In-silico models based on: • Lipophilicity • H-bond capacity • Molecular size • Polar Surface Area (PSA) • Quantum properties Rule of five - Lipinski Permeability Measurements Methods approved for the Biopharmaceutical Classification System* • In-vivo intestinal perfusion studies in humans or animals • In-vitro permeation experiments using excised human or animal intestinal tissues • In-vitro permeation experiments across a monolayer of cultured human intestinal cells (e.g Caco-2 cells) Can any of these methods be adapted for high throughput measurements? *2000, www.fda.gov.cder/guidance/index.html

  5. human colonic cell line apical Drug basolateral Caco-2 Permeability Assay Because they are made from cultured cells, Caco-2 membranes express all mechanisms of transport; Therefore, if a drug goes through a Caco-2 membrane, it will probably be absorbed by the GI tract Can Caco-2 assay be used for permeability screening? Yes, but… It takes several days to create membranes* and requires cell culture skills * normally 21-25 days 4-day culture was recently reported: Int. J. Pharm. 2000, 200, 41.


  6. Permeability through lipid membranes ?! Transcellular Absorption Structure of the cell membrane Phospholipid Head Lipophilic Tail Protein 80-95% of commercial drugs (a) • Lipid membranes are quickly and easily made by robots; easily automated for high throughput permeability assays. a) Artursson P., Book of Abstracts, PAMPA 2002 Conference, 2002, San Francisco; Mandagere A.K., Thompson T.N., J. Med. Chem. 2002, 45, 304.

  7. PAMPA: Permeability through lipid membranes 1998 - PAMPA was initially introduced by Kansy from Hoffmann-La Roche 2005 – 95 hits in Pubmed Membrane: egg lecithin in hydrophobic filter Good correlation between PAMPA flux and % HIA Active transport of polar compounds with low Mw Kansy, M.; Senner, F.; Gubernator, K., J. Med. Chem. 1998, 41, 1007.

  8. PAMPA: Parallel Artificial Membrane Permeability Assay Passive Diffusion- Pe (cm/s) Acceptor Membrane (20% (W/V) phospholipid mixture in dodecane) Drug Donor

  9. SDS micelles GastroIntestinal Tract (GIT) Double-Sink Conditions (pH= 5-8) PAMPA: Parallel Artificial Membrane Permeability Assay Passive Diffusion- Pe (cm/s) Acceptor (pH=7.4) Membrane Drug Unstirred Water Layer Donor

  10. PAMPA: workstation

  11. sink (acceptor at top) phospholipid cocktail stirrer (donor at bottom) PAMPA: Sandwich plates

  12. Solubility-Diffusion Model / pH-Partition Theory LogP Passive diffusion: pKa, solubility and lipophilicity are important!

  13. Acid Base The effect of the pH on Permeability Faller, B., Wohnsland, F., J.Med.Chem., 2001, 44, 923; Ruell, J.A., Tsinaman, K.L., Avdeef, A., Eur.J.Pharm.Sci., 2003, 20, 39; Kerns, E.H., Di, L., Jupp, P., Pharm.Sci., 2004, 93, 6, 1440;

  14. unionisable The effect of the pH on Permeability Permeability of ionisable compounds is pH dependent!

  15. Unstirred Water Layer and Ionisation BBB UWL: In GIT: 40 mm In BBB: no UWL GIT pKaflux pH where 50% of the resistance to transport comes from the UWL and 50% from the membrane UWL can be reduced by stirring the donor solution

  16. Pe is UWL limited! Log P ≥ 2 Log P < 2 Compounds with a log P ≥ 2, follow the protocol with stirring. PAMPA Assay: DOUBLE-SINK and Stirring

  17. Permeability improves with increase in lipophilicity The Effect of lipophilicity on Permeability

  18. Pe is UWL limited Paracellular transport The Effect of lipophilicity on Permeability Caco-2 Assay

  19. Caco-2 versus CLogP/D 2 y = -0.2183x + 0.8639x + 0.4508 2 R = 0.5362 2 1.5 Membrane Retention 1 logPapp 0.5 0 -1 0 1 2 3 4 5 -0.5 cLogD7.4 AstraZeneca database Organon database What is the Permeability hurdle, lipophilicity or solubility? Riley et al, Curr. Drug Metab., 2002, 3, 527

  20. The Effect of Solubility on Permeability: Co-Solvent PAMPA Assay (b) SOl (ketoconazole)ACN:H2O=53 (pH 5); 37(pH 7.4); SOl (danazol)ACN:H2O=1 (pH 5); 3(pH 7.4)

  21. The Effect of Solubility on Permeability-Co-Solvent PAMPA Assay Pe is solubility limited (Low Sol., but high Pe) (High Sol., high Pe) (High Sol., Low Pe) Pex10-6 (cm/s) Membrane keeps its integrity Sugano, K., Hamada, H., Machida, M., Ushio, H, Int. J. Pharm.2001, 228, 181; Ruell, A.J., Tsinman, O., Avdeef, A., Chem. Pharm. Bull.2004, 52, 561

  22. The Effect of Solubility on Permeability-Co-Solvent PAMPA Assay (b)

  23. Ionisable compounds display Permeability- pH profile • Lipophilicity ↔ Permeability Reducing the UWL is important for lipophilic compounds. Highly lipophilic compounds display a high membrane retention. • Low aqueous solubility might be a limiting factor in Permeability measurements: Use of co-solvent method allows to differentiate compounds from classes III and IV in the BCS system. 20% of ACN doesn’t interfere with the integrity of the membrane. a)- Kern, E. et al., Pharm. Sci., 2004, 93, 6, 1440; b)- Bermejo, M. et al., Eur. J. Pharm. Sci., 2004, 21, 429; d) Avdeef, A., et al., Chem. Pharm. Bull., 2004, 52, 561; Sugano, K., et al., Int. J. Pharm.,2001, 181. PAMPA Assay in Organon

  24. low permeability high permeability Max-Pe PAMPA Model for Prediction of Human Intestinal Absorption a)- Avdeef, A., Absorption and Drug Development, 2003, Hoboken, NJ: Wiley-Interscience, b)- Avdeef, A. Curr. Top. Med. Chem., 2001, 1, 277.

  25. Factors Determining Intestinal Drug Absorption Fraction of drug absorbed (Fa) is governed by several processes: • Dose/Dissolution ratio, • Chemical degradation and/or metabolism in the lumen, • Complex binding in the lumen, • Intestinal Transit, • Effective Permeability across the Intestinal Mucosa (HJP) • Effective Permeability across the Intestinal Mucosa (HJP) Winimater, S., Bonham, N. M., Lernnernas, H., J. Med. Chem., 1998, 41, 4939.

  26. PAMPA Model for prediction the Human Jejunal Permeability (HJP)(a) Double–Sink (pH=5.0/7.4) a)- Avdeef, A., Absorption and Drug Development, 2003, Hoboken, NJ: Wiley-Interscience, b)- Karlsson, J. P., Artursson, P., Int. J. Pharm., 1991, 7, 55; Karlsson, J. P., Artursson, P., Eur. J. Pharm. Sci., 1999, 9, 47.

  27. Permeability and Molecular Properties Pe is a physicochemical process that depends on physicochemical properties of a molecule and its interactions with a membrane.

  28. Passive Diffusion Transport Caco-2 vs. PAMPA BCS compounds Absorptive Transport Secretory transport Kerns, E.H., Di, L., Petusky, S., J. Pharm. Sci., 2004, 93, 6,1440.

  29. Comparison of PAMPA and Caco-2 Permeability Assay Characteristics PAMPA is a good choice for Screening on Permeability.

  30. Strategy for Combined Use of PAMPA and Caco-2 Passive Diffusion Passive, active, influx, efflux and paracellular Mechanistic Information PAMPA Caco-2 PAMPA + Caco-2 Exploratory Discovery Pre-Development Development Kern, E., (Wyeth Research), J. Pharm. Sci. 2004, 93, 6, 1440.

  31. Acknowledgements Medicinal Chemistry Maarten Honing Marcel Hermkens Michiel Scheffer Department of Medicinal Chemistry

  32. PSA Model for Prediction of Human Intestinal Absorption

  33. Pex10-6 (cm/s) Co-Solvent PAMPA Assay Membrane keeps its integrity

  34. Physicochemical properties of 309 NCEs with low and high bioavailability in rats Bad two or more properties out the preferred range Moderate one property out the preferred range Most important properties are logP, Mw and rotatable bonds