Absorption/Transport

Contract Research | Absorption/Transport | Metabolism | Induction | Physico-Chemical | Toxicity | Sponsor-Initiated Protocols

Drug Permeability Measurement in Caco-2, LLC-PK1 or MDCK Cell Monolayers
Drug permeability through cell monolayers correlates well with intestinal permeability and oral bioavailability. Several mammalian cell lines are appropriate for this measurement (Pharm Res. 12:693 [1995]; J. Pharm. Sci. 88:28 [1999])^1,2. Apical to basolateral diffusion is measured using a standard set of time points and drug concentrations or adapted to a high throughput mode. Analysis is by LC/MS or LC/MS/MS. Alternatively, the incubations can be returned to the sponsor for analysis. FDA recommended permeability comparators and controls for membrane integrity are included. Data are reported as apparent permeability (Papp) or percent flux under fixed conditions.

Human P-glycoprotein (PGP) Screen
Determine if your compounds interact with the xenobiotic transporter PGP. ATP hydrolysis is required for in vivo drug efflux by PGP. The in vitro PGP-ATPase assay measures the phosphate liberated from drug-stimulated ATP hydrolysis in our human PGP membranes (Cat. No. 453228, formerly K228). The assay screens compounds in a high throughput mode using single concentration determinations compared to the ATPase activity of a known PGP substrate, or with a more detailed approach by determining the concentration-dependence and apparent Km of the drug-stimulated ATPase activity, or inhibitory interaction with PGP.

PGP-Mediated Drug Transport in Polarized Cell Monolayers
PGP (encoded by MDR1) is a member of the ABC transporter superfamily and is expressed in the human intestine, liver and other tissues. Localized to the cell membrane, PGP functions as an ATP-dependent efflux pump, capable of transporting many structurally unrelated xenobiotics out of cells. Intestinal expression of PGP may affect the oral bioavailability of drug molecules that are substrates for this transporter. Determine if your compounds are PGP substrates by a direct measure of transport across polarized cell monolayers. Bidirectional transport (apical to basolateral and basolateral to apical) is measured in Caco-2 cells, or in LLC-PK1 cells expressing human PGP cDNA and corresponding control cells. The concentration-dependence of intrinsic activity of human PGP is analyzed for saturation of PGP-mediated transport, and apparent kinetic parameters (Papp, Km, Vmax) are calculated. Alternatively, the incubations can be returned to the sponsor for analysis. Controls for membrane integrity and comparator compounds are included.

New: BD Biosciences has developed LLC-PK1 cell lines expressing mouse, dog, cynomolgus and rhesus monkey PGP cDNA. Determine if your compounds are substrates for PGP transport in control and polarized cell monolayers.

Inhibition of PGP-Mediated Drug Transport in Polarized Cell Monolayers
Drugs that inhibit PGP can alter the absorption, disposition and elimination of co-administered drugs and can enhance bioavailability or cause unwanted drug-drug interactions. Determine if your compounds are PGP inhibitors by a direct measure of inhibition of PGP-mediated transport of a model substrate across polarized cell monolayers. Typically, multiple test compound concentrations are examined. Data are reported as IC50 values or percent inhibition. Positive controls for PGP-inhibition and membrane integrity are included.

Transporter-Mediated Drug Uptake in Xenopus laevis Oocytes
Membrane transporters play a key role in determining the exposure of liver, kidney, brain and other tissues to a variety of solutes, including nutrients, cellular by-products, environmental toxins, drugs and other xenobiotics. Human Organic Cation Transporter 1 (hOCT1, SLC22A1) exists in liver, kidney and other tissues. It transports TEA, NMN, MPP and many other cations (J. Pharmacol. Exp. Ther. 286(1):354 [1998])³. Human Na+-Taurocholate Cotransport Protein (hNTCP, SLC10A1) is found in liver and localized to the sinusoidal membrane. It transports many bile salts (J. Clin. Invest. 93(3):1326 [1994])⁴. Human Organic Anion Transporting Polypeptide 1 (hOATP1, SLC21A3) is predominantly found in brain. Human Organic Anion Transporting Polypeptide 2 (hOATP2, SLC21A6) is a liver-specific transporter and localized to the sinusoidal membrane. Both hOATP1 and hOATP2 have broad substrate selectivity, including conjugated and unconjugated bile salts, steroids and steroid conjugates, organic compounds, thyroid hormones (J. Biol. Chem. 274(24):17159 [1999])⁵. Rat Organic Anion Transporting Polypeptide 1 (rOATP1, SLC21A1) is localized to the sinusoidal membrane in liver, or to the brush border membrane in kidney. It also has broad substrate selectivity (Am. J. Physiol. 271:G231 [1996]; J. Biol. Chem. 274(52):37161 [1999])^6,7. All of these transporters have been expressed in Xenopus laevis oocyte. Determine if your compounds are substrates of these transporters by a direct uptake assay. In the direct uptake assay, the tested compound is incubated with transporter-cRNA injected oocytes and the compound taken up inside the oocytes is quantified by scintillation counting or by LC/MS. The concentration-dependence is analyzed for saturation of transporter-mediated transport, and apparent kinetic parameters calculated. Time dependence of transporter-mediated transport is analyzed by incubating the test compound with oocytes for different time periods. Negative controls are performed under the same conditions by using water injected or un-injected oocytes.

Inhibition of Transporter-Mediated Drug Uptake in Xenopus laevis Oocytes
Drugs that inhibit membrane transporters can alter the absorption, disposition and elimination of co-administered drugs and can enhance bioavailability or cause unwanted drug-drug interactions. Inhibition of transporter-mediated drug uptake can be measured with indirect assays. In this test, we determine if your compounds can inhibit the transporter-mediated uptake of a model substrate. Typically, multiple test substance concentrations are examined. Data are reported as IC50 values, or percent inhibition when using only one or two concentrations of test compound.

Service Information/References

1. Stewart, B.H., Chan, O.H., Lu, R.H., Reyner, E.L., Schmid, H.L., Hamilton, H.W., Steinbaugh, B.A. and Ta y l o r, M.D. Comparison of intestinal permeabilities determined in multiple in vitro and in situ models: relationship to absorption in humans. Pharm. Res.12:693 [1995].
   
2. Irvine, J.D., Takahashi, L., Lockhart, K., Cheong, J., Tolan, J.W., Selick, H.E. and Grove, J.R. MDCK (Madin-Darby Canine Kidney) cells: A tool for membrane permeability screening. J. Pharm. Sci. 88:28 [1999].
   
3. Zhang, L., Schaner, M.E. and Giacomini, K.M. Functional characterization of an organic cation transporter (hOCT1) in a transently transfected human cell line (HeLa). J. Pharmacol. Exp.Ther. 286:354 [1998].
   
4. Hagenbuch, B. and Meier, P.J. Molecular cloning, chromosomal localization and functional charaterization of a human liver Na+/bile acid cotransporter. J. Clin. Invest. 93(3):1326 [1994].
   
5. Abe, T., Kakyo, M., Tokui, T., Nakagomi, R., Nishio, T., Nakai, D., Nomura, H., Unno, M., Suzuki, M., Naitoh, T., Matsuno, S. and Yawo, H. Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1. J. Biol. Chem. 2 7 4 (24):17159 [1999].
   
6. B e rgwerk, A.J., Shi, X., Ford, A.C., Kanai, N., Jacquemin, E., Burk, R.D., Bai, S., Novikoff, P.M., Stieger, B., Meier, P.J., Schuster, V.L. and Wolkoff, A.W. Immunologic distribution of an organic anion transport protein in rat liver and kidney. Am.J.Physiol. 271(2 Pt 1):G231 [1996].
   
7. Hsiang, B., Zhu, Y., Wang, Z., Wu, Y., Sasseville, V., Yang, W.P. and Kirchgessner, T.G. A novel human hepatic organic anion transporting polypeptide (OATP2). Identification of a liver-specific human organic anion transporting polypeptide and identificaiton of rat and human hydroxymethylglutaryl-CoA reductase inhibitor transporters.
   J. Biol.Chem. 274(52):37161 [1999].

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The complete FDA guidance document can be viewed on the web at:
http://www.fda.gov/cder/guidance/clin3.pdf