Using Ex Vivo Porcine Jejunum to Identify Membrane Transporter Substrates: A Screening Tool for Early—Stage Drug Development
Abstract
1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Porcine Intestine
2.3. Experimental Protocol for Permeation Studies
2.4. Analytical Methods
2.5. Data Analysis
2.5.1. Permeability Calculations
- dc/dt: change in the acceptor concentration calculated from the slope of the concentration–time curve between 20 and 80 min
- V: volume of the buffer in the donor compartment (7 mL)
- A: exposed surface area (1.26 cm2)
- C0: initial concentration of the substrate in the donor compartment (100 µM, in case of digoxin: 50 µM)
2.5.2. Calculating Drug Deposition (QDEP) and Drug Permeation (QPERM)
- mint 2h: amount of drug in the intestinal membrane at the end of the experiment (t = 2 h)
- macc 2h: amount of drug permeated into the acceptor compartment at the end of the experiment (t = 2 h)
- mdonor 0h: amount of drug in the donor compartment at the beginning of the experiment (t = 0 h)
2.6. Statistical Analysis
3. Results and Discussion
3.1. SLC Transporters
3.1.1. PEPT1
3.1.2. OATP2B1
3.1.3. OCT1
3.2. ABC Transporters
3.2.1. P-gp
3.2.2. BCRP
3.2.3. MRP2
3.2.4. MRP3
3.3. Localization of Membrane Transporters in Porcine Jejunum
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Transport Protein | Substrate (BCS/BDDCS) | Km (µM) | MW (g/mol) | log P [49] | log D a Octanol/H2O, pH 7.0 | Solubility in KBR (n) (mg/mL) | Inhibitor (Ki/IC50) (µM) |
---|---|---|---|---|---|---|---|
SLC Transport Proteins | |||||||
PEPT1 | Cefadroxil (-/III [50]) C16H17N3O5S | n.d. | 363.39 | −0.4 | −3.15 | 45.03 ± 1.10 (4) | Losartan (24/52) [51] |
OATP2B1 | Rosuvastatin (III [52]/III [50]) C22H28FN3O6S | 2.4 [53] | 481.54 | 0.13 | −1.91 | 0.42 ± 0.16 (4) | Rosiglitazone (-/5.2) [54] |
OCT1 | Ranitidine (III [55]/III [50]) C14H22N2O3 | 70 [56] | 314.40 | 0.27 | −1.44 | 19.64 ± 2.78 (3) | Atropine (-/1.2) [57] |
ABC Transport Proteins | |||||||
P−gp | Digoxin (II [55]/IV [50]) C41H64O14 | 73 [58] | 780.94 | 1.26 | 1.29 | 0.04 ± 0.01 (3) | Verapamil (-/10.7) [59] |
Fexofenadine (III [55]/III [50]) C32H39NO4 | n.d. | 501.66 | 5.6 | 1.23 | 0.42 ± 0.16 (4) | ||
BCRP | Sulfasalazine (IV [60]/II [50]) C18H14N4O5S | 0.7 [61] | 398.39 | 0.4 | −0.10 | 3.31 ± 0.14 (5) | Fluvastatin (5.43/-) [62] |
MRP2 | Valsartan (III [55]/III [50]) C24H29N5O3 | 30.4 [63] | 435.5 | 3.68 | −0.68 | 3.71 ± 0.68 (5) | Indomethacin (-/0.06) [64] |
MRP3 | Fexofenadine (III [55]/III [50]) C32H39NO4 | n.d. | 501.66 | 5.6 | 1.23 | 0.42 ± 0.16 (4) | Indomethacin (-/-) |
Drug | Transporter | Papp,pig (10−6 cm/s) | QDEP (%) | QPERM (n)(%) | QDEP (%) | QPERM (n)(%) | Papp,rat (10−6 cm/s) | Papp,human (10−6 cm/s) | |
---|---|---|---|---|---|---|---|---|---|
(−INH) (n) | (+INH) (n) | (−INH) | (+INH) | (−INH) | (−INH) | ||||
Cefadroxil | PEPT1 | 2.82 ± 0.20 (4) | 1.91 ± 0.55 (6) | 1.31 ± 0.40 | 0.50 ± 0.18 (4) | 1.47 ± 0.25 | 0.31 ± 0.12 (6) | 4.99 ± 0.50 [70] | - |
Rosuvastatin | OATP2B1 | 0.91 ± 0.64 (7) | 2.25 ± 0.85 (6) | 3.10 ± 1.46 | 0.17 ± 0.09 (7) | 1.30 ± 0.14 | 0.29 ± 0.16 (6) | - | 6.95 ± 1.50 [30] |
Ranitidine | OCT1 | 5.07 ± 0.83 (12) | 1.96 ± 0.28 (5) | 1.11 ± 0.46 | 0.62 ± 0.17 (12) | 0.43 ± 0.12 | 0.21 ± 0.03 (5) | 4.00 [72] | 5.50 [72] |
Digoxin | P-gp | 0.38 ± 0.23 (4) | 1.64 ± 0.79 (3) | 0.62 ± 0.17 | 0.21 ± 0.12 (4) | 0.40 ± 0.19 | 0.12 ± 0.04 (3) | 6.4 ± 1.9 [14] | 1.44 ± 0.72 [30] |
Sulfasalazine | BCRP | 0.01 ± 0.00 (5) | 0.63 ± 0.43 (9) | 3.23 ± 0.56 | 0.00 ± 0.00 (5) | 2.47 ± 1.24 | 0.12 ± 0.07 (9) | 2.76 ± 0.19 [73] | 0.09 ± 0.06 [30] |
Valsartan | MRP2 | 1.20 ± 0.10 (4) | 0.93 ± 0.41 (4) | 0.44 ± 0.02 | 0.17 ± 0.02 (4) | 0.57 ± 0.07 | 0.24 ± 0.10 (4) | - | - |
(−IND/−VER) (n) | (−IND/+VER) (n) | (+IND/−VER) (n) | (+IND/+VER) (n) | |
---|---|---|---|---|
Papp,pig (10−6 cm/s) (n) a | 2.11 ± 0.73 (6) | 4.48 ± 3.29 (5) | 0.64 ± 0.20 (6) | 0.84 ± 0.52 (6) |
QDEP (%) (n) | 1.00 ± 0.24 (6) | 0.54 ± 0.09 (5) | 0.81 ± 0.61 (6) | 1.50 ± 0.33 (6) |
QPERM (%) (n) | 0.06 ± 0.01 (6) | 0.36 ± 0.20 (5) | 0.27 ± 0.05 (6) | 0.22 ± 0.10 (6) |
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Arnold, Y.E.; Kalia, Y.N. Using Ex Vivo Porcine Jejunum to Identify Membrane Transporter Substrates: A Screening Tool for Early—Stage Drug Development. Biomedicines 2020, 8, 340. https://doi.org/10.3390/biomedicines8090340
Arnold YE, Kalia YN. Using Ex Vivo Porcine Jejunum to Identify Membrane Transporter Substrates: A Screening Tool for Early—Stage Drug Development. Biomedicines. 2020; 8(9):340. https://doi.org/10.3390/biomedicines8090340
Chicago/Turabian StyleArnold, Yvonne E., and Yogeshvar N. Kalia. 2020. "Using Ex Vivo Porcine Jejunum to Identify Membrane Transporter Substrates: A Screening Tool for Early—Stage Drug Development" Biomedicines 8, no. 9: 340. https://doi.org/10.3390/biomedicines8090340
APA StyleArnold, Y. E., & Kalia, Y. N. (2020). Using Ex Vivo Porcine Jejunum to Identify Membrane Transporter Substrates: A Screening Tool for Early—Stage Drug Development. Biomedicines, 8(9), 340. https://doi.org/10.3390/biomedicines8090340