口服药物的体外吸收模型

Intestinal Mucosa: A Physical and Biochemical Barrier for Oral Drug Absorption
E L
Light micrograph of monkey small intestinal mucosa illustrating the three layers of the mucosa: the epithelium (E), the lamina propria (L), and the muscularis mucosa (M). (taken from J. L. Madera and J. S. Trier in Physiology of the Gastrointestinal Tract (L. R. Johnson, Ed.), 2nd Edition, Raven Press, New York, 1987, p.1210)
•Immunosuppressive Drugs •Anti-gout agent
Colchicine
•Steroids
Dexamethasone Progesterone Cortisol
•Antibiotic
Erythromycin
•Anti-tuberculous agent
Rifampin
Schematic Illustration of MDR1, MRP1, MRP2, MRP3, MRP5 and BCRP in Transfected Polarized Epithelial Cells
Correlation between Oral Absorption in Humans and Membrane Permeability (Papp) in Caco-2 Cells
P. Artursson et al., Adv. Drug Del. Rev. 46: 27-43 (2001)
History of Cell Culture Models Used to Predict Drug Permeation across the Intestinal Mucosa
Late 1980s-Mid 1990s: Caco-2 cells Mid 1990s: Caco-2 cells vs. MDCK cells
Ref.: Tang, F., Horie, K, and Borchardt, R.T. (2002) Are MDCK cells transfected with human MDR1 gene a good model of the human intestinal mucosa? Pharm. Res. 19 765-772 Tang, F., Horie, K, and Borchardt, R.T. (2002) Are MDCK cells transfected with human MRP2 gene a good model of the human intestinal mucosa? Pharm. Res. 19 773-779
In Vitro Models for Oral Drug Absorption
Outline



Physical and Biochemical Barrier for Oral Drug Absorption and In Vitro models for drug absorption Evaluation of the Method Caco -2 Cell Suitability Use of Caco–2 and Factor Affecting Permeation of NCE Continuous Dissolution/Caco-2 System for Formulation Screening of NCEB
•Cancer drugs
Vinblastine Vincristine Taxol Daunorubicin Etoposide Cyclosporin A FK506
•HIV protease inhibitors
Amprenavir Indinavir
•Cardiac drugs
Digoxin Quinidine
Intestinal Perfusion Studies in Humans
Correlation between Fraction Absorbed of d-Glucose and Phenazone in Human Regional Perfusion Study
Drug Absorption Study in situ and in Conscious Rat
A
C D
Basolateral
Multidrug Resistance Transporters and CYP3As Form a Concerted Barrier to Drug Absorption
Apical side
Metabolized
CYP3As Basolateral side
Substrate drug
Expression of MDR1 in Caco-2, MDCK-WT, and MDCK-MDR1 Cells
KD M 1A 1B 2A 2B 250
3A 3B
4A 4B
148
M: marker, A: 5 µg protein B: 10 µg protein. 1: Caco-2, 2: MDCK-WT, 3: MDCKMDR1 (low passage), 4: MDCK-MDR1 (high passage).
Apical side
Tight junction
MDR1 MRP1 MRP2 MRP3 MRP5 BCRP
Basolateral side
Reference: P. Borst et al., BBA 1461,347-357 (1999) J. Jonker et al., JNCI 92 (20), 1651-1656 (2000)
Drug metabolite
Multidrug Resistance Transporters (MDR1, MRP2, BCRP)
MDR1 (P-glycoprotein, P-gp)
Substrates e.g. Digoxin Inhibitors e.g. GF120918
A
B
Examples of Drugs That are Substrates for MDR1
DQ Papp =
PA to B
Apical (A)
DQ/Dt: The linear appearance rate of mass in the receiver solution.
A: Cell monolayer surface area.
Dt · A ·C0
Cell Monolayer
Basolateral (B)
Late 1990s: Caco-2 cells vs. MDCK cells vs. MDCK-MDR1 vs. MDCK-MRP2
Caco-2 Cell Monolayer System
Advantages Cells permit estimation of: • both cellular uptake and drug transepithelial transport • drug transport in the presence of metabolic reactions • polarized efflux Cells are: • of human origin (colon adenocarcinama) • viable for long periods Disadvantages • Relative long-period of time for culture (3 weeks) • Underexpression of key drug-metabolizing enzymes (e.g.,CYP3A4) • Variable expression levels of efflux transporters (e.g., MDR1, MRP2)
In Situ Rat Intestinal Perfusion
0.2ml/min Pump Donor Blood
• Animals: MaleSpragueDawley rats weighing 350400g were used. • Perfusion solution: NaH2PO4 (57.9mM), Na2SO4 (79.6mM), pH 7.5.
Models Used to Determine the Intestinal Mucosal Permeation Characteristics of a Drug Candidate
•Oral dosing of animals or humans •Intestinal perfusion studies in humans •Dosing of animals through a chronic cannula in the intestinal lumen •In Situ intestinal perfusion studies in animals •Efflux transporter-gene knockout mice •In Vitro permeation studies using excised human or animal intestinal mucosal tissues •In Vitro permeation studies using cell culture models •PAMPA, IAM •Gastro Plus
C0: The initial concentration in the donor solution. Apically localized efflux transporter (e.g. MDR1) Substrate drug (e.g. Digoxin)
PB to A
Ratio of PB to A/PA to B : An indicator of efflux activity of transporter in cell monolayer
Jugular vein
Extraction for LC/MS/MS Analysis Mesenteric vein
Blood
Centrifugation HPLC Analysis
Ileum
Perfusate
Filtration
Bi-directional Transport Experiment Using Caco-2 Cell Monolayers and Calculation
M
View of Intestinal Mucosa, Pre-1990 A
? Current View of Intestinal Mucosa
Apical Paracellular Diffusion Transcellular Diffusion Metabolism A B Efflux Transport
MDCK Cell Monolayer System
J. D. Irvine et al., JPS 88, 28(1999)
Papp values in MDCK cells correlated well with those in Caco-2 cells The correlation between MDCK Papp values and human intestinal absorption is similar to the results observed with Caco-2 MDCK grow faster than Caco-2 (1 week vs 3 weeks)
Questions Concerning MDCK-MDR1/MDCK-MRP2 Cells as the Model of the Intestinal Mucosa
MDR1/MRP2 cDNA
MDCK cLeabharlann Baidulls
MDCK-MDR1/MRP2 cells
When compared with Caco-2 cells, do these cell lines : express the same protein ? exhibit polarized efflux ? exhibit the same kinetics and affinity for substrates ?