Xenobiotic Bioavailability:Xenobiotic Bioavailability: Role of IntestinalRole of Intestinal
DispositionDisposition
Thomas J. CookDepartment of Pharmaceutics
Ernest Mario School of Pharmacy Rutgers, The State University of New Jersey
Bioavailability and Fraction DoseBioavailability and Fraction Dose Absorbed
• Bioavailability (F) – measurement of the rate andextent of therapeutically active drug that reachesextent of therapeutically active drug that reaches the systemic circulation and is available at thethe systemic circulation and is available at the site of actionsite of action ((ShargelShargel & Yu, 1999)& Yu, 1999)
• Fraction Dose Absorbed ( fa – fraction of oral)dose that traverses the intestine intactdose that traverses the intestine intact
Intestinal DispositionIntestinal Disposition
• Intestinal permeability, metabolism,solubilitystability and dissolution of a xenobiotic
• Inhibition of membrane transporters and/ormetabolizing enzymesmetabolizing enzymes
transporters and/or metabolizing enzymestransporters and/or metabolizing enzymes • Modulation of the expression of membrane
Factors Affecting Oral BioavailabilityFactors Affecting Oral Bioavailability
PhysicochemicalPhysicochemical PhysiologicalPhysiological • Solubility • GI Transit• Ionization • Bile Secretion• Dissolution Rate •• Chemical Stability ••
• Polymorphism ofTransporters/Enzymes
Diffusion (intra-lumenal) • Regional Effects
Transport MechanismsMetabolism
Oral Bioavailability ComparisonOral Bioavailability Comparison
0 20 40 60 80 100 0
20
40
60
80
100
Rodents Dogs Primates
Hu
man
Bio
avai
lab
ility
(%
)
Animal Bioavailability (%)
Adapted from: W.K. Sietsema, Int. J. Clin. Pharmocol. Ther. Toxicol., 27 :179-211 (1989)G. Grass
FaFa –– Permeability ComparisonPermeability Comparison
From: L. Salphati, et. al., J. Pharmacy Pharmocol., 53:1007-1013 (2001)
PermeabilityPermeability –– PermeabilityPermeability ComparisonComparison
From: L. Salphati, et. al., J. Pharmacy Pharmocol., 53:1007-1013 (2001)
Intestinal Transport andIntestinal Transport and MetabolismMetabolism
Figure from Eur J Pharm Sci 21: 25, 2004
Proteins Involved in IntestinalProteins Involved in Intestinal DispositionDisposition
• Influx Transporters • Metabolizing Enzymes • Peptide, bile acid,
nucleoside, amino acid,nucleoside, amino acid, etc.etc.
• Efflux Transporters • P-gp, MRP2, BCRP, etc. • Nuclear Hormone
Receptors
sulfotransferases
• Phase I –– CYP isoforms(primarily 3A4, 2D6,(primarily 3A4, 2D6,
• Phase II –– GSTs, UGTs
C AR, PXR, PPAR, RXRetc.etc.
Intestinal DispositionIntestinal Disposition
• Permeability• Metabolism
Also• Inhibition• Induction
figure fro m Drug Metab Dispos 31: 1507, 2003.
Permeability
Low Class 4 Mixed
Class 2 Solubility Limited
Class 3 Permeability
Limited
Class 1 Dissolution Rate
Limited
High
LowHigh
Solu
bilit
y
Biopharmaceutical Classification SystemBiopharmaceutical Classification System
Decomposition
Release from dosage form
Xenobiotic in solution in GI tract
Transit
Metabolism in liver
Metabolism in the gut wall
Xenobiotic in systemic circulation
Rate of dissolution
Efflux
Permeability across the membrane
BCS
Factors affecting rate and extent of oralFactors affecting rate and extent of oral absorptionabsorption
S. Agrawal
Clinical Intestinal Metabolism DrugClinical Intestinal Metabolism Drug InteractionsInteractions
(adapted from: Doherty, M.M. and Charman, W.N., Clin Pharmacokinet, 41:235-253, 2002)
Preclinical Methods for Intestinal Disposition
• Intestinal permeability studies • Perfusion• Diffusion chamber (excised tissue or cultured cells)• Everted gut sac • PAMPA
• Intestinal metabolism • Perfusion • Microsomes
• Oral PK studies • P-gp, CYP inhibitors • Knockout animals
ChlorpyrifosChlorpyrifos
• Organophosphate pesticide • Potential exposure routes • Limited human bioavailability studies• Goals
• Determine intestinal permeability as a function of region and concentration• Determine effect of chlorpyrifos on expression and function of membrane transporters
ChlorpyrifosChlorpyrifos
• Single-pass Intestinal Perfusion (SPIP)• Regional permeability as a function of concentration
• Exposure studies in Caco-2 cells• Competitive PCR assay for MDR1• Effect on membrane efflux function
Single Pass Intestinal PerfusionSingle Pass Intestinal Perfusion
• Permeability determinedby loss fromby loss from perfusateperfusate
−Q ⎛CP out ⎞eff = ln⎜ ⎟⎜ ⎟2πrl ⎝ Cin ⎠
• Correct for adsorption,stability, accumulationstability, accumulation
Figure from: Salphati, et al, JPP 2001, 53: 1007–1013
⎟⎟⎠
⎞ ⎜⎜ ⎝
⎛− =
in
out eff C
C rl QP ln
2π
ResultsResults –– PermeabilityPermeability
T.J. Cook and S.S. Shenoy, Toxicology, 184:125-133, 2003
Duodenum Jejunum Ileum
P eff
(cm
/s)
0.00
2.00e-5
4.00e-5
6.00e-5
8.00e-5
1.00e-4 Chlorpyrifos 0.1 µM Chlorpyrifos 2 µM Chlorpyrifos 10 µM Propranolol
* **
ResultsResults –– Effect of CPF on MDR1Effect of CPF on MDR1 Expression in CacoExpression in Caco--2 Cells2 Cells
S. Agarwala, W. Chen and T.J. Cook, Toxicol. In Vitro, 18:403-409 (2004)
0.00E+00
2.00E+04
4.00E+04
6.00E+04
8.00E+04
1.00E+05
1.20E+05
1.40E+05
4 8 24 Time (hours)
Cop
y N
umbe
rs
Control CPF
*
*
*
Results Effect of CPF on Efflux Function in Caco-2 Cells
VL VC VH
S. Agarwala, W. Chen and T.J. Cook, Toxicol. In Vitro, 18:403-409 (2004)
Control 2.87 3.40 4.44
8 hr CPF pre- 3.65 4.18 5.01
incubatio n
Increase 27% 23% 13%
SulforaphaneSulforaphane 3 2 2
S CH2 CH2
O
• Isothiocyanate from cruciferous vegetables• Potential chemopreventive agent• Mechanism of action
• Induction of Phase II metabolizing enzymes and effluxtransporters, e.g., MRP2transporters, e.g., MRP2
• Goal: Determine intestinal disposition and effect ofSFN on expression of Phase II enzymes and MRP2 in intestine
H C CH CH N C S
SPIP-MV
• PermeabilityDeterminationDetermination • Lumenal • Blood
• Bioanalytical
Figure from Cummings, et al, JPET, 305:306, 2003.
SFN and SFNSFN and SFN--GLU in MesentericGLU in Mesenteric BloodBlood
Agrawal, S., Tsao, Y., Hu, P., and Cook, T.J., Intestinal Disposition of Sulforaphane, In Preparation, 2005.
Permeability of SFN and MetabolitesPermeability of SFN and Metabolites
Pe-SFN
Pb-SFN
Pb-SFN-G
SH
Pb-SFN-N
AC
SP
IP-M
V P
erm
eabi
lity
(cm
/sec
)
0.00
1.00e-4
2.00e-4
3.00e-4
4.00e-4 2.73 x 10-4
7.09 x 10-6
1.96 x 10-4
6.13 x 10-7
Agrawal, S., Tsao, Y., Hu, P., and Cook, T.J., Intestinal Disposition of Sulforaphane, In Preparation, 2005.
Effect of SFN on GST Expression in RatEffect of SFN on GST Expression in Rat
IleumIleum
0.0%
20.0%
40.0%
60.0%
80.0%
100.0%
120.0%
140.0%
0 5 10 15 20 40
SFN Conc (uM)
Perc
ent C
hang
e in
Exp
ress
ion
GST A1 GST M1 GST P1 GST A3
** **
**
** **
** * **
*
** **
**
**
**
**
*
** ** **
**
Tsao, Y., Hu, P., and Cook, T.J., AACR Frontiers in Cancer Prevention Research, #A133, 2004.
LumenEnterocyteBlood LumenEnterocyteBlood
Model of SFN Intestinal DispositionModel of SFN Intestinal Disposition
SFNSFNSFN
SFN-NAC SFN-NAC
SFN-GLU SFN-GLUSFN-GLU
Agrawal, S., Tsao, Y., Hu, P., and Cook, T.J., Intestinal Disposition of Sulforaphane, In Preparation, 2005.
RelevanceRelevance
• Depends on: • Metabolic pathways• Therapeutic index of drug• Toxicity of xenobiotic• Variability in intestinal metabolism
• Xenobiotic – Drug Interactions• Induction of expression• Relative affinity for transporter/enzyme• Concentration, etc• Exposure
SummarySummary • Intestinal disposition is critical for the bioavailability of orally
administered compounds (but may not be the limiting factor)• Interactions with transporters/enzymes (modulation of
expression and/or function) should be consideredexpression and/or function) should be considered • Dietary factors (e.g., grapefruit juice) can contribute to
variability in oral drug bioavailabilityvariability in oral drug bioavailability • “Baseline” expression of patients may change based on
dietary factorsdietary factors • Potential contribution of unidentified transporters and
enzyme isoformsenzyme isoforms
AcknowledgementsAcknowledgements
• Group Members • Collaborators • Dr. Tony Kong• Dr. Harold Newmark• Dr. Eric Weyand
• Financial Support• U.S. E.P.A.. • Charles and Johanna Busch
Grant• National Cancer Institute• McKesson Bioservices
• Dr. Shruti Agrawal• Shilpi Agarwala• Joseph Desiderio • Peidi Hu• Lemuel Liou• Tamira Mullarkey• Smriti Shenoy • Yvonne Tsao• Yan Xu• Brian Yeagy