Strategic Use of Preclinical Pharmacokinetic Studies and In Vitro Models in Optimizing ADME Properti

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Strategic Use of Preclinical Pharmacokinetic
Studies and In Vitro Models in Optimizing ADME
Properties

• Dhiren R. Thakker
• School of Pharmacy
• UNC-CH
• AAPS Workshop September 19-22, 2004

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ADME Processes are Important Determinants of
Therapeutic Efficacy and Adverse Effects
Distribution
Systemic Circulation
Target
Non-target tissues
Metabolism
Absorption
Excretion
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Optimizing ADME Properties of the Lead

• Assess ADME properties and identify properties
  that need to be improved
• Identify appropriate in vitro model
• Screen compounds for the selected ADME properties
• Develop structure-property relationship and
  provide feedback to chemistry

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Optimum ADME in Clinic How do you get there?
In Vitro
In Vivo
Animals
Humans
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Pharmacokinetic Parameters

• Top Level Parameters
• Bioavailability
• Half-life

• Experimental Parameters
• Plasma concentration
• CT
• AUC
• Clearance
• Metabolic
• Biliary
• Renal
• Volume of Distribution

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Determinants of Bioavailability

Bioavailability
Absorption
First Pass Clearance
Solubility
Permeability
Hepatic
Intestinal
Biliary
Metabolic
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Assessing Sites of Presystemic Elimination
Stomach
Portal Vein
Systemic Circulation
Duodenum
Liver
Jejunum Ileum
Sampling
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Determinants of Clearance

Clearance
Excretion
Metabolism
Biliary
Renal
Metabolic Enzymes
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Intestinal Absorption at the Cellular and
Molecular Level
Transcellular
Paracellular
Apical (mucosal)
P-gp
Tight Junction
Metabolism
Intracellular Sequestration
Basolateral (serosal)
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In Vitro Models for Intestinal Absorption/Transpor
t

• Cell culture models
• Caco-2, MDCK, MDCK-MDR, conditionally
  immortalized 2/4/A1, HT-29
• Everted intestinal sacs
• Intestinal rings

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In Vitro Studies for Intestinal Transport
Transwell? System

• Screening
• Mechanism of transport
• Saturable vs. passive diffusion
• (Papp vs. concentration)
• Efflux transporter
• (Papp BL to AP /Papp AP to BL)
• (Papp /- Pgp inhibitor)

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Effect of P-gp on Absorptive and Secretory
Transport of Digoxin Caco-2 cells
AP to BL Papp BL to AP Papp
3.50E-05
3.07E-05
2.50E-05
Papp (cm/sec)
1.46E-05
1.36E-05
1.50E-05
5.00E-06
1.71E-06
DigoxinGW918 (0.5 mM)
Digoxin (Control)
Troutman and Thakker, Pharm Res, 2003
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Effect of P-gp on Absorptive and Secretory
Transport of Rhodamine 123 (and Doxorubicin)
across Caco-2 cells
AP to BL Papp BL to AP Papp
2.00E-05
1.62E-05
Papp (cm/sec)
1.00E-05
1.52E-06
1.93E-06
1.42E-06
0.00E00
Rhodamine 123 GW918 (0.5 mM)
Rhodamine 123
(Control)
Troutman and Thakker, Pharm Res, 2003
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Assessing the Attenuation of Absorptive Transport
by P-gp
3.50E-05
2.00E-05
GW918
control
Papp (cm/sec)
1.3E-05
1.9E-06
1.4E-06
1.7E-06
Rhodamine 123
Digoxin
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Results of a Transport Screen (Caco-2 Cells) of
Candidate Compounds

• Molecular descriptors of transport/permeability
• log P / log D
• volume
• polar surface area
• H-bond
• solvation energy

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Correlation between Calculated vs. Experimental
Permeability of 1,5-Benzodiazepines across Caco-2
Cell Monolayers

Log (Papp) 8.05 - 0.027 (H-Bond ) - 0.0065
(Surface Area) 0.03 (Solvation Energy)
Gan et al., unpublished
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Frequencies of High, Medium and Low Papp Values
Before and After Application of the
Structure-Transport Relationship to
1,5-Benzodiazepines
Gan et al., unpublished
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Metabolism Effect on Drug Therapy

• Inadequate therapeutic efficacy
• Low oral bioavailability due to rapid first pass
  metabolism
• Short duration of action due to rapid metabolism
• Inadequate drug exposure due to Induction of
  metabolic enzymes
• Adverse effects
• Undesired pharmacological activity of metabolites
• Covalent modification of macromolecules
• Over exposure to co-administered drugs due to
  inhibition of metabolism

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Metabolism-related Screens

• Metabolic stability
• Inhibition of metabolic enzymes
• Induction of metabolic enzymes
• Formation of Reactive metabolites

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Metabolic ReactionsChemical Processes

• Oxidation
• Reduction
• Hydrolysis
• Conjugation

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Hepatic Drug Metabolizing Enzymes

• Cytochromes P450 (CYP)
• Flavin monooxygenase (FMO)
• Peroxidases
• Amine Oxidases
• Xanthine Oxidase
• Dehydrogenases
• Cytochrome P450 Reductase
• Keto Reductase
• DT Diaphorase
• Azo Reductase

• Proteases/Peptidases
• Esterases
• Glucuronidases
• Sulfatases
• Phosphatases
• Glucuronosyl transferases
• Glutathione transferases
• Sulfotransferases
• Methyl transferases
• Acetyl transferases

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In Vitro Models for Drug Metabolism Studies

• Metabolic Stability
• Microsomes (CYP), S9
• Hepatocytes (suspended or cultured)
• cDNA expressed or purified enzymes
• CYP Inhibition
• cDNA expressed enzymes
• Microsomes selective CYP inhibitors
• CYP Induction
• Sandwich-cultured hepatocytes
• Formation of Reactive Metabolites
• Microsomes/trapping agent

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Biliary Excretion A Schematic View
sinusoidal membrane
blood flow
intracellular sequestration
hepatocyte
tight junction
bile
bile
canalicular membrane
reabsorption
biliary excretion
metabolism
egress
uptake
sinusoidal membrane
blood flow
protein binding
Courtesy Kim Brouwer
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Collagen Sandwich-Cultured (SC) Hepatocytes
24 hours post culture
72-96 hours post culture
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Quantification of Substrate in Bile Canaliculi
and In Vitro Biliary Clearance (B-CLEAR?)
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Substrate in
-free Buffer
Ca
Standard Buffer
Bile Canaliculi
(bc)
bc
bc
cells
cells
cells
cells
cells
cells
cells
cells
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Renal Excretion
Active Reabsorption
Passive Reabsorption
Filtration
Secretion
www.columbia.edu/cu/biology/ courses/w2501/nephro
n.jpg
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Kidney (Proximal Tubule) Transporters
CATION TRANSPORTERS
ANION TRANSPORTERS
Courtesy Kim Brouwer
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Models To ExamineRenal Transport Processes

• Intact kidney in vivo
• Isolated perfused kidney
• Isolated perfused or nonperfused tubules
• Cultured renal cells
• Isolated plasma membrane vesicles
• (basolateral or brush border)

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Distribution A Black Box!

• Processes Affecting Distribution
• Plasma protein binding
• Filtration across capillary endothelium
• Transport across capillary endothelium (e.g.
  blood-brain barrier)
• Diffusion through intercellular matrices
• Transport across cell membranes
• Brain penetration a special case
• Capillary endothelial cell monolayers
• Perfusion models
• Brain-to-plasma ratio (In vivo PK)

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When In Vitro Models FailOptimize PK by Cassette
Dosing

• Determine pharmacokinetic parameters for
  compounds after cassette dosing
• Approach
• Determine if the approach works with a test set
• Screen new compounds as mixtures
• Include appropriate controls
• Retest the lead with desired PK as a single
  compound

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Questions???