Part 5 of Ohio State's Drug Development Bootcamp

1. IN VITRO ADMET CONSIDERATIONS
FOR DRUG DISCOVERY AND
LEAD GENERATION
28 March 2017
James Drug Development Institute Symposium
Ohio State University
EVERY STEP OF THE WAY
EVERY STEP OF THE WAY1

2. Overview
• Overview of In Vitro ADMET (ADME/Tox; DMPK)
• Attractive Compound Properties
• Approaches for Obtaining In Vitro Data
• Metabolic Transformations
• Permeability (Transport, Efflux)
• Protein Binding

3. In Vitro Assays to Mimic In Vivo
In vitro assays are designed to mimic fate of compound in vivo
(for oral pill, usually preferred)
• General aqueous solubility
• Absorption from gut into bloodstream
• Binding to plasma proteins and RBCs (free drug fraction
for efficacy)
• Degradation in bloodstream or tissue
• Metabolism (liver, GI tract, etc.)
• Possible drug-drug interactions (drug as substrate or
inhibitor of enzymes, transporters)
• Possible toxicity to cells
• Excretion/elimination

4. Uses of In Vitro Metabolism (ADMET) Data
ADMET of a drug = Absorption
Distribution
Metabolism
Excretion
Toxicity
Used at many stages of drug discovery/development process:
 Compound library design
 Prioritizing screening “hits” and chemotypes during lead generation
 Lead optimization
 Compound selection for in vivo studies
 IND-enabling studies (even NDA)
 Development of in silico (computer) models
 Key = eliminate “dud” compounds unlikely to succeed (drug
discovery/development is like very high-stakes legalized gambling)

5. Types of In Vitro ADMET Assays
In Vitro ADMET
Excretion/ToxicityMetabolismDistributionAbsorption
Cell Viability (cytotoxicity)
CYP450 Inhibition
(incl. time-dependent TDI)
Metabolic Stability
(microsomes, S9, hepatocytes),
metabolite monitoring
Caco-2 Cell Permeability
Reaction Phenotyping (CYP450
and UGTs)
Ultracentrifugation
Equilibrium Dialysis
Protein Binding
(plasma, serum, tissues,
microsomes, pure proteins)
Melanin Binding
RBC Partitioning
(Blood/Plasma Ratio)
Matrix Stability
(e.g., plasma, buffer, tissue)
CYP450 Induction
Hemolysis
(blood compatibility)
Aqueous Solubility
Metabolite Profiling/ID/Mass
Balance (cold or radioactive)
LogD (Octanol/Water
Partitioning)
MDCK & MDCK-MDR1 Cell
Permeability
Transporter Substrate and
Inhibition (assay panels)

6. How In Vitro ADMET Assay Data Help Early Drug Discovery
Assay Deliverable What question does assay answer?
Aqueous solubility Limit of solubility (µM) Is compound soluble? Soluble under assay conditions?
Octanol/water partitioning LogD How hydrophobic is the compound?
Plasma stability % remaining or T1/2 How rapidly is compound degraded in plasma/blood?
Metabolic stability % remaining or T1/2 How rapidly is compound metabolized?
Permeability Papparent, efflux ratio Is compound likely to be absorbed from gut into bloodstream?
Protein binding % free or % bound or Kd Is compound highly protein bound?
Tissue binding % free or % bound or Kd Is compound highly tissue bound?
RBC partitioning (blood/plasma ratio) Blood/plasma ratio, Kp
CYP450 inhibition % inhibition or IC50 or Ki Will compound have drug-drug interaction issues?
CYP450 induction Fold induction Will compound have drug-drug interaction issues?
Metabolite profiling/ID Masses found over time What does compound get metabolized to?
CYP450 reaction phenotyping T1/2 +/- inhibitors Which CYP450(s) metabolize compound?
Hemolysis (blood compatibility) % lysis Does the compound lyse RBCs?
Melanin binding % bound, Kd, Bmax Is the compound likely to accumulate in pigmented tissues?
Cell viability % inhibition or IC50 Does compound kill cells?
Cell proliferation % inhibition or IC50 Does compound interfere with normal cell growth?
HERG inhibition % inhibition or IC50 Is HERG-related cardiotoxicity a potential problem?
Ames/genotox Does compound cause genetic mutations?

7. Example of Compound Properties “Sweet Spots”
[Personal opinions, no single industry-accepted criterion; always exceptions]
• Overall goal is selective, potent & safe in target, cell-based, in vivo models
• Lipinsky’s rules
• High aqueous buffer solubility (e.g., >100 µM)
• High permeability (Papp>1E-06cm/sec), low efflux (<2)
• IC50 >1µM for multiple CYP450s (potent 2D6 and 3A4 inhibition could be an issue)
• Stability in plasma
• Moderate metabolic T1/2 (~1 hr LMs), hepatic clearance (CLint, CLhep)
• Moderate plasma protein binding (~70-90%), preferably not just to AGP
• No unique human metabolite
• Patentable
• Ease of chemical synthesis & kg scale up
• Other intangibles (no bad smell, color, etc.)

8. In Vitro ADMET Assay Details/Parameters
Non-GLP is fine (not required to be GLP)
Cytochrome P450 inhibition
• Drug-drug interactions (DDIs)
• 7 Major human isozymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4)
• Drug substrates (typically LC/MS/MS detection vs. fluorescence)
• Time-dependent inhibition (TDI; IC50 shift +/- 30 min pre-incubation)
Metabolic stability (% remaining)
• Microsomes (liver, intestinal) and S9 (multiple species)
• Hepatocytes (multiple species, cryopreserved)
Matrix stability (plasma, serum, blood, buffer, SIF, SGF)
Cytochrome P450 and UGT reaction phenotyping
• Selective chemical inhibitors or enzymes to determine which enzymes involved
Protein binding:
• Equilibrium dialysis (e.g., Thermo RED device)
• Ultra-centrifugation (500,000 x g for 2.5 hours)
• Ultra-filtration (has non-specific binding problems)
Tissue binding
• Tissue homogenates (e.g., brain, kidney, tumor, etc.)

9. In Vitro ADMET Assay Details (cont.)
RBC partitioning (blood/plasma ratio)
• To determine if compound binds RBCs vs. stays in plasma
Melanin binding (synthetic melanin pigment)
CYP450 induction
• Drug-drug interactions (DDI)
• Measures if compound increases CYP450 enzyme activity or mRNA levels in hepatocytes (flip
side of CYP inhibition)
Permeability (Caco-2, MDCK, MDCK-MDR1)
• Tests if compound can be absorbed or transported across intestinal cell layer, or is effluxed
(e.g., by Pgp) out of cell (“escort” vs. “bouncer”)
Aqueous solubility
• Kinetic: measures turbidity using plate reader (cloudy solution)
• Thermodynamic: equilibrium (overnight), measure concentration by LC/MS/MS
Metabolite profiling/ID (LC/MS/MS using Q-Trap or high-resolution instrument)
Hemolysis (blood compatibility)
LogD (octanol/water partitioning)
• Measures how greasy a molecule is (lipophilic or hydrophilic)
Cell viability (cytotoxicity)
• Typically fluorescent or absorbance readouts (plate reader)
• Generic cell lines (e.g., L929, HepG2, HEK293, HeLa, Hepatocytes)

10. In Vitro Study Strategies, Considerations
In the early discovery stages, minimize costs & time (e.g., Excel results
should be fine; don’t need to pay for Word study reports until later stage).
Simplest assay is most cost-effective; only pay for needed options:
• Single time point vs. T1/2; single compound concentration vs. IC50
• Can be helpful to do bi-directional permeability (to assess efflux);
concurrent matrix stability control
• Testing compounds side-by-side gives more robust comparisons
• Standard curves not needed for early stage discovery
• Identify and focus on critical path experiments (avoid “interesting” or
“bells and whistles” experiments); try to prioritize/kill compounds early.

11. What to Consider re. Internal vs. Collaborator vs. CRO Lab
• Who already has assay established and is experienced?
• Are you going to run assay routinely or once-in-a-while?
• Need fast initiation and turnaround time (3-5 days) to keep lead
optimization cycles short
• Client responsiveness, attention to detail
• Direct access to scientist/Study Director for suggestions and questions
• If outsourced, need competitive price (~mid-point, not cheapest)
• “Good, fast, cheap – pick 2!”
• “You get what you pay for”

12. Drug-Drug Interactions
• One drug can interfere with metabolism of another drug via:
• Competition for metabolic pathway (inhibition)
• Up-regulation of metabolic enzymes (induction)
• Effect of transporters or efflux pumps
• The FDA and EMA offer guidance for metabolism data needed for regulatory submission (IND)

13. Types of Metabolic Transformations
Types of metabolic transformations:
• Phase 1 (oxidation, hydrolytic, reductive)
• Usually more hydrophilic
• Enzymes: Cytochrome P450s, aldehyde
oxidase (AO), xanthine oxidase (XO),
monamine oxidases (MAOs), flavin-
containing monooxygenases (FMOs)
• Phase 2 (Conjugative: glucuronidation, sulfation,
acetylation, methylation, glutathione, amino acid)
• Usually more polar and less toxic
• Enzymes: UGTs, SULTs, GSTs, NATs
• Metabolites may possibly be more potent or
toxic than parent
• CYP and UGT levels can vary dramatically
between patients, populations (polymorphisms)
Description Mass Shift (amu)
Oxidation (Phase 1)
N-dealkylation
Deethylation -28
Loss of water -18
Demethylation -14
Dehydrogenation -2
Oxidative deamination +1
Hydrogenation +2
Hydroxylation/N,S-oxidation +16
Hydration +18
Di-oxidation +32
Acetylation +42
Conjugation (Phase 2)
Glycine +57
Sulfation (sulfonation) +80
Taurine +107
Cystein conjugation +121
Glutamine +145
N-acetyl-cysteine conjugation +163
Glucuronidation +176
Oxidation with O-glucuronidation +192
Glutathione conjugation +305

14. Importance of Metabolic Transformations
• Acetaminophen (Tylenol, APAP, paracetamol; metabolite of phenacetin which was withdrawn due to
nephrotoxicity and carcinogenesis); typical dose 1-2 g/day, max 4 g/day.
• Mainly glucuronidated and sulfated before elimination in bile.
• At >7 g/day (in adult; >150 mg/kg in child), phase 2 pathways are saturated and phase 1 (CYP450
3A4 and 2E1) pathways generate NAPQI (N-acetyl-p-benzoquinone imine) via hydroxylation and
dehydration. NAPQI is a nephrotoxic and hepatotoxic metabolite.
• NAPQI can be detoxified by glutathione pathway, but pathway can be overwhelmed.
• Acetaminophen overdose is most common drug-related toxicity reported to poison centers, and is
main cause of acute liver failure in U.S. Lethal dose is 10-15 g. Chronic alcohol consumption
depletes glutathione levels.

15. Microsomal Stability: Example of Species Differences
SPECIES PROFILING OF IMIPRAMINE
HEPATIC MICROSOMAL METABOLIC STABILITY
-10
0
10
20
30
40
50
60
70
80
90
100
-15 0 15 30 45 60 75 90
INCUBATION TIME (min)
%TestCompoundRemaining
RAT
MOUSE
DOG
GUINEA PIG
CYNO. MONKEY
RHESUS MONKEY
HUMAN
SPECIES PROFILING OF
HEPATIC MICROSOMAL METABOLIC STABILITY
0
20
40
60
80
100
Verapamil Testosterone Labetalol Imipramine
%TestCompoundRemaining@15minutes
RAT
MOUSE
GUINEA PIG
DOG
CYNO. MONKEY
RHESUS MONKEY
HUMAN
• Metabolic rates can vary dramatically between species
• In vitro species comparisons facilitate selection of appropriate animal
models

16. Metabolite Mass Monitoring
• Leverages metabolic stability assays
• Can concurrently monitor level of parent + putative metabolite masses vs. time
• Detect common and abundant phase 1 (NADPH-dependent; oxidation, dealkylation) or
phase 2 (glucuronidation, sulfonation) metabolites

17. Transporters, Permeability, Efflux
The FDA and EMA also offer guidance for transporter & efflux data needed for regulatory submission (IND)

18. Permeability (Caco-2 and MDCK)
Caco-2
• Human colon cancer cell line
• Takes 21 days to grow before ready to use in assay
• Has full range of human transporters (including Pgp, BCRP, MRP2, etc.)
• Test unidirectional (A-B, SDL-1) or bidirectional (B-A/A-B, efflux ratio, SDL-2)
• Multiple control drugs [warfarin (high), ranitidine (low), talinolol (efflux)]
• Deliverable: default Excel summary; Papp and/or efflux ratio
MDCK & MDCK-MDR1 [MDR1 = Pgp = P-glycoprotein]
• Dog cell line (mimics human Caco-2; Madin-Darby Canine Kidney)
• Takes only 5-7 days to grow before ready to use in assay
• MDCK wild type has full range of dog transporter enzymes
• MDCK-MDR1 transfected cell has full range of dog transporter enzymes plus human MDR1 (Pgp)
To study human Pgp involvement, options are: 1) Caco-2 ± Pgp inhibitor; or 2) MDCK-MDR1 ± Pgp
inhibitor; or 3) MDCK wild type vs. MDCK-MDR1.

19. Pgp (MDR1): Caco-2 vs. MDCK-MDR1
Assay:
Apical side (A)
Basolateral side (B) Caco-2
(human)
hPgp
Drug 1 Drug 2 Drug 1 Drug 2
hPgp dPgp
MDCK-MDR1
(dog with human)

20. Protein Binding: Equilibrium Dialysis Approach
• Protein binding can help interpret differences in potency between target and cell-
based assays (e.g., presence of 10% FBS)
• A white paper will be released for protein binding, but may not have many specifics
• Pierce Teflon RED (Rapid Equilibrium Device) unit or HTDialysis Teflon plate
• Plasma, serum, whole blood, pure proteins (HSA, AGP)
• Multiple species, anticoagulants
• Steps: Spike test article into inner dialysis tube; dialyze for 6 hours at 37°C with
shaking in CO2; matrix-match aliquots from inner and outer chambers; determine
levels by LC/MS/MS
• Calculate % Free, % Bound
Plasma to Plasma Equilbrium Ratio
versus
Percent Plasma Protein Bound
Percent Plasma Protein Bound
0 20 40 60 80 100 120
PlasmatoPlasmaEqulibriumRatio
0
10
20
30
40
50 20 hours of dialysis
Equilibrium Ratio Issues?

21. Protein Binding: Ultracentrifugation Approach
• Advantages:
• Doesn’t rely on equilibrium 
• Shorter exposure time to plasma (2.5 hours) 
• Smaller sample volume
• Cons:
• % bound lower than expected (but more accurate)
• Not automatable
• Ignores binding to “floating” chylomicrons and
lipoproteins
• Steps: Spike test article into matrix (plasma, etc.);
separate protein-bound compound from free by
sedimenting plasma proteins via approx. 500,000 x g for
2.5 hours, 37°C; aliquot supernatant (free drug) below
lipid layer; matrix-match samples; determine levels by
LC/MS/MS
• Calculate % Free, % Bound
Beckman table-top ultracentrifuges
Centrifuge
BoundDrugFreeDrug
Total:Drug:PlasmaMixture
Plasma
Water
Centrifuge
BoundDrugFreeDrug
Total:Drug:PlasmaMixture
Plasma
Water

22. Extra
Extra slides (& examples of some in vitro data)

23. Cytochrome P450 Reaction Phenotyping
DEXTROMETHORPHAN
Reaction Phenotyping
Incubation Time (min)
0 15 30 45 60 75 90 105 120 135 150 165 180
%ParentRemaining(±NSD)
0
20
40
60
80
100
Control A
Furafylline/CYP1A2
Omeprazole/CYP2C19
Ketoconazole/CYP3A4
Quinidine/CYP2D6
Sulfaphenazole/CYP2C9
Tranylcypromine/CYP2A6
Control B
MIDAZOLAM
Reaction Phenotyping
Incubation Time (min)
0 15 30 45 60 75 90 105 120 135 150 165 180
%ParentRemaining(±NSD)
0
20
40
60
80
100
Control A
Furafylline/CYP1A2
Omeprazole/CYP2C19
Ketoconazole/CYP3A4
Quinidine/CYP2D6
Sulfaphenazole/CYP2C9
Tranylcypromine/CYP2A6
Control B
HYDROXY-MIDAZOLAM
Formation
C
ontrolAFU
R
A
/C
Y
P
1A
2
O
M
E
P
/C
Y
P
2C
19K
E
TO
/C
Y
P
3A
4Q
U
IN
/C
Y
P
2D
6S
U
P
H
/C
Y
P
2C
9TR
C
Y
/C
Y
P
2A
6
C
ontrolB
MetaboliteFormed@15min
(%ofControl±NSD)
0
20
40
60
80
100
.
DEXTRORPHAN
Formation
C
ontrolAFU
R
A
/C
Y
P
1A
2
O
M
E
P
/C
Y
P
2C
19K
E
TO
/C
Y
P
3A
4Q
U
IN
/C
Y
P
2D
6S
U
P
H
/C
Y
P
2C
9TR
C
Y
/C
Y
P
2A
6
C
ontrolB
MetaboliteFormed@120min
(%ofControl±NSD)
0
20
40
60
80
100
.
• Determines which CYP450 isozyme(s) involved with test article metabolism and/or metabolite formation.
• Perform metabolic stability assay ± specific CYP450 inhibitors

24. Summary of Protein Binding Approaches
• Equilibrium Dialysis (RED):
• Best known method and widely accepted method
• But may over-estimate % bound for highly bound compounds due to lack of equilibrium.
• Not suitable for compounds unstable in matrix (RED assay takes 4-24 hours)
• Ultracentrifugation (UC):
• Well known and accepted method
• Most suitable for highly bound compounds and/or compounds with limited stability in matrix
(UC assay only takes 2.5 hours)
• Ultrafiltration (UF):
• Easy method to perform
• Very significant non-specific binding (NSB) problems and other limitations

25. 1. Equilibrium dialysis approach
• Free drug is separated from protein-bound drug by dialysis membrane (e.g., 6-8K
MWCO)
• Plasma, serum, whole blood, purified proteins
• Multiple species, anticoagulants
• Method:
• Test article is spiked into plasma in “donor” side
• Dialyzed against buffer (“receiver” side) for 6+ hours at 37°C with agitation
• Aliquots from each side are sampled and matrix-matched
• Concentrations determined by LC/MS/MS
• Deliverable = % protein bound [= 100% - (Free/Total)]; also % recovery, equilibrium
assessment can be checked

26. Equilibrium dialysis apparatus
Pierce/Thermo Teflon R.E.D. (Rapid Equilibrium Device) insert
• Disposable insert contains dialysis membrane
• Donor “tube” surrounded by buffer (“receiver side”) for higher surface area:volume ratio (faster
equilibration times?)
• Automation-friendly

27. Pros/cons of equilibrium dialysis approach
• Pros
• “Gold standard” and well-known, accepted
• Newer, easy-use apparatus commercially available
• Lower NSB (Teflon)
• Lower volume required
• Higher throughput (automatable)
• Cons
• Long equilibration times (even >24 hours?)
• Potential matrix stability issues
• Potential volume shifts
• May need dialyzed matrix for matrix matching

28. Is equilibrium dialysis really at equilibrium?
• If % plasma protein binding is
high, need to check for
equilibration
• Very high affinity
• Slow off-rate
• Irreversible binding
• Small differences in % bound
values can be large differences in
% free:
• 99.5% vs. 99.9% bound
equates to a 5-fold difference
in free concentration
Plasma to Plasma Equilbrium Ratio
versus
Percent Plasma Protein Bound
Percent Plasma Protein Bound
0 20 40 60 80 100 120
PlasmatoPlasmaEqulibriumRatio
0
10
20
30
40
50 20 hours of dialysis
Equilibrium Ratio Issues?

29. 2. Ultracentrifugation approach
• Advantages:
• Doesn’t rely on equilibrium
• Shorter exposure time to plasma (2.5 hours)
• Steps:
•Spike test article into plasma (serum, purified proteins)
• Separate protein-bound compound from free by sedimenting plasma proteins
by ultracentrifugation
• Approx. 500,000 x g for 2.5 hours, 37°C
• Free drug is in supernatant below lipid layer
• Matrix-match samples +/- centrifugation; determine concentrations by
LC/MS/MS
• Deliverable = % protein bound, % recovery
Beckman TL100
table-top
ultracentrifuge

30. Ultracentrifugation approach
• Pros
• Short plasma exposure, “equilibration” time
• Simple approach
• Minimizes recovery/stability issues
• Limits aqueous solubility, NSB issues
• Moderate sample volume (2 ml)
• Minimal sample handling
• Cons
• % Bound values may be lower than expected (but more accurate!)
• Very slight residual (small) plasma proteins/peptides in supernatant
• Ignores binding to “floating” chylomicrons and lipoproteins
• Dynamic protein concentration during sedimentation
• Needs expensive ultracentrifuge equipment
Centrifuge
BoundDrugFreeDrug
Total:Drug:PlasmaMixture
Plasma
Water
Centrifuge
BoundDrugFreeDrug
Total:Drug:PlasmaMixture
Plasma
Water

31. Ultracentrifugation method an accepted approach
• “Indeed, our technique accurately determined the plasma protein binding ratios of a wide range of
compounds and could be used to evaluate protein binding kinetics.”
“…our results indicate the reliability of this micro-scale ultracentrifugation technique for the
evaluation of the protein binding of drugs….” (Nakai 2003 J Pharm Sci 93, 847)
• “The UC method was confirmed to be comparable with the ED method in terms of reliability and
rather superior in terms of reproducibility, especially at low drug concentrations.” (Yasuo 2008
Chem Pharm Bull, 2948)
• Extensively used at several large pharmas

32. 3. Ultrafiltration approach
• Separation of bound drug from free drug using UF membrane
• Pros
• Rapid equilibration time (minimal recovery/stability issues)
• New methods/apparatus (improved NSB)
• Lower volume required
• Higher throughput
• Cons
• Significant NSB (particularly for hydrophobic compounds)
• Oncotic issues during concentration of protein