1. Overview
• Discuss systems and assays that can be reasonably
implemented by academic groups for CNS drug
discovery.
• Assumes existing target-specific potency and selectivity
assays (e.g., related receptors or enzymes).
• Discussion topics:
– ADME (Absorption, Distribution, Metabolism and Excretion)
• Solubility
• Pharmacokinetics
• Metabolism
– Toxicology
• In Vitro assays
• Rodent tolerability studies

2. Probe Compound vs. Drug Candidate
• The extent of compound characterization will be dictated
by whether the molecule is being developed as a
research “probe” for POC in animal models or as a
potential IND candidate.
– Probe Compound Requirements:
• Adequate ADME properties for valid in vivo assessment
(solubility, clearance, half-life)
• Demonstration of adequate (free) drug levels in brain.
• Tolerability in animal species of choice (mouse/rat) at projected efficacy
doses.
• Evidence that compound metabolism doesn’t change upon repeated dosing.
– IND Candidate Requirements: all of the above, plus
• In vitro safety pharmacology, including hERG and human CYP450 inhibition
profiling.
• Human microsome studies to determine predicted clearance, CYP
metabolism, and ideally preliminary metabolite identification.
• Ultimately, IND-enabling studies, including GLP respiratory, CV, and safety
toxicology in two species.

3. Probe Compound Characterization

4. Compound Solubility
• Compound solubility is affected by many factors (salt
forms, pH, buffer systems, etc.)
– Typical objective in academic drug discovery is to ensure compounds are
sufficiently soluble in assay buffer systems to allow interpretable results.
• For animal studies, need sufficient solubility to allow adequate
dosing.
• Generally want solubility >60 μg/ml (Lipinski et al., Adv. Drug Disc. Rev.
23:3-25).
• Two basic types of solubility determinations
– DMSO stock dilutions: measure compound precipitation
(typically a kinetic measurement)
– Solid compound: measure compound dissolution (typically an
equilibrium measurement)
• We typically conduct kinetic solubility measurements of compounds
dissolved in DMSO.

5. Compound Solubility
• Multiple simple kinetic solubility methods exist (e.g., see Pan et al., J.
Pharm. Sci. 90:521-29 and Hoelke et al., Anal. Chem. 81:3165-72).
• One method accessible to most labs is solubility determination
based on light scatter of precipitates using a UV-Vis plate reader.
0.45
Sum Abs 550,600,650,700
0.40 quercetin 1
0.35
quercetin 2
0.30
0.25
quercetin 3
nm
0.20
0.15
0.10
0.05
0.00
-0.05 -6 -5 -4 -3
log conc (M)

6. ADME
• LC-MS/MS – A key investment
~$300K

7. ADME & LC-MS/MS
• Key aspects of ADME are enabled with a LC-
MS/MS
– Compound plasma pharmacokinetics, including
clearance and half-life determinations.
– Compound brain penetration. ~98% of compounds do
not equilibrate across the BBB.
– Estimation of free drug levels in plasma and brain
through equilibrium dialysis studies.
– Approximation of human metabolism using liver
microsomes, including identification of major CYP450
isozymes involved in compound metabolism.

8. Example PK
Tau Fibrillization Inhibitor (CNDR-51362)
dose
CL
AUC
CL plasma 108 mL/hr 51 mL/min/kg
Mouse hepatic blood flow ~ 90 mL/min/kg
Compound CL= 57% HBF
<30% HBF = low clearance
30-70% HBF= moderate clearance
>70% HBF= high clearance

9. Compound BBB Penetration
• A B/P ratio of ≥1 is indicative of full BBB penetration, but
compounds with B/P <1 might still equilibrate across the
BBB.
• It is free (unbound) compound that crosses the BBB. At
true equilibrium, B/Pfree =1, where
B/Pfree = B/P x fu(brain))/fu(plasma)
• The unbound fraction in plasma and brain can be
approximated by equilibrium dialysis.

10. Equilibrium Dialysis
CNDR-51362
AUC (0.5-16h), brain = 469 ng-hr/ml
B/P = = 1.58
AUC (0.5-16h), plasma = 297 ng-hr/ml
fu, brain = 0.011
fu, plasma = 0.019
B/Pfree = B/P x fu(brain))/fu(plasma)= 0.91

11. Projected Dosing
Effect of Unbound Fraction
Dose = [(Cl x Ct x T)/F]/fu
Where
Cl = Clearance (L/h) Assuming:
Ct = Target drug level Ct = 50 nM
T = Dosing interval (hours) Drinking interval = 1 hour
F = Bioavailability Mouse Weight = 25 g
Compound 51362 51397
CL,brain (mL/hr) 66 50
CL, plasma (mL/hr) 108 78.6
fu,brain 0.011 0.043
fu,plasma 0.019 0.069
F 0.64 0.63
Predicted Daily Dose (mg/kg) 160 30

12. Rodent Tolerability Studies
• Dr. John Sagartz will provide a more detailed overview of
animal toxicology.
• Academic research centers are typically equipped to
conduct rodent efficacy studies and non-GLP preliminary
rodent toxicological assessments.
• Our laboratory will typically conduct two types of mouse
tolerability studies for novel lead compounds that appear
to have appropriate potency and ADME properties.
– Tier 1: Maximum Tolerated Dose (acute)
– Tier 2: Repeated Dose Tolerability

13. Rodent Tolerability Studies
• MTD Design
– Normal mice (n=4) are dosed via oral gavage (0.5%
methylcellulose) starting at 1-3 mg/kg, with 3X dose-escalation
– Dosed every two days until two or more mice show signs of
intolerance (altered locomotor activity, sedation, ataxia, hypo- or
hypertonia, salivation or excitation).
• 1-Month Tolerability
– Normal mice (n=6/dose) are dosed at 0.1x-, 0.3x- and 1x-MTD
via oral gavage for 2 weeks or 1 month.
– Assessments include
• Behavioral observations
• Body weights
• Organ weights at study completion
• Complete blood counts at study completion
• Plasma and brain compound levels at study completion (compared to
separate group receiving drug for 3 days)

14. IND Candidate Characterization

15. Human Liver Microsomes
• Drug metabolism:
– Phase I (oxidation) - CYP450 isozymes and FMOs
– Phase II (conjugation) - UDP-glycosyltranferases, glutathione transferases and
sulfotransferases.
• Human metabolic rate can be estimated through use of human liver microsomes
(contain CYP450s, FMOs and UGTs).
– ~80% of drugs metabolized by CYP450s.
• Compound-metabolizing CYP450 isozymes can be indentified with liver microsomes.
– Significant CYP2D6 metabolism is a flag due to allelic variation in humans (5-10% of
Caucasians are poor 2D6 metabolizers).
• Compound metabolites can be identified with microsomes and LC/MS-MS.
CNDR-51362/hLM with CYP3A4
10.8 Inhibitor
ln(Peak Area)
10.6
10.4 y = -0.004x + 10.74
10.2
10 y = -0.013x + 10.65
9.8
0 20 40 60
51362 + hLM
51362 + KETO + hLM Minutes
Clint = ke (slope) X (mg microsomes/g liver) x (g liver/kg BW) x (1/ mg/ml microsomal protein)
Human HBF = 20 ml/min/kg Compound CL = 2.15 ml/min/kg (11% HBF)

16. In Vitro Safety/Toxicology
CYP450 Inhibition
• CYP450 inhibition profiling can be assessed via
– LC-MS/MS (measure inhibition of known CYP450 substrates using baculozomes).
– Commercial assay kits (e.g. Invitrogen Vivid fluorescent kits).
• Although there are >50 CYP450 isozymes, most drugs are metabolized by
just 7 isozymes (3A4/5, 2C9, 2D6, 2C19, 1A2, 2C8 and 2E1).
• Significant inhibition of major CYP450 isozymes can result in drug-drug
interactions.
• FDA guidance suggests that in vivo CYP450 inhibition studies are needed if
plasma Cmax is >10% of CYP450 Ki.
• We flag compounds if CYP450 inhibition is >75% at 10 μM.

17. In Vitro Safety/Toxicology
• At least 11 drugs have been withdrawn from the U.S. as a result of
their likely hERG cardiac channel inhibition (long QT intervals).
• Compound interaction with the hERG channel can be readily
estimated with commercial ligand binding kits (e.g., Invitrogen
Predictor hERG FP assay kit).
• hERG Binding assays have generally good correlation with more
definitive patch-clamp analyses.
Want ~100-fold window between
effective plasma drug
concentrations and hERG Ki/IC50.
We flag compounds that show
>50% inhibition at 10 μM.

18. Conclusions
• It is important that compounds intended for use in animal
efficacy models undergo sufficient characterization to
ensure adequate exposure at doses that are well
tolerated.
• Academic labs can also perform preliminary safety
pharmacological assessments at relatively little expense
which will provide important information about whether a
compound (or compound series) has drug candidate
potential.
• Academic labs should seek CRO assistance in planning
and conducting IND-supporting studies.

19. Acknowledgements
CNDR Drug Discovery CNDR Drug Discovery
(Biology/Pharmacology) (Chemistry)
Andrea Asimoglou Amos B. Smith, III
Jenna Carroll Carlo Ballatore
Alex Crowe Francesco Piscitelli
Julia Durante Longchuan Huang
Edward Hyde
Michiyo Iba
Michael James
CNDR Directors
Katie Robinson
Virginia Lee
Laurel Vana
John Trojanowski
Sharon Xie
Mandy Yao
Drug Discovery
Bin Zhang
Director
Kurt Brunden
Funding Sources: NIA/NIH, Astra-Zeneca, Bristol-Myers Squibb, AHAF
CART, Marian S. Ware AD Foundation, Nathan Bilger Alzheimer
Drug Discovery Initiative

20. IND-Enabling Safety Evaluation
John E. Sagartz, DVM, PhD, DACVP
President, Seventh Wave Laboratories LLC

21. “What is there that is not
poison? All things
are poison and nothing (is)
without poison.
Solely the
dose determines that a thing is
Philippus Aureolus Theophrastus
Bombastus von Hohenheim not a poison.”
Paracelsus
(1493 - 1541)

22. “The Poison Squad”
 „Hygenic Table Studies‟ initiated December 20, 1902
 US Congress appropriated funds
 “Whether preservatives should ever be used or not, if so, what preservatives
and in what quantities?”
 Teams of 12 ate food containing larger doses of „preservatives‟ such as
borax, sulfuric acid and formaldehyde
 Preceded pure Food and Drugs Act (1906) and 1938 Food Drug and Cosmetic Act

23. Nonclinical Safety Assessment
• International Conference on Harmonization
(ICH) Guidelines
– Harmonization of expectations between
Europe, Japan, and US
– Expectation for evaluation of
• Genetic toxicity: ICH S2
• Safety pharmacology: ICH S7A,B
• General toxicology with exposure assessment: ICH
M3(R2), ICH S3

24. ICH S2: Genetic Toxicity
• Genetic toxicology studies include in vitro and in vivo
systems
• Evaluate the potential to induce mutations and
chromosomal damage
– bacterial mutation
– cytogenetics
– mammalian gene mutation

25. Genetic Toxicology
• “Standard Battery” for Genotoxicity
– A test for gene mutation in bacteria
• Ames assay (bacterial reverse
mutation assay)
• Identifies comparatively subtle
effects on chromosomes (point
mutations, substitutions, frame
shifts)
• Tester strains of E. coli and
Salmonella typhimurium
– An in vitro test with cytogenetic
evaluation of chromosomal damage
with mammalian cells (Chromosomal
aberrations assay)
– An in vivo test for chromosomal
damage using rodent hematopoietic
cells