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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

Session 2 part 2

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Page 1: Session 2 part 2

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

Page 2: Session 2 part 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.

Page 3: Session 2 part 2

Probe Compound Characterization

Page 4: Session 2 part 2

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.

Page 5: Session 2 part 2

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.

-6 -5 -4 -3-0.050.000.050.100.150.200.250.300.350.400.45

quercetin 1quercetin 2quercetin 3

log conc (M)

Su

m A

bs

550,

600,

650,

700

nm

Page 6: Session 2 part 2

ADME

• LC-MS/MS – A key investment

~$300K

Page 7: Session 2 part 2

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.

Page 8: Session 2 part 2

Example PK Tau Fibrillization Inhibitor (CNDR-51362)

mL/min/kg 51 mL/hr 108 CLAUCdose

CL

plasma

Mouse hepatic blood flow ~ 90 mL/min/kgCompound CL= 57% HBF

<30% HBF = low clearance30-70% HBF= moderate clearance>70% HBF= high clearance

Cl

NN

S

NH

O

NH2

O

Page 9: Session 2 part 2

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.

Page 10: Session 2 part 2

Equilibrium Dialysis CNDR-51362

fu, brain = 0.011fu, plasma = 0.019

AUC (0.5-16h), brain = 469 ng-hr/mlAUC (0.5-16h), plasma = 297 ng-hr/ml

= 1.58B/P =

B/Pfree = B/P x fu(brain))/fu(plasma) = 0.91

Page 11: Session 2 part 2

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

NH

N

N

S

Cl

NH2

O

OCNDR-51362Molecular Weight: 360.82

NH

N

N

S

F

NH2

O

OCNDR-51397Molecular Weight: 344.36

Dose = [(Cl x Ct x T)/F]/fu

WhereCl = Clearance (L/h)Ct = Target drug levelT = Dosing interval (hours)F = Bioavailability

Assuming:Ct = 50 nMDrinking interval = 1 hourMouse Weight = 25 g

Projected Dosing Effect of Unbound Fraction

Page 12: Session 2 part 2

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

Page 13: Session 2 part 2

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)

Page 14: Session 2 part 2

IND Candidate Characterization

Page 15: Session 2 part 2

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.

0 10 20 30 40 50 609

9.5

10

10.5

11

f(x) = − 0.0042279878451676 x + 10.7461003591935f(x) = − 0.0130116426303008 x + 10.6498995842283

CNDR-51362/hLM with CYP3A4 Inhibitor

51362 + hLMMinutes

ln(P

eak

Are

a)

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)

Page 16: Session 2 part 2

In Vitro Safety/ToxicologyCYP450 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.

Page 17: Session 2 part 2

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.

Page 18: Session 2 part 2

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.

Page 19: Session 2 part 2

Acknowledgements

Funding Sources: NIA/NIH, Astra-Zeneca, Bristol-Myers Squibb, AHAFCART, Marian S. Ware AD Foundation, Nathan Bilger Alzheimer

Drug Discovery Initiative

CNDR DirectorsVirginia Lee

John Trojanowski

Drug Discovery Director

Kurt Brunden

CNDR Drug Discovery (Chemistry)

Amos B. Smith, IIICarlo Ballatore

Francesco PiscitelliLongchuan Huang

CNDR Drug Discovery (Biology/Pharmacology)

Andrea AsimoglouJenna CarrollAlex Crowe

Julia DuranteEdward HydeMichiyo Iba

Michael JamesKatie Robinson

Laurel VanaSharon XieMandy YaoBin Zhang

Page 20: Session 2 part 2

IND-Enabling Safety EvaluationJohn E. Sagartz, DVM, PhD, DACVP

President, Seventh Wave Laboratories LLC

Page 21: Session 2 part 2

Philippus Aureolus Theophrastus Bombastus von Hohenheim

Paracelsus(1493 - 1541)

“What is there that is not poison? All things are poison and nothing

(is) without poison.

Solely the dose determines that a thing is not a poison.”

Page 22: Session 2 part 2

“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

Page 23: Session 2 part 2

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

Page 24: Session 2 part 2

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

Page 25: Session 2 part 2

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