DM/PK-guided Lead OptimizationA Historical Perspective of a Paradigm Shift

Dhiren R. ThakkerSchool of Pharmacy

UNC-Chapel Hill

NEDMDGGerald Miwa Retirement Symposium

April 9, 2007

Current ParadigmOptimizing Biology and ADME Synchronously

C. Breneman, Krisitn Bennett, J. Bi., M. Song, M. Embrechts– Rensselaer Polytechnic Institute www.drugmining.com

DM/PK – Research Interface at Glaxo Late 1980’s – Early 1990’s

Preclinical DM/PK Research

Safety Assessment

Clinical DM/PK

Research DM/PK Group

Ultra Short Acting OpioidRemifentanil -

Rapid Offset of Action Within 5 to 10 minutes after the discontinuation of ULTIVA, no residual analgesic activity is present.

Source: Ultiva® product labelChemical vs. Enzymatic Hydrolytic Rates

Use of in vitro metabolism screen to identify

“Metabolic Hot Spot”

Rationale for Incorporating Metabolic Studies Early in the Discovery Program

NH

N

O

HN

NH

O

O

Cl O OHNC

DihydropyridazinoneDHP-1

PhosphodiesteraseInhibition

β-Blockade

Potential metabolic hot spot

PK of Parent and the Amine Metabolite Following Oral Administration of DHP-1 (5 mg/kg) to Male Beagle

Dog Suggests First Pass Metabolism

ParentAmine

10 20 30 40

Time (hrs)

0.1

1.0

Seru

m C

onc .

(μg/

ml)

Major Metabolite of DHP-1

NH

N

O

HN

NH

O

O

Cl O OHNC

The Amine Metabolite

Relative Rates of In Vitro Metabolism for Dihydropyridazinone Leads

10 20 30 40

Incubation Time (min)

50

100

Rem

aini

ng S

ubst

rate

(%)

DHP-1

DHP-2

DHP-3

Improvedmetabolicstability

Functional Group Contributing to the Metabolic Hot Spot for Dihydropyridazinones

NH

N

O

HN

NH

O

O

Cl O OHNC

NH

N

O

HN

NH

O

O

Cl O OH

DHP-1-like compounds

DHP-2,3-like compounds

Stabilizing a metabolic hot spot can uncover a secondary hot spot

NH

N

O

HN

NH

O

O

R3R3

R2 O OHNC

R1

(CH2)n

Metabolic Hot Spots

primarysecondary

Rationally designed in vitro metabolism study, based on a good understanding of likely metabolic pathways, could lead to a

rapid solution

Background• Phospholipase A-2 Inhibitors

for Inflammatory Diseases (Arthritis, Asthma)

• Glycerophospholipid derivatives

• Major problem – Poor bioavailability in rats

• First pass metabolism was implicated – absorption (permeability) was not a problem (Caco-2 studies, in vivo studies with radiolabeled compounds)

Likely Metabolic Hot Spots

ROO

OPO

O-

Hydrolysis

Oxidation

Evidence for Oxidative and Hydrolytic Metabolismof the Lead Compound

Oxidative metabolism

Incubation with liver homogenates + NADPH

Hydrolytic metabolism

Incubation with liver homogenates - NADPH

In Vitro Metabolism of the Lead Compound by Rat Liver Enzymes and Caco-2 Cells

R O

O

PO O-

O2) fatty acid oxidation1) ω−hydroxylation

LIVER HOMOGENATE

ester + phosphonate cleavageLIVER HOMOGENATE & CACO-2 CELLS

ester cleavage

Modification of Metabolic Hot Spots

RNO

OPO

O-

F F

FF

F

In vitro studies do not provide guidance for improving in vivo metabolic clearance

A case for n-in-one in vivo PK studies

Dutasteride® for BPH

The terminal elimination half-life of dutasteride® is 5 weeks at steady stateFrank Lee et al.

Profile of the Lead Series

• Alpha-1 antagonistsfor BPH

• Once-a-day dosing• MW: 400-600, basic, clogP

5-7• Well-absorbed• Short half-life• Elimination predominantly by

metabolism• Highly protein bound

N

O

N

O

F FF

SO2NHR

R1

Dog Microsomal Metabolism Screen

• Poor correlation between in vitro metabolism rate and in vivo T1/2 or CL

• Several possible reasons considered» Sequential metabolism» Phase II metabolism» Protein binding

• Follow-up studies showed that none of these reasons could explain the lack of correlation between in vitroand in vivo studies

PK of Mixtures?????Origin of Cassette Dosing

The Challenge• Screen over 150 compounds to find a lead

appropriate for once-a-day dosing• The only predictive screen was in vivo PK in dogs• In vivo screen would be too slowThe Solution• In vivo PK studies of mixtures!!!

Judd Berman

Cassette Dosing “Validation” (5-in-One)

• Five compounds of known CL, VSS, and T1/2administered concomitantly to a dog

• Dose=0.25 mg each compound/kg IV » 1/4th the dose of compounds administered individually

• Plasma samples assayed by LC/MS/MS» Assay for simultaneous analysis of 5 compounds

without interference from any of the metabolites

• Pharmacokinetic parameters compared to those previously obtained

5-in-One Dog vs. Individual DosingHalf-life comparison

y = 0.8342x + 0.2875R2 = 0.9021

0

1

2

3

4

5

0 1 2 3 4 5Half-life (hr)--5-in-One Dosing

Hal

f-life

(hr)

--Ind

ivid

ual D

osin

g

N-in-one in vivo screen worked

• Good correlation in PK parameters.• Useful for ranking compounds• 125 compounds screened in 10 dog studies

in 2 months.• Approximate structure-PK relationships developed• Compounds with a wide range of T1/2 could be

identified from which leads could be selected for further evaluation

Half-lives of 125 Compounds in Dogs as Determined by Cassette Dosing

0

2

4

6

8

10

12

14

16

18H

alf-l

ife (h

)

N

O

N

O

F FF

SO2NHR

R1

Acknowledgments• Kim Adkison• Charley Boehlert• Ken Brouwer• Lawrence Gan• Kathy Halm• Frank Lee• Diane Levesque• Doug Rickert• Archie Sinhababu• Bob St. Claire• Cosette Serabjit-Singh• Tim Tippin• Steve Unger• John Walsh• Souzan Yanni

• Judd Berman • Paul Feldman• Steve Frye• Jeff Leighton• Joel Shaffer• Elizabeth Sugg

• Peter Myers• Leslie Hudson

Gerald Miwa

DM/PK-guided Lead Optimization�A Historical Perspective of a Paradigm Shift��Current Paradigm�Optimizing Biology and ADME SynchronouslyDM/PK – Research Interface at Glaxo �Late 1980’s – Early 1990’sUltra Short Acting Opioid�Remifentanil - Rationale for Incorporating Metabolic Studies Early in the Discovery ProgramPK of Parent and the Amine Metabolite Following Oral Administration of DHP-1 (5 mg/kg) to Male Beagle Dog Suggests First Pass Major Metabolite of DHP-1Relative Rates of In Vitro Metabolism for Dihydropyridazinone LeadsFunctional Group Contributing to the Metabolic Hot Spot for DihydropyridazinonesStabilizing a metabolic hot spot can uncover a secondary hot spot BackgroundLikely Metabolic Hot Spots In Vitro Metabolism of the Lead Compound by Rat Liver Enzymes and Caco-2 CellsModification of Metabolic Hot Spots Dutasteride® for BPHProfile of the Lead SeriesDog Microsomal Metabolism ScreenPK of Mixtures?????�Origin of Cassette DosingCassette Dosing “Validation” (5-in-One)5-in-One Dog vs. Individual Dosing�Half-life comparisonN-in-one in vivo screen workedHalf-lives of 125 Compounds in Dogs as Determined by Cassette DosingAcknowledgments