THE PATH FROM CHEMICAL TOOL TO APPROVABLE DRUGAn Integrated Approach to Drug Discovery

EVERY STEP OF THE WAY

EVERY STEP OF THE WAY

The Path to a Clinical Candidate

‘END TO END’ INTEGRATED DRUG DISCOVERY

3 EVERY STEP OF THE WAY

B/DMolecular Biology

Cell LineGeneration

FBDDStructuralBiology

CRISPRAdenoviral

Platform Human 1o Cells

Functional Genomics

Library Design

Analytical &Purification

ProcessChemistryCADD

Synthetic Chemistry

Formulation

Pharmaceutics

Chemo-genomics

D/PS/I

Safety Assessment

Safety Pharmacology

Non-GLP/GLP Toxicology

Anatomic &Clinical Pathology

Imaging

Animal ModelDevelopment

Large Animal Efficacy Models

DiscoveryPathology

In Vivo Efficacy

In Vivo Validation

PK/PD

Dose to Human Predictions

ADME

Bioanalysis

Targets Clinical Candidate

Pharmacologyin vitro/in vivo

Hit Finding:HTS, HCS

IND EnablingStudies

MedicinalChemistry

BiomarkerDevelopment

Target Discovery

& Validation

DP DP

Ch

Discovery Pathway

Chemistry

Biology/ Discovery Technologies

DMPK/Pharmacology/Safety/ In vivo models

4 EVERY STEP OF THE WAY

Are we there yet?

BEGIN WITH THE END IN MIND!Properties of Drug-Like Molecules

5 EVERY STEP OF THE WAY

• Potent• Modulate the target in a predictable way

• Selective• How selective is selective enough?

• Formulatable• Reasonable synthetic route or method of

production

• Good safety profile• Understanding of toxicity• No such thing as a “magic” therapeutic index

• Well-behaved pharmacokinetics• PK relates to the pharmacodynamics• Amenable to QD dosing (BiD acceptable in

some cases)• Rapid, predictable onset of action• Consistent metabolism• Clearance is important• No accumulation• Understand drug-drug interactions

• Available biomarker

THE FIRST PART OF THE JOURNEY

6 EVERY STEP OF THE WAY

• A chemical tool is a fit for purpose compound.

• “Good enough” with respect to potency, selectivity and PK

• Will never become a drug

• Should serve as a starting point for a synthetic chemistry strategy• Must be modifiable in a sensible manner

• Shouldn’t have serious red flags (e.g. highly reactive intermediates)

• Should be able to provide proof of concept for the project

Identification of a Chemical Tool

Inhibition of Cyclin-Dependent Kinase-4: A Targeted Approach to Cancer Therapy

A Case Study in Cancer Drug DiscoveryPeter Toogood, David Fry, W. R. Leopold

CASE STUDYRoad from Tool to Approved Drug

Cyclin DCdk4/6

Cdk2Cyclin E

E2F/DP1

Rb

The Eukaryotic Cell Cycle

Growth FactorsG0

PP

G1

SG2

M

Transcription stops,DNA replication starts

E2F/DP1

Rb

PPRb

PP E2F/DP1 Transcription

E2F/DP1P

PPRb

PPCyclin A

PPRb

PP

PPRb

PP

Cdk1Cyclin B

p16

p27

C. J. Sherr Science 1996, 274, 1672.S. A. Ezhevsky et al. Mol. Cell. Biol. 2001, 21, 4773.

(Kip1)

(INK4a)

Cdk1

Cdk3

Cyclin ACdk2

HDAC

HDAC

RATIONALE FOR TARGETING CDK4/6Beginning with the End in Mind

• Most human tumors are deregulated in some part of the Cyclin D/CDK4/p16/Rb/E2F pathway:• e.g. p16 deletion, cyclin D over-expression, Rb phosphorylation, Rb deletion

• Inhibition of CDK4/6 will restore cell cycle control at the G1 checkpoint

• CDK4/6 inhibition anticipated to be cytostatic not cytotoxic

• Rb-deleted tumors not anticipated to be sensitive

N

N N OHN

N

N N OHN2

6

26

8

PD 0168553

Cdk4/D IC50 > 10 µMCdk1/B IC50 > 10 µMCdk2/A IC50 > 10 µMCdk2/E IC50 > 10 µMPDGFr IC50 = 0.184 µMFGFr IC50 = 1.479 µMcSrc IC50 = 1.754 µM

PD 0166447

Cdk4/D IC50 = 0.620 µMCdk1/B IC50 = 1.015 µMCdk2/A IC50 = 0.129 µMCdk2/E IC50 = 0.410 µMPDGFr IC50 = 1.785 µMFGFr IC50 = 3.295 µMcSrc IC50 = 21.50 µM

S/T Kinases

Tyr-Kinases

IDENTIFICATION OF NOVEL INHIBITORSTransitioning From Tyr Kinase Inhibition to Ser/Thr Kinase Inhbition

Crystal Structure of PD 0172803 Bound to Cdk2 0.00 0.10 0.20 0.30

40 nM

20 nM10 nM

control

1 / [ATP](mM)

1Rate of pRb

phosphorylation

Increasing[Inhibitor]

0.5

1.0

1.5

2.0

INHIBITORS ARE ATP-COMPETITIVEUnderstanding Mechanism

Electron donating, polar substituents provide optimal

potency and physical properties

Cycloalkyl groups preferred over alkyl- or aryl-

substituents

Are any substituentstolerated here?

M. Barvian et al. J. Med. Chem. 2000, 43, 4606.

N

N NHN O

N

NH

56

28

PD 0205606

INITIAL OPTIMIZATION FOR POTENCYA Rational Synthetic Strategy

N

N NHN O

N

NH

2 8

0205606

Cdk4/D:Cdk2/A:

FGFr:HCT-116:

IC50 (µM)0.0060.0150.0810.138

PD 0205606 is a potent Cdk inhibitor with reasonable solubility (0.12 mg/mL)

But….

CL = 142 mL/min/KgAUC (PO) = 145 ng/hr/mLt 1/2(PO) = 1.6 h

Loss of selectivity!

Poor Pharmacokinetics!

THE FIRST CHEMICAL TOOL

PD 0202020

Cdk4/D IC50 = 0.720 µM Cdk1/B IC50 = 0.193 µMCdk2/A IC50 = 0.012 µMCdk2/E IC50 = 0.218 µMPDGFr IC50 = 10.82 µMFGFr IC50 = 3.900 µMcSrc IC50 = 7.150 µM

A potent inducer of apoptosis in HT-29 colon carcinoma cells

Metabolized at C5-C6 double bond

N

N NHN O

N

5 6

O O

Op450

Proposed biotransformation

ENGINEERING OUT METABOLIC LIABILITY

N

N NHN O

N

NH

56 N

N NHN O

N

NH

0205606Cdk4/D IC50 = 0.006 µMCdk2/A IC50 = 0.024 µMFGFr IC50 = 0.081 µMPDGFr IC50 = 0.5 µM

0217048 (R = H)Cdk4/D IC50 = 0.014 µMCdk2/A IC50 > 5 µMFGFr IC50 = 4.23 µMPDGFr IC50 = 1.81 µM

2

R

• Substituents at C5 larger than methyl are not tolerated• Effect of the C5 methyl group is modified by the C6 substituent

R Cdk4/D IC50(µM)

Cdk1/B IC50(µM)

Cdk2/A IC50(µM)

Cdk2/E IC50(µM)

Et 0.025 4.119 1.538 1.650

Cl 0.016 >5 1.625 1.500

Br 0.005 2.615 0.439 0.950

Ac 0.002 >5 0.230 1.15

CO2Et 0.006 >5 >5 >5

ATTACKING THE C5 – C6 POSITIONC5-Methylation Affords Cdk4 Selectivity

N

N NHN O

N

NH

O

PD 0326562

Enzyme IC50 (µM)

Cdk4/DCdk1/BCdk2/ACdk2/EFGFrPDGFrVEGFrLck

MDA-MB-435

0.002>50.231.153.393.101.691.744

IC50 = 0.03 µM

PD 0326562 is efficacious at 75 mg/Kg but has a narrow therapeutic index with significant toxicity at 100 mg/Kg

HOW SELECTIVE IS SELECTIVE ENOUGH ?

0174413Cdk4/D:Cdk2/A:

0.210 µM0.012 µM

0204661Cdk4/D:Cdk2/A:

0.092 µM0.002 µM

0205783Cdk4/D:Cdk2/A:

0.145 µM5.010 µM

N

N NHN O

N

N NHN O

N

N

N NHN O

N

PD 0204661 is toxic…..but PD 205783 is somewhat selective for Cdk4

UNDERSTANDING TOXICITYA Re-examination of the Pharmacophore

N

N NHN O

N

NH

2 8

6 BrN

N NHN O

N

N

NH

Br

Rat PKCL = 53 mL/min/KgIV t1/2 = 2 h F = 10%

Enzyme IC50 (µM)

Cdk4/DCdk1/BCdk2/ACdk2/EFGFrPDGFr

0.016>5>5>51.164.36

0325056 causes a clean G1 arrest up to 10.0 µM

0221933 0325056

DISCOVERY OF EXQUISITE SELECTIVITY OF CDK4

PD Number

022193303250560324275033272503265620332991

Cdk4/D

0.0020.0160.0060.1230.0020.011

Cdk1/B

0.281>53.530->5>5

Cdk2/A

0.042>50.556>50.23>5

FGFr

0.0801.160.535-3.39>5

Cdk2/E

0.172>53.500-1.15>5

PDGFr

0.0564.360.300-3.10> 5

Potency and selectivity for Cdk4: IC50 (µM)

1 2 3

Structure

112233

A

CHNCHNCHN

N

N NHN O

A

N

NH

BrN

N NHN O

A

N

NH

O

N

N NHN O

A

N

NH

O

THE SELECTIVITY EFFECT IS GENERAL

Protein Kinase IC50 (mM)

Cdk4/D1Cdk4/D3Cdk6/D2Cdk2/E2Cdk2/ACdk1/BCdk5/p25Gsk3βEGFrFGFrPDGFrVEGFrLckInsulin ReceptorAMPKChk1

0.0110.0090.015>10>10>10>10>10>10>10>10>10>10>10>10>10

Protein Kinase IC50 (mM)

CK-1SGKC-srcJNKMAPKAP-K1aMKKp70S6KPDK1PHKSAPK3MAPK2p70S6KPKAPKBPKCDYRK 1A

>10>10>10>108.0>10>10>10>10>10>10>10>10>10>102.0

PD0332991 IS SELECTIVE FOR CDK4/6 VS OTHER KINASES

Cell Line Cell Type Rb IC50 (µM)MDA-MB-435 Breast Carcinoma + 0.16

ZR-75-1 Breast Carcinoma + 0.17

T-47D Breast Carcinoma + 0.04

MCF-7 Breast Carcinoma + 0.10

H1299 Lung Carcinoma + 0.12

Colo205 Colon Carcinoma + 0.13

CRRF-CEM Acute lymphoblastic leukemia (ALL)

+ 0.25

K562 CML + 0.40

MDA-MB-468 Breast Carcinoma - >3 (20%)

H2009 Lung Carcinoma - >3 (0%)

IN VITRO ACTIVITY OF PD0332991Dependence on Rb

Biological Effect

Rb phos. (Ser780)Cellular ProliferationInitiate G1 arrest

IC50 (µM)

0.0660.0320.040

Data obtained using MDA-MB-435 and MDA-MB-453 breast carcinoma cell lines

RELATING TARGET MODULATION TO BIOLOGICAL EFFECTSTarget Biomarker Correlates with Biological Effects

1

10

100

1000

10000

0 5 10 15 20 25 30Time (Hours)

Subject Rat05

Subject Rat06

Subject Rat07

Subject Rat08

Mean Data

Individual and Mean Plasma 0332991 Concentration-Time Profiles Following a Single 20 mg/kg PO Dose to Fasted Male Sprague-Dawley Rats

Time (h)

Conc

entr

atio

n (n

g/m

L) t1/2 = 2.8 hF = 56%

PD 0332991 IS ORALLY BIOAVAILABLEWell Behaved PK Profile

Days Post Tumor Implant20 30 40 50

Med

ian

Tum

or M

ass

(mg)

150

200

250

300

400

500

600

700

800900

1500

100

1000

control36 mg/kg58 mg/kg93 mg/kg150 mg/kg240 mg/kg

Rx days:12-39Route: OralSchedule: Q1DDuration of therapy

PD0332991 INHIBITS TUMOR GROWTH IN VIVOMDA-MB-435 Breast Cancer Line

Days Post Tumor Implant20 25 30 35 40 45 50 55 60 65 70 75 80 85

Med

ian

Tum

or M

ass

(mg)

60

708090

150

200

250

300

400

500

600

700800900

100

1000

control12.5 mg/kg37.5 mg/kg75 mg/kg150 mg/kg

Rx days:18-31Route: OralSchedule: Q1D

Limit of palpation

Duration of therapy

PD0332991 INHIBITS TUMOR GROWTH IN VIVOColo-205

Med

ian

Tum

or M

ass

(mg)

Days Post Implant

Daily PO Therapy

MDA-MB-468

60708090

100

200

300

400

20 25 30 35 40 45 50 55 60

Control

240 mg/kg (3/10 NSD)

150 mg/kg

75 mg/kg mg/kg

37.5 mg/kg

PD0332991 IS INACTIVE IN Rb NEGATIVE TUMORSPredicted by Hypothesis

DEVELOPMENT OF PD 0332991 FOR CLINICAL USEObserved Clinical Toxicity is Exaggerated Pharmacology

PD 0332991 was nominated for development in 2002An Investigational New Drug application was filed in 2003Phase I clinical trials commenced in 2004

2007 ASCO Annual Meeting Abstract (J. Clin. Oncology 2007, 25, no. 18S)

A Phase I Dose Escalation Trial of Daily Oral CDK4/6 Inhibitor PD 0332991O’Dwyer et al

- Principal and dose-limiting toxicity is myelosuppresion. - Of patients receiving PD 0332991 for 21 days in a 28 day cycle, MTD = 125 mg QD.- 6 patients achieved stable disease, 3 of them for at least 20 cycles (breast, colon, ovarian)

CLINICAL ACTIVITY AGAINST ER-POSITIVE BREAST CANCERPromising Early Results

ASCO Annual Meeting, Abstract no. 3060 (J. Clin. Oncology 2010, 28, 15s)

Phase I/II Study of PD 0332991 and Letrozole in ER-Positive Breast CancerSlamon et al

PD 0332291 (125 mg) QD for 21 days of a 28 day cycle with letrozole 2.5 mg QD

• 12 Patients enrolled (post-menopausal, ER-positive, HER-2 negative)• 3 Patients discontinued due to disease progression• DLT = grade 4 neutropenia

• 3 Patients with partial response• 9 Patients with stable disease

PHASE I CLINICAL DATA IN MULTIPLE MYELOMAOther Indications

52nd ASH Annual Meeting, Abstract 860. Lentzsch et al.

• A Phase I Study of PD 0332991: Complete CDK4/6 Inhibition and Tumor Response in Sequential Combination with Bortezemib and Dexamethasone for Relapsed and Refractory Multiple Myeloma

• Schedule B: Patients received PD 0332991 (100 or 125 mg) QD days 1-12 of a 21 day cycle, with (IV) bortezomib and (oral) dexamethasone administered on days 8, 11, 15 and 18.

“Encouraging antitumor activity was observed in this heavily treated MM population”

Of 12 patients receiving schedule B , there was one partial response , three patients with stable disease (≥ 3 months) and one very good partial response with a patient who had relapsed on lenalidomide, bortezomib, and carfilzomibtherapies and a stem cell transplant

WE HAVE A DRUG!!!

30

Pfizer’s Palbociclib (PD-0332991) Receives Food And Drug Administration Breakthrough Therapy Designation For Potential Treatment Of Patients With Breast CancerWednesday, April 10, 2013 8:00 am EDT

The U. S. Food and Drug Administration granted accelerated approval to palbociclib (IBRANCE, Pfizer, Inc.) for use in combination with letrozole for the treatment of postmenopausal women with estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer as initial endocrine-based therapy for their metastatic disease.February 3, 2015

OTHER TUMOR TYPES IN CLINICAL STUDY

31

Development Continues

•Various Stages (I-II)• Mantle Cell Lymphoma• Refractory GIST• NSCLC• Glioblastoma• Liver

Timeline

September 2002

1996

Project Initiated PD 0

3329

91 e

nter

s De

velo

pmen

tDISCOVERY

December 2003

IND

September2004

FIH

PRECLINICALSAFETY ASSESSMENT

PHASE I

Mul

tiple

ong

oing

Phas

e I/

II tr

ials

June2007

2011

1stPh

ase

Ida

ta p

ublis

hed

PHASE I/II

Multiple Myeloma (+Velcade and Dex)Mantle Cell Lymphoma

MCL (+Velcade)Non-Hodgkins Lymphoma

LiposarcomaGlioblastoma MultiformeBreast Cancer (+Letrozole)

Solid Tumors

2013 2014

FDA

Decl

ares

Br

eakt

hrou

gh S

tatu

s

PFE

anno

unce

sIt

will

subm

it N

DA

PHASE III

2015

FDA

Gra

nts A

ccel

erat

edAp

prov

al fo

r Pal

boci

clib

Summary• Selective CDK4/6 inhibition was hypothesized to present a safer

approach to treating Rb-positive tumors with a deregulated cell cycle• PD 0332991 was discovered as a result of a concerted effort to identify

CDK4/6-selective kinase inhibitors by employing a number of chemical tool compounds throughout the process.

• PD 0332991 was advanced to human clinical trials on the basis of broad-based antitumor efficacy vs Rb-positive human xenograft tumors in mouse models and its preclinical safety profile

• FDA has granted breakthrough status to the compound for the treatment of breast cancer.

• Additional studies ongoing for treatment of other malignancies.

CONTACT USJ. A. Cornicelli, Ph.D., F.A.H.A.

251 Ballardvale StreetWilmington, MA01887

askcharlesriver@crl.com

www.criver.com

877.CRIVER.1