Ronald J. Christopher, Ph.D., D.A.B.T.

, FCP Compound Selection & Preclinical Studies

Preclinical Research & Development Overview

Pharmaceutical R&D Paradigm Compound Selection Preclinical R&D Activities
Pharmacology Drug Metabolism & Pharmacokinetics Drug Safety

Case Example & Clinical Translation

Discovery To Market The Economics

Time: Discovery IND: IND NDA/BLA: Review/Approval Time: 1-5 years ~ 6 years 1.1 years avg

Expense: The cost of developing a new drug is higher than ever about $1.3 billion

Success Rate of Drug Development

Candidates for a new drug to treat a disease might

include from 5,000 to 10,000 chemical compounds. On average about 250 of these show promise for further development About 10 of these will progress to human clinical trials Research to Market Success rate: ~1 in 1,000 compounds

R&D Focus on Validation State of Targets

Low validation state In Vitro (e.g. cell)
In Vivo (animal) In Vivo

High validation state

In Vivo (animal) Clinical In Vivo (animal) Launched

(animal)

(e.g. Ph II)

Drug

>70% of all marketed drugs result from R & D efforts on previously clinically validated targets

Compressing the Drug Discovery Process?

Average Industry R& D Timeline: >12 Years
Target Identification (1 3 y) Lead Generation (1 2 y) Lead Optimization (1.5 2.5 y) Pre-clinical Development (1 y) Formal Development (4 8 y)

Iteration (can add years)

Desired R& D Timeline: < 7 years

Lead Gen. (0.7 y) Lead Opt. (0.9 y) Pre-clinical Development (1 y) Formal Development (4 y)

Taking Shortcuts
We cant afford to collect extra data in the R&D Process Therefore, optimal efficiency in R&D is critical CORPORATE FINANCE

You wanna spend WHAT?!?

How Do We Improve?
Drug Co B

Drug Co D

Drug Co C

Drug Co E

Drug Co A

http://www.fda.gov/oc/initiatives/criticalpath/stanski/stanski.html

The new paradigm for drug R&D

Integration of skills Joint ownership/responsibility

Discovery

Preclinical

Clinical

Input from Business Development, Regulatory Affairs, Project Management, Legal

Compound Selection

Target Choice

A good target has distinctly different meaning to

biology and chemistry personnel

In a biology sense, a good target is a biological

pathway that can be intercepted in some way to give a useful therapeutic outcome

In a chemistry sense, a good target is a biological

pathway that can be intercepted in a useful sense by an orally active small organic molecule

Interplay of the disciplines leads to success

11

Typical Compound Criteria in Research

v Focus on First-in-Class or Best-in-Class v Structurally unique molecule v Solid Pharmacology
v Potency that meets or exceeds Gold standard v Target selectivity >1,000 fold selective vs. closely related target v Efficacy in relevant animal models (durability of response important)

v Excellent Drug Metabolism & Pharmacokinetic Properties

v No DDI liabilities v Suitable for Q.D dosing (if oral) v Limited metabolism, etc.

v Robust Efficacy in rodent autoimmune disease models v Excellent Safety profile (in vitro, in vivo)

Lead Generation Strategy

Biochemical/Cell-based Screening

Shapes & Fragments

Scaffold Morphing Virtual screening De Novo Design Biased libraries

Co-Crystal Complexes

Iterative Structure-Based Drug Design

Drug Design & Compound Synthesis

Known Inhibitors Compound Screening

In Vivo DMPK In Vivo PD Developability Criteria (P450, hERG, etc)

Novel TSD Leads

Research Testing Cascade

Metrics
Tier 1
1,000

First tier screens

Receptor Binding [EC50 < 10 nM] Solubility [0.1 - 0.2 mg/mL aq pH7] HLM / MLM / RLM T1/2 stability > 60 hERG binding [3H]-Astemizole > 10 M HepG2 cytotoxicity panel IC50 > 100 M Human 5 major P450s [microsomal] Protein binding [human / rodent] < 95 % Mini AMES [+/- S9]

Tier 2
100

Second tier studies

Oral efficacy & Dose Response [ED50 ~ 1 mpk] Mouse PK / PD Single dose iv/po, SD rat and mouse [PK/PD ) Cardiotox.: hERG CEREP panel Ames mutagenicity [+/- metabolic activation]

Tier 3

Third tier studies

Primary disease model : Mouse, Rat efficacy in vitro metabolism, metabolite ID Dose escalation PK Single dose IV/PO dog, monkey PK/PD Dog CV / rodent telemetry Pharm. Sci. [solid state testing, preformulation]

Typical Research Assay Flow Scheme

Chemistry Enzyme/Ligand Assays
IC50 < 100 nM

Target

Ligand Selectivity
(off-targets or closely related targets)

Selectivity > 1000-fold

Rat PK/PD (iv and po) Ligand inhibition (PD) Plasma conc. timecourse

Lead Series Declaration

Safety pharmacology

Rat/Dog Plasma DPP4 Inhibition Human/Rat/Dog microsome stability CYP450 inhibition Solubility Protein binding

e.g., Cerep initial rodent toxicology Non-rodent PK hERG Channel Genetic toxicity

Candidate Selection

Development (IND enabling studies)

Case Example

Structural Biology in Drug Discovery

An increasing role in understanding disease and in the design of new medicines

Viracept Agenerase

Gleevec

Actos Nesina

17
Reprinted with permission from Time magazine. Authorization expires April 18th, 2010

Atomic Structure of DPP-4 Protein

-propeller domain Peptidase domain

Propeller opening

Catalytic triad Side opening

Structural Biology adopts the use of Xrays in the design of new medicines But how can X-rays be used to take a picture of a protein target involved in a human disease and then design a drug for that target?

X-Ray Crystallography
Diffraction Pattern

Crystal

Source

X-Rays
X-Ray Wavelength: 1 C C bond length: 1.5"

+ Phases

Structure

DPP-4 Inhibitor Program Critical Success Factors

Absolute Criteria:
Highly selective and very potent No CYP450 interactions Once-daily dosing Orally active Superior Efficacy and safety profiles

Relative Criteria:
Equivalence or superiority to best known competitor DPP-4 inhibitors on all significant parameters Key comparator compounds: Novartis (Galvus) and Merck (Januvia)

Overall Goal: A Best-in-Class DPP-4 Inhibitor

Vildagliptin

Alogliptin

Structures of DPP-4 Inhibitors

F F NH 2 O N N N N CF3

non-coval ent
O
H 3C

CN N N

MK-431 (Sitagliptin/Januvia) Januvia (Sitagliptin) launched Oct 2006

NH3 + PhCO2 -

SYR-322 Alogliptin (Alogliptin benzoate)

covalent ( cyanopyr r ol idine)

N NC O

N H HO

LAF-237 (Vildagliptin/Galvus) Galvus (Vildagliptin)

H

H NH 2 N O

covalent ( cyanopyr r ol idine)

NC

HO

BMS-477118 (Saxagliptin)

Saxagliptin

DPP-4 Related Enzymes Inhibitors thought to be specific for DPP-4 may inhibit other enzymes in the DPP-4 activity and/or structural homologue (DASH) family Include:

FAP/Seprase DPP-2 DPP-8 DPP-9 PREP Tryptase

Biological role of related proteases:
T-cell apoptosis Attenuating T-cell activation Inactivation of regulatory neuropeptides Pathogenesis of cancer (promoting growth & metastasis)

Alogliptin: DPP-4 Potency & Selectivity Comparisons

20 15 10 5 0 Alogliptin Galvus Januvia DPP-4i 6.9 23.8 12.1

DPP4

Alogliptin is a potent DPP-4 inhibitor with high selectivity against related serine proteases Potency
++++ + ++

DPP-8 and DPP-9 activity appear to correlate with toxicities in animals and may be a key liability

IC50 (nM) for each enzyme

Compound Alogliptin Galvus (Vildagliptin) Januvia (Sitagliptin) DPP-2
> 100,000 > 100,000

DPP-8
> 100,000 1,400

DPP-9
> 100,000 81.5

FAP
> 100,000 73,000

PREP
> 100,000 > 50,000

Tryptase
> 400,000 > 200,000

> 50,000

19,000

62,000

> 100,000

> 100,000

> 400,000

Plasma Concentrations and DPP-4 Inhibition in Monkeys on Alogliptin (PO Dosing)

Plasma Concentration (ng/ml) 100,000 10000 1000 100 10 1 0 4 8 12 16 time (hour) 20 24 2 mg/kg 10 mg/kg 30 mg/kg

Dose linear pharmacokinetics T1/2 (PO) = 6 hours %F = >80%

110 100 % Inhibition of DPP-4 activity 80 60 40 20 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours) 2 mg/kg 10 mg/kg 30 mg/kg

0 -10
21

Inhibition initiated at 0.25 hours post dose Maximum DPP-4 inhibition at 2 to 3 hours post dose (90% to 91%)

Pharmacology

Alogliptin Reduces DPP-4 Activity and Increases Active GLP-1 Levels Non-Obese/Diabetic N-STZ-1.5 Rats
120 DPP-4 Activity (%) Active GLP-1 (pM) 100 80 60 40 20 0 Control 0.1 0.3 1 Alogliptin (mg/kg) # # # 60 50 40 30 20 10 0 Control 0.1 0.3 Alogliptin (mg/kg) 1 # # #

16

Alogliptin orally administered 1.5 h before meal load. Mean and SD, N=8, #P0.025

Alogliptin Lowers Plasma Glucose and Increases Plasma Insulin ( OGTT in N-STZ-1.5 Rats )
350 Incremental Glucose AUC (0-120min)(mg/dLmin) Plasma Glucose (mg/dL) 300 250 200 150 100 50 0 Pre 0 Time (min) 60 120 Control 0.03 mg/kg 0.1 mg/kg 0.3 mg/kg 16000 14000 12000 10000 8000 6000 4000 2000 0 Control 0.03 0.1 Alogliptin (mg/kg) 0.3

4 Plasma (ng/mL) PlasmaInsulin Insulin (ng/mL) 3.5 3.5 3 2.5 2.5 2 1.5 1.5 1 0.5 0.5 0 Pre Pre -60

4 Plasma Insulin at 10 min (ng/mL)

Control Control 0.03 mg/kg 0.03 mg/kg 0.1 mg/kg 0.1 mg/kg 0.3 mg/kg 0.3 mg/kg

3.5 3 2.5 2 1.5 1 0.5 0 Control 0.03

* *

0 Time (min) Time (min)

60 60

120 120

0.1 Alogliptin (mg/kg)

0.3

17

Mean and SD, N=6, *P0.025

Effects on Fasting Plasma Glucose in Normal SD Rats

120 100 Plasma Glucose (mg/dL) 80 60 40 20 0 0 30 60 Time (min) 90 120
Control Alogliptin 30 mg/kg Alogliptin 100 mg/kg Nateglinide 30 mg/kg Nateglinide 100 mg/kg

3 2.5 Plasma Insulin (ng/mL) 2 1.5 1 0.5 0 0 30 60 Time (min) 90 120

2000 1000 Incremental Glucose AUC (mg/dL 0-120 min) 0 Control -1000 -2000 -3000 -4000 -5000 Alogliptin 30 mg/kg Nateglinide 30 mg/kg Nateglinide 100 mg/kg

Alogliptin 100 mg/kg

Fasting Sprague-Dawley rats (7 wks old, male) were orally administered alogliptin or nateglinide at 0 min. Mean SD, N=6. *P0.025

* *

18

Alogliptin In Vivo Pharmacology (multiple doses)

Alogliptin in db/db mice

db/db (N=8) and db/+ (n=5) mice (8 week-old)

Dietary admixture: CE-2 powder diet containing 0.01%, 0.03%, 0.1% of Alogliptin for 2 days 0.01% 14 mg/kg 0.03% 42 mg/kg 0.1% 140 mg/kg

Plasma DPP-IV activity and GLP-1 levels

Alogliptin on DPP-IV and GLP-1 in db/db Mice

DPP-IV activity
120

GLP-1 level
40 35

Plamsa DPP-4 activity (% of control db/db mice)

Plasma active GLP-1 (pM)

100 80 60 40 20 0 0.01 Control 0.03 SYR-322 (%) 0.1 lean

30 25 20 15 10 5 0 0.01 Control 0.03 SYR-322 (%) 0.1 lean

* *

Alogliptin dose-dependently inhibited plasma DPP-IV activity. *p 0.025 vs. control by one-tailed Shirley-Williams test.

Alogliptin increased plasma active GLP-1 levels. *p 0.025 vs. control by one-tailed Williams test.

Chronic (4 Week) Study in ob/ob (obese) mice

Study-1; Dose-dependent efficacy of Alogliptin
Study design Control ob/ob SYR-322 ob/ob SYR-322 ob/ob lean ?/+ n=8 n=8 n=8 n=4 Untreated 0.002% in diet (2.80.3 mg/kg/d) 0.01% in diet (14.1 0.8 mg/kg/d)

Four weeks treatment of Alogliptin admixture with diet.

Study-2; High dose efficacy of Alogliptin

Study design Control ob/ob SYR-322 ob/ob lean ?/+ n=7 n=7 n=4 Untreated 0.03% in diet
(42.2 4.0 mg/kg/d)

Four weeks treatment of Alogliptin admixture with diet.

Mice; ob/ob/Crj and lean(Charles River Laboratories Japa

Plasma DPP-IV Activity and Active GLP-1 Levels after 4-week Treatment of Alogliptin in ob/ob Mice
DPP-IV activity
Plasma DPP-IV activity (%)
120

GLP-1
16

Plasma GLP-1 (pM)

100 80 60 40 20 0 Control

* *

14 12 10 8 6 4 2 0 Control 0.002%

0.002%

0.01%

?/+

0.01%

?/+

Mean and SD, n=8 *p<0.05 vs control by one-tailed Williams test. #p<0.05 vs control by one-tailed Shirley Williamstest.

Alogliptin Reduces DPP-4 Activity and Increases GLP-1 Levels

DPP-4 Activity
Plasma DPP-4 activity (%)
120

16

GLP-1
#

* *

Plasma GLP-1 (pM)

100 80 60 40 20 0 Control

14 12 10 8 6 4 2 0 Control 0.002% 0.01% ?/+

0.002%

0.01%

?/+

Alogliptin

Alogliptin

ob/ob Mice Mean and SD, n=8 *p<0.05 vs control by one-tailed Williams test. #p<0.05 vs control by one-tailed Shirley Williams test.

Alogliptin Increases Plasma Insulin and Decreases Plasma Glucagon

Plasma Insulin
140

Plasma Glucagon
600

100 80 60 40 20 0 0.002% Control

* *

Plasma glucagon (pg/mL)

Plasma insulin (ng/mL)

120

500 400 300 200 100 0

0.01% ?/+

0.002% Control

0.01% ?/+

SYR-322 Alogliptin

SYR-322 Alogliptin

Mean and SD, n=8 *p<0.05 vs control by one-tailed Williams test.

Pancreatic Insulin Content Restored with Drug

Control (Ob/Ob) Control

Alogliptin Treated (Ob/Ob)

Intense insulin staining was observed in islets of ob/ob mice treated with alogliptin Insulin staining in islets of alogliptin-treated ob/ob mice was comparable to that in vehicletreated non-diabetic ?/+ mice

Moritoh et al, 511-P, ADA 2007

Drug Metabolism & Pharmacokinetics

In Vivo Pharmacokinetic/Pharmacodynamic Profiles

Sprague-Dawley rats Beagle dogs Cynomolgus monkeys

Plasma Concentrations and DPP-IV Inhibition in Monkeys for Alogliptin (po)

Plasma concentrations
100000

% Inhibition of DPP-IV activity

110 100 90 80 70 60 50 40 30 20 10 0 -10

plasma concentration (ng/ml)

% Inhibition of DPP IV activity

10000 1000 100 10 1

2 mg/kg 10 mg/kg 30 mg/kg

2 mg/kg 10 mg/kg 30 mg/kg

12

16

20

24

time (hour)

8 10 12 14 16 18 20 22 24

Time (hours)

Dose linear pharmacokinetics T1/2 (oral) = 6 hours F = >80%

Inhibition Initiated at 0.25 hours post dose Maximum DPP-IV inhibition at 2 to 3 hours post dose (90% to 91%) Inhibition still apparent at 24 hours post dose (81% to 84%)

Drug Metabolism Profile

CYP Isoforms involved in metabolism CYP-2D6 (N-demethylated metabolite) CYP-3A4 also involved in metabolism CYP induction/inhibition Minimal induction of CYP3A4/5 (up to 5.88X) Minimal inhibition of CYP2D6 (27% at 100 mol/L) Low protein binding No drug-drug interactions (in vitro) when co-administered with other diabetic agents

Pharmacokinetic Profile
Pharmacokinetic Parameters Following a Single Oral Dose

Parameter
Cmax AUC(0-) T1/2 (hours) (IV) Tmax (hours) F (%) Excretion Route

Rats (20 mg/kg)

1,192 2,821 1.4 1.7 42 Urine, feces

Dogs (2 mg/kg)
278 699 2.9 0.75 71 Urine, feces

Monkeys (10 mg/kg)

3,208 15,859 5.7 1.0 87 --

Units: Cmax= ng/mL; AUC= nghr/mL

Drug Safety Evaluation

Alogliptin Drug Safety Profile Overview

Safety Pharmacology: No CNS, Cardiovascular or Pulmonary toxicities noted.

Genetic Toxicology: Not mutagenic or clastogenic.

Chronic Toxicology: doses up to 900 mg/kg (rat) and 200 mg/kg (dog)

Clinical Translation

Human Safety Margins

Endpoints From Oral Toxicity Studies 6 Month NOAEL Dose (mg/kg/day) AUC (nghr/mL) Exposure Multiples* 25 12.5 mg mg

Chronic Toxicity Study in Rats 400 258,579

362

181

9 Month NOAEL

Chronic Toxicity Study in Dogs 200 400,140

560

280

*Plasma AUC0-24h values determined based on data obtained in the multiple repeat dose (14 day) study in patients with type 2 diabetes mellitus.

NOAEL - No Observable Adverse Effect Level = nontoxic

Single Dose in Healthy Volunteers: Pharmacokinetics

10000

Alogliptin Concentration vs Time

1000

25

100

50 100

10

200 400 800

10

20

30

40 Time (hr)

50

60

70

80

Single Dose in Healthy Volunteers: DPPIV Inhibition

DPP-4 Inhibition vs Time
100

80

60
25 50 100 200 400 800 Placebo

40

20

-20 0 10 20 30 40 Time (hr) 50 60 70 80

Alogliptin Single Dose in Healthy Volunteers: Conclusions

No dose-limiting adverse events
25 mg to 400 mg to 800 mg

Alogliptin was absorbed rapidly Total exposure (AUC) and peak exposure (Cmax) increased with increasing dose Pharmacokinetics consistent with once daily dosing DPP-4 inhibition consistent with once daily dosing No significant metabolites Plasma and urine concentrations of M-I (Ndemethylated) and M-II (N-acetylated) metabolites were <5% of the parent drug
6

Thank you for your Attention