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: 1-5 years
IND → NDA/BLA: ~ 6 years
Review/Approval Time: 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

Ø  Candidates for a new drug to treat a disease might

include from 5,000 to 10,000 chemical compounds.

Ø  On average about 250 of these show promise for

further development

Ø  About 10 of these will progress to human clinical

trials

Ø  Research to Market Success rate: ~1 in 1,000

compounds
R&D Focus on Validation State of Targets
Low validation state High validation state
In In In
Vivo Vivo Vivo
In Vitro In Vivo
(animal) Clinical
(animal) Launched
(animal)
(e.g. cell) (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 Lead Lead Pre-clinical Formal

Identification Generation Optimization Development Development
(1 – 3 y) (1 – 2 y) (1.5 – 2.5 y) (1 y) (4 – 8 y)

Iteration (can add years)

Desired R& D Timeline: < 7 years

Lead Lead Pre-clinical Formal
Gen. Opt. Development Development
(0.7 y) (0.9 y) (1 y) (4 y)
 Taking Shortcuts
•  We can’t 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

Iterative Virtual screening

Co-Crystal Structure-Based
Complexes Drug Design De Novo Design

Biased libraries
Drug Design &
Known Inhibitors
Compound
Synthesis Compound Screening

In Vivo DMPK
In Vivo PD
Developability Criteria Novel TSD Leads
(P450, hERG, etc…)
Research Testing Cascade Metrics
Ø  First tier screens
Ø  Receptor Binding [EC50 < 10 nM]
Ø  Solubility [0.1 - 0.2 mg/mL aq pH7]
Ø  HLM / MLM / RLM T1/2 stability > 60’ Tier 1
hERG binding [3H]-Astemizole > 10 µM
Ø 
Ø  HepG2 cytotoxicity panel IC50 > 100 µM
1,000
Ø  Human 5 major P450s [microsomal]
Ø  Protein binding [human / rodent] < 95 %
Ø  Mini AMES [+/- S9]
Tier 2
Ø  Second tier studies 100
Ø  Oral efficacy & Dose Response [ED50 ~ 1 mpk]
Ø  Mouse PK / PD
Ø  Single dose iv/po, SD rat and mouse [PK/PD )
Ø  Cardiotox.: hERG
CEREP panel
Ø 
Ø  Ames mutagenicity [+/- metabolic activation] Tier 3
5
Ø  Third tier studies
Ø  Primary disease model : Mouse, Rat efficacy
Ø  in vitro metabolism, metabolite ID
Ø  Dose escalation PK
Ø  Single dose IV/PO dog, monkey PK/PD
Ø  Dog CV / rodent telemetry
Ø  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) •  Rat/Dog Plasma DPP4 Inhibition

•  Ligand inhibition (PD)
•  Plasma conc. timecourse
Lead Series
Declaration
•  Human/Rat/Dog microsome stability
•  CYP450 inhibition
•  Solubility
•  Protein binding

•  Safety pharmacology
Candidate
•  e.g., Cerep Selection Development
•  initial rodent toxicology (IND enabling studies)
•  Non-rodent PK
•  hERG Channel
•  Genetic toxicity
Case Example
 Structural Biology in Drug Discovery

An increasing role in understanding disease and in

the design of new medicines

Viracept Gleevec Actos

Agenerase Nesina

17
Reprinted with permission from Time magazine.
Authorization expires April 18th, 2010
Atomic Structure of DPP-4 Protein
Peptidase domain
β-propeller domain

Propeller
opening

Catalytic
triad

Side opening
Structural Biology adopts the use of X-
rays 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: 1Å
C – C bond length: 1.5Å"

Structure
+ Phases
 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
non-coval ent
F N N
O CN N
N
H 3C
N N CF3
MK-431 (Sitagliptin/Januvia)
O N Januvia (Sitagliptin)
launched Oct 2006

NH3 + PhCO2 -
covalent N
N
SYR-322 ( cyanopyr r ol idine)
Alogliptin
(Alogliptin benzoate) NC O
H
HO

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

H
H NH
2
covalent N
( cyanopyr r ol idine) NC O
HO

Saxagliptin
BMS-477118 (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:
–  FAPα/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
Ø  Alogliptin is a potent DPP-4
DPP4 10 inhibitor with high selectivity
against related serine
5 proteases
0
DPP-4i Potency Ø  DPP-8 and DPP-9 activity
appear to correlate with
Alogliptin 6.9 ++++ toxicities in animals and may
be a key liability
Galvus 23.8 +
Januvia 12.1 ++

IC50 (nM) for each enzyme

Compound DPP-2 DPP-8 DPP-9 FAP PREP Tryptase

Alogliptin > 100,000 > 100,000 > 100,000 > 100,000 > 100,000 > 400,000

Galvus
> 100,000 1,400 81.5 73,000 > 50,000 > 200,000
(Vildagliptin)
Januvia
> 50,000 19,000 62,000 > 100,000 > 100,000 > 400,000
(Sitagliptin)
 Plasma Concentrations and DPP-4 Inhibition in Monkeys
on Alogliptin (PO Dosing)

100,000
Plasma Concentration

2 mg/kg
10000 10 mg/kg •  Dose linear pharmacokinetics
30 mg/kg
(ng/ml)

1000 •  T1/2 (PO) = 6 hours

100 •  %F = >80%
10
1
0 4 8 12 16 20 24
time (hour)

110
100
•  Inhibition initiated at
0.25 hours post dose
DPP-4 activity
% Inhibition of

80
2 mg/kg
60
40
10 mg/kg
30 mg/kg
•  Maximum DPP-4
20 inhibition at 2 to 3 hours
0
-10
post dose (90% to 91%)
0 2 4 6 8 10 12 14 16 18 20 22 24
21 Time (hours)
Pharmacology
 Alogliptin Reduces DPP-4 Activity and
Increases Active GLP-1 Levels

Non-Obese/Diabetic N-STZ-1.5 Rats

#
120 60
#
#
100 # 50

Active GLP-1 (pM)

DPP-4 Activity (%)

80 # 40

60 30
#
40 20

20 10

0 0
Control 0.1 0.3 1 Control 0.1 0.3 1
Alogliptin (mg/kg) Alogliptin (mg/kg)

Alogliptin orally administered 1.5 h before meal load.

16
Mean and SD, N=8, #P≤0.025
 Alogliptin Lowers Plasma Glucose and Increases Plasma
Insulin ( OGTT in N-STZ-1.5 Rats )

350 16000

Incremental Glucose AUC

Control 14000
300

(0-120min)(mg/dL・min)
Plasma Glucose (mg/dL)

0.03 mg/kg 12000

250 0.1 mg/kg
10000 *
0.3 mg/kg
200
8000
150 6000
100 4000
50 2000
0 0
Pre 0 60 120 Control 0.03 0.1 0.3
Time (min) Alogliptin (mg/kg)

4 4
Control
Control
3.5
*
3.5
Plasma Insulin at 10 min
3.5 0.03 mg/kg
0.03 mg/kg
(ng/mL)

*
Insulin(ng/mL)

3 0.1 mg/kg
0.1 mg/kg 3
2.5
2.5 0.3 mg/kg
0.3 mg/kg 2.5
(ng/mL)

2
PlasmaInsulin

2
1.5
1.5 1.5
Plasma

1 1
0.5
0.5 0.5
0 0
Pre
Pre
-60 0 60
60 120
120 Control 0.03 0.1 0.3
Time (min)
Time (min) Alogliptin (mg/kg)

17
Mean and SD, N=6, *P≤0.025
 Effects on Fasting Plasma Glucose in Normal
SD Rats
120 3

100 2.5

Plasma Insulin (ng/mL)

80 2
Plasma Glucose (mg/dL)

60 1.5

40 Control 1
Alogliptin 30 mg/kg
Alogliptin 100 mg/kg
20 Nateglinide 30 mg/kg 0.5
Nateglinide 100 mg/kg
0 0
0 30 60 90 120 0 30 60 90 120
Time (min) Time (min)

2000
Nateglinide Nateglinide
1000 30 Fasting Sprague-Dawley rats (7 wks old,
mg/kg
Incremental Glucose AUC

100 mg/kg
0 male) were orally administered alogliptin
(mg/dL 0-120 min)

Control
Alogliptin
or nateglinide at 0 min.
-1000
30 mg/kg Alogliptin Mean ± SD, N=6. *P≤0.025
100 mg/kg
-2000

-3000
*
-4000

-5000 *
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 GLP-1 level

40 * * *
120
35
(% of control db/db mice)
Plamsa DPP-4 activity

100

Plasma active GLP-1

30
80 25

(pM)
60 20
*
* 15
40
* 10
20
5
0 0
0.01 0.03 0.1 0.01 0.03 0.1
Control SYR-322 (%) lean Control SYR-322 (%) lean

Alogliptin dose-dependently inhibited Alogliptin increased plasma active

plasma DPP-IV activity. GLP-1 levels.

*p ≤ 0.025 vs. control by one-tailed *p ≤ 0.025 vs. control by one-tailed

Shirley-Williams test. Williams’ test.
Chronic (4 Week) Study in ob/ob (obese) mice

Study-1; Dose-dependent efficacy of Alogliptin

Study design
Control ob/ob n=8 Untreated
SYR-322 ob/ob n=8 0.002% in diet (2.8±0.3 mg/kg/d)
SYR-322 ob/ob n=8 0.01% in diet (14.1± 0.8 mg/kg/d)
lean ?/+ n=4
Four weeks treatment of Alogliptin admixture with diet.

Study-2; High dose efficacy of Alogliptin

Study design
Control ob/ob n=7 Untreated
SYR-322 ob/ob n=7 0.03% in diet (42.2± 4.0 mg/kg/d)
lean ?/+ n=4
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
GLP-1
120 16
#
Plasma DPP-IV activity (%)

100 * 14

Plasma GLP-1 (pM)

12
80
10
60
* 8
#
6
40
4
20
2
0 0
Control 0.002% 0.01% ?/+ Control 0.002% 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 Williams’test.
 Alogliptin Reduces DPP-4 Activity and
Increases GLP-1 Levels
DPP-4 Activity GLP-1
120 16
#
Plasma DPP-4 activity (%)

14
100

Plasma GLP-1 (pM)

* 12
80
10
60 8
#
40
* 6
4
20
2
0 0
Control 0.002% 0.01% ?/+ Control 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 Plasma Glucagon
140 600

120 500

Plasma glucagon (pg/mL)

Plasma insulin (ng/mL)

*
*
100
400
80
300
60
200
40

20 100

0 0
0.002% 0.01% 0.002% 0.01%

Control Alogliptin
SYR-322 ?/+ Control 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) Ø Intense insulin staining was

observed in islets of ob/ob mice
treated with alogliptin

Ø Insulin staining in islets of

alogliptin-treated ob/ob mice was
comparable to that in vehicle-
treated 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 % Inhibition of DPP-IV activity
100000 110
2 mg/kg 100
plasma concentration

10000 10 mg/kg 90

DPP IV activity
80

% Inhibition of
30 mg/kg
70 2 mg/kg
1000
(ng/ml)

60
50 10 mg/kg
100 40 30 mg/kg
30
10 20
10
1 0
0 4 8 12 16 20 24 -10
0 2 4 6 8 10 12 14 16 18 20 22 24
time (hour)
Time (hours)

Inhibition Initiated at 0.25 hours post dose

Dose linear pharmacokinetics
T1/2 (oral) = 6 hours Maximum DPP-IV inhibition at 2 to 3 hours post
dose (90% to 91%)
F = >80%
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 µmol/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

Rats Dogs Monkeys

Parameter (20 mg/kg) (2 mg/kg) (10 mg/kg)
Cmax 1,192 278 3,208
AUC(0-∞) 2,821 699 15,859
T1/2 (hours) 1.4 2.9 5.7
(IV)
Tmax (hours) 1.7 0.75 1.0
F (%) 42 71 87
Excretion Urine, feces Urine, feces --
Route
Units: Cmax= ng/mL; AUC= ng·hr/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 Exposure
From Oral Multiples*
Toxicity Dose AUC 12.5 25
Studies (mg/kg/day) (ng·hr/mL) mg mg

6 Month Chronic Toxicity Study in Rats

NOAEL 400 258,579 362 181

9 Month Chronic Toxicity Study in Dogs

NOAEL 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

Alogliptin Concentration vs Time

10000

1000

25

100
50
100

10

200

400

800

1
0
10
20
30
40
50
60
70
80

Time (hr)

4
Single Dose in Healthy Volunteers: DPPIV Inhibition

DPP-4 Inhibition vs Time

100

80

60

25
50
40 100
200
400
800
20
Placebo

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

5
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 (N-
demethylated) and M-II (N-acetylated) metabolites
were <5% of the parent drug

6
Thank you for your
Attention