The Impact of Genomics on Drug Discovery

1
The Impact of Genomics on Drug Discovery

• Functional Genomics andPharmacology
  B. Monia (11/14 11/21)
• New Approaches to Drug Discovery B.Monia (11/21)
• Evaluation of Drugs in the Clinic J. Tami (11/28)
• Pharmacogenomics J. Tami (12/5)

2
Functional Genomics and Pharmacology- Agenda
from Last Time -

• The Human Genome Project (HGP)
• Paradigm shifts in Drug Discovery resulting from
  the HGP and other Genome Projects.
• Target Validation The new unmet need for Drug
  Discovery
• Correlative Approaches to Target Validation
• Comparative Genomics
• Microarrays
• Proteomics

3
Drug Discovery in the Post-Genomics Era Part
II- Agenda for Today -

• Functional Genomics (cont.)
• Causative Approaches to Target Validation
• Overexpression Systems
• Gene Ablation/Transgenic Animals
• Small Molecule Inhibitors
• Antisense
• Interference RNA
• New Approaches to Drug Discovery
• High-Throughput Screening and Structure-based
  Design
• Antibodies
• Protein Therapeutics
• Antisense

4
Causative Approaches for Target Validation-
Reverse Genetics -
mAb
GeneKnockouts
OverexpressionSystems
DNA
RNA
Small Molecule Inhibitor
Protein
?
RNA Degradation (e.g., RNase H)Modulation of
SplicingArrest of Translation
?
RNAi
AntisenseOligonucleotides
5
Table 1. A comparison of different experimental
approaches for modulating the function of cell
signaling molecules
ProbabilityofSuccess
Relevanceto DrugDiscovery
Potentialfor DrugDevel.
RequiredResources
Method
Versatility
Specificity
Cost
OverexpressionSystems
High
Low toModerate
Moderate
Low
Low toModerate
Low
Low
Gene Knockouts(Mammalian)
High
High
High
High
Moderate
Low
None
Gene Knockouts(Non-Mammalian)
High
High
Moderate
Moderate
High
Low
None
Small MoleculeInhibitors
Low
Low
High
High
Low
High
Yes
MonoclonalAntibodies
Low
High
Moderate
High
Moderate
High
Yes
AntisenseOligonucleotides
High
High
Low toModerate
Low toModerate
High
High
Yes
InterferenceRNA
High
High
Low toModerate
Low toModerate
?
?
None (?)
6
Mouse Transgenic and Gene Knockout Models

• Transgenic technology involves expressing foreign
  genes in the mouse.
• Foreign DNA is introduced into fertilized mouse
  eggs by microinjection.
• The gene knockout procedure involves inactivating
  (Knocking out) genes in the mouse.
• Genes are isolated, inactivated, and introduced
  into embryonic stem (ES) cells.
• ES cells are derived from early mouse embryos and
  have the capacity to contribute genetically to
  the complete development of the animal (both
  somatic and germ-cell contributions).
• Mice resulting from transgenic and gene knockout
  experiments develop, are usually born, bred and
  analyzed for the effects of gene
  overexpression/inactivation.
• May require the animals to be heterozygous for
  the gene of interest in knockout studies.
• New developments using specialized gene
  regulatory elements
• Conditional gene expression (e.g., diet-induced).
• Tissue-specific expression (e.g., liver).

7
Additional Genetic Models for Functional Genomics

• A variety of other model systems are being
  employed using gene knockout approaches.
• Yeast (e.g., S. cerevisiae)
• Fly (e.g., Drosophila)
• Plants (e.g., Arabidopsis)
• Worm (C. elegans)
• Caenorhabditis elegans (roundworm) in particular
  has received a great deal of attention. Offers
  many advantages
• Multicellular organism with a defined cell number
  at maturity (959).
• Genome sequencing completed (2769 genes, 6
  chromosomes).
• Shares many of the essential biological
  characteristics of humans.
• Sexual reproduction
• Development
• Nerve function (with a brain)
• Signaling pathways
• Simple to use
• Grows on petri dish (traditional genetic
  selection procedures)
• Transparent body visible with microscope
• Average life span 2-3 weeks
• Systematic gene ablation is straightforward.

8
Genetic Models for Functional Genomics-
Limitations -

• Labor intensive/long periods of time required
  (for mouse).
• Phenotypic results may not reflect human biology.
• Role of a gene in development vs
  post-development.
• Role of a gene in model organisms vs humans.
• Phenotypic results may not reflect pharmacology.
• Drugs do not Knockout gene products.
• Drugs modulate protein activity not protein
  levels.
• Structural vs enzymatic roles of proteins.

9
Antisense Technology is Based on Simple
Watson-Crick Hybridization Mechanisms
Protein
Transcription
Antisense Hybridization
Antisense Suppression
Uracil
Adenine
Guanine
Cytocine
mRNA/OligonucleotideDuplex
DNADuplex
mRNA
10
Watson-Crick Binding of Antisense Inhibitor to RNA
RNA Target
Antisense Oligonucleotide
11
History Lesson

• The starting point for antisense technology
  occurred in the mid 1970s with the discovery of
  small RNA molecules in bacteria and viruses that
  regulate DNA replication and RNA processing
  through antisense hybridization mechanisms
  (Tomizawa and colleagues at the NIH).
• A few hundred base pairs in length.
• Transcribed from endogenous DNA sense strand.
• The initial work of Tomizawa led to the concept
  of antisense RNA expression vectors that were
  first studied by Izant and Weintraub in the mid
  1970s.
• Recombinant DNA technology exploited.
• Expression vectors that produce antisense RNA in
  cells.
• Isolated antisense RNA injected into cells.
• The concept of using short (15-25 bases),
  synthetic strands of complementary DNA to
  hybridize with RNA and block mRNA function was
  first tested in the late 1970s by Zamecnik and
  Stephenson.
• DNA synthesizers as key breakthroughs.
• Viruses as targets followed by cellular targets.

12
What were the initial major hurdles for antisense
technology?

• Understanding how oligonucleotides suppress gene
  expression (Mechanisms of action).
• Medicinal chemistry of oligonucleotides.
• Metabolism
• Affinity
• Synthesis
• Understanding oligonucleotide pharmacokinetics.
• Toxicology of oligonucleotides.
• Understanding anomalous behavior of
  oligonucleotides.

13
Antisense Mechanisms of Action
eIFs
60S
5? UTR
60S
Intron
Coding
Coding
AAAAAn
40S
40S
AUG
3? UTR
CAP
RNaseH-MediatedDegradationof (pre)/mRNA
SplicingModulation
TranslationalArrest

• Regulate
• Inhibit

14
Medicinal Chemistry of Antisense Oligonucleotides
4
'
-
P
o
s
i
t
i
o
n

(
O
,

S
,

C
)
5
'
H
e
t
e
r
o
c
y
c
l
e
H
A
,
C
,
G
,
T
5
'
C
H
X
S
u
g
a
r
3
'
R
O
O
2
'
-
P
o
s
i
t
i
o
n
L
i
n
k
a
g
e
P
O

S
O
C
o
n
n
e
c
t
i
o
n

s
i
t
e
s
4
'
P
h
o
s
p
h
o
r
o
t
h
i
o
a
t
e
A
,
C
,
G
,
T
O
3
'
R
P
e
n
d
a
n
t
s
R
e
p
l
a
c
e

s
u
g
a
r
-
p
h
o
s
p
h
a
t
e
(
e
.
g
.
,

a
m
i
d
e

l
i
n
k
a
g
e
,

P
N
A
)
15
Antisense Specificity and Breadth - RNase H
Dependent Mechanism -
Control
Control
21959
18259
c-Jun
None
None
c-Fos
A-raf
B-raf
C-raf
c-Jun
A-raf mRNA
JNK-1
c-Fos
B-raf mRNA
JNK-2
G3PDH
C-raf mRNA
G3PDH
Kinases (RAF)
Transcription Factors
Kinases (JNK)
Control
Control
Control
Control
Control
Control
TRAF2
TRAF6
None
H-ras
K-ras
?i2
?i3
?i1
G?i1
Ha-rasmRNA
TRAF2
G?i2
Ki-rasmRNA
TRAF6
G?i3
G3PDHmRNA
G3PDH
G3PDH
Heterotrimeric G-Proteins
Adaptor Proteins
Low MW G-Proteins
16
Antisense Specificity and Versatility In Vivo-
Intravenous Administration -
Control ASO
JNK 1 ASO
JNK 2 ASO
p38 ASO
No ASO
JNK 1
JNK 2
p38a
120
120
JNK-1
100
JNK-2
100
p38?
80
80
Target mRNA Levels( control)
60
60
40
40
20
20
0
0
CTL
JNK1ASO
JNK2ASO
p38?ASO
CTL
JNK-1ASO
JNK-2ASO
P38?ASO
Liver
Adipose Tissue
17
Antisense Technology for Functional Genomics

• Advantages
• Offers excellent specificity (in general)
• All targets are approachable
• Fast, efficient
• Acts pharmacologically
• Works in cell culture and in animals
• Drugable
• Disadvantages
• Anomalous behavior under certain circumstances.
• Not all cell types are easily transfectable in
  vitro.
• Not all tissues/cell types in animals
  approachable.
• Not inexpensive. Advanced chemistries not easily
  accessible.

18
RNA Interference A New Functional Genomics Tool

• RNA Interference (RNAi) is the process where the
  introduction of double-stranded RNA into a cell
  inhibits gene expression in a sequence-dependent
  fashion.
• First discovered in C. elegans in the mid 1990s.
• RNA duplexes 20-30 bases in length injected into
  worms or ingested by worms results in the
  complete silencing of a homologous gene.
• Soaking worms in RNAi also induces gene
  silencing.
• RNAi activities have now been demonstrated in
  many other organisms inlcuding plants, flies and
  mammals.

19
RNA Interference A New Functional Genomics Tool

• Mechanism of RNAi silencing poorly understood.
• Requires target RNA synthesis which becomes
  cleaved in the region corresponding to the RNAi.
• Input RNAi gets processed to 19-20 nucleotide
  guide RNAs (siRNAs) with 2 nucleotide 3?
  overhangs.
• siRNAs may be amplified by RNA-dependent
  polymerases.
• siRNA duplexes then bind to a nuclease complex to
  form a RNA-Induced Silencing Complex (RISC).
• Responsible for target RNA cleavage.
• Many of the key genes/proteins involved in the
  RNAi process are being rapidly identified in C.
  elegans.
• RNAi as a tool for functional genomics is
  receiving a great deal of attention.
• One-third of the genes in the C. elegans genome
  have been subjected to RNAi silencing.
• Can RNAi be a new approach for therapeutics?

20
A Model for RNA Interference
Protein or RNPcofactor
dsRNA
Cleavage(shortening)
siRNA
Target RNA
RISC
Cleaved ormodifiedtarget RNA
Additional cellulardegradation mechanisms?
Degraded target RNA
21
New Approaches to Drug Discovery- Why the need? -

• Progress in the discovery of new therapeutic
  agents for the treatment of chronic,
  life-threatening diseases has been disappointing.
• Poor understanding of the molecular basis of
  diseases.
• Inadequate technologies available to maximize
  drug discovery (innovation).
• New approaches based on a firm molecular
  understanding of disease was greatly needed.
• New technologies that could produce drugs in
  non-traditional ways was greatly needed.

22
483
23
Small Molecule Drugs- Drugable vs
Non-Drugable Protein Targets -
Drugable AcceptableSpecificity
Non-Drugable

• Some proteins with intrinsicenzymatic activity.
• Some cell surface/nuclearhormone receptors.

• Everything else

5
Drugable withSmall Molecules
24
High-Throughput Screening for Drug Discovery - I

• High-throughput screening (HTS) is the process by
  which large numbers of compounds can be tested,
  in an automated fashion, for activity as
  inhibitors (antagonists) or activators (agonists)
  of a particular target.
• Result of advances in instrumentation
  engineering(e.g., robotics) and bioinformatics.
• Ten-twenty thousand compounds per day.
• Typically performed on 96-well micortitre plates.
• The primary goal of HTS is to identify
  high-quality leads and to provide directions
  for their optimization. Requires follow-up
  optimization (SAR).
• HTS provides leads only because it cannot
  evaluate
• bioavailability
• pharmacokinetics
• toxicity
• specificity

25
High-Throughput Screening for Drug Discovery - II

• HTS requires four basic elements
• suitable compound libraries
• assay method configured for automation
• robotics workstation
• computerized system capable of handling the data
• Two general types of assays are employed
• Cell-free assays
• ligand-receptor (peptide) interactions
• proximity-dependent fluorescence transfer
• Cell-based assays
• 2nd-messenger levels (e.g., Ca2, cAMP)
• Receptor gene expression
• Successes
• Cyclosporin A (organ transplant)
• Mevastation (Lipoprotein metabolism)

26
High-Throughput Screening for Drug Discovery - III

• Structure-based Drug Design
• Following identification of a lead from HTS,
  applying techniques to determine the
  3-dimensional structure of the target protein
  bound to the lead drug is being commonly employed
  today for lead optimization.
• Used mainly to improve binding affinity and
  specificity.
• Major advances in technology are making
  structure-based drug design a routine component
  to drug discovery.
• Improved X-ray detectors
• Better computers and graphics
• Multidimensional NMR
• Limitations of HTS
• The likelihood of success in finding a lead that
  eventually evolves into a drug is still low.
• Expensive and time-consuming to develop HTS
  assays and to perform follow-up lead
  optimization.
• Few biological processes are amenable to HTS.

• Lead Optimization
• Structure-based
• SAR

TargetValidation
Optimized Drug
HTS
EstablishHTS Assays
27
Monoclonal Antibody Therapy - I

• General Aspects
• Monoclonal Antibody Therapies (MAT) is based on
  the breakthrough findings of Kohler and Milstein
  that monoclonal antibodies (mAbs) of a given
  specificity could be obtained by fusing an
  antibody producing B-cell with an immortalized
  myeloma cell.
• Hybridoma cells
• Produce specific mAbs in unlimited quantities
• Has revolutionized the biological sciences
  (diagnostics/therapeutics).
• MAT was touted as The Magic Bullet for human
  therapeutics gt 25 years ago.
• Therapeutic results were disappointing until
  recently.
• Two major hurdles were encountered.
• Biologic activity in humans severely limited due
  to the hosts immune response to foreign mAbs
  (production of human antimouse Abs). Causes
  neutralization of activity and toxicity.
• Large-scale production

28
Hybridoma Technology
Immunizationwith Ag
Antibody Response/ Characterization
Isolation/Clonal Selection of B-Cells (spleen)
Immortalizationby fusion withmyeloma cells
Hybridoma
mAb Production
29
Monoclonal Antibody Therapy - II

• Approaches to reduce or eliminate human
  anti-mouse antibodies (mAb engineering).
• Chimeric Antibodies
• Cloned antibody genes are generated in which the
  variable regions from the original mouse mAb are
  combined with human antibody-constant regions.
• Highly effective in reducing human anti-mouse
  antibodies (HAMA). However, the variable
  (Ag-recognition) domains are still foreign HAMA
  often still occurs neutralizing efficacy.
• Primatized Antibodies
• Chimeric antibody approach in which
  primate-derived variable regions with human
  antibody constant regions.
• Very time-consuming. Reduces immunogenicity to
  human host further, but host reactions still
  occur.

30
Monoclonal Antibody Therapy - III

• Humanized mAbs
• All portions of the mAb not required for antigen
  binding, including framework residues in the
  variable region, are replaced with human
  sequences.
• Gene engineering, expression, evaluation
• lt 10 of optimized mAb retains mouse sequence
• Highly-effective (but time-consuming) approach
• New approaches under development
• Phage-display libraries expressing human VH and
  VL chains are assembled into libraries and
  expressed as combinatorial matrices.
• Assayed for immunoreactivity against antigen of
  interest.
• Transgenic Mouse Approach
• A procedure in which the endogenous mouse Ig gene
  loci is replaced by homologous recombination with
  their human homologs.
• Immunization of transgenic mouse followed by
  standard hybridoma procedures produces a fully
  humanized mAb.

31
Monoclonal Antibody Therapy - Successes I

• Organ Transplantation
• Rejection of allografts is an immunological
  response exerted primarily by T-cells. mAb
  approaches against T-cell antigens has proven
  effective.
• CD25 (IL2-Receptor ? chain). Highly effective
  approach for the prevention of graft rejection
  episodes and graft loss (e.g., renal).
• basiliximab (chimeric)
• dacliximab (humanized)
• Coronary Artery Disease
• Atherosclerotic lesions (plaques) involve
  platelet aggregation followed by coagulation and
  thrombus formation. Platelet receptors involved
  in early steps of plaque formation.
• basis of one-aspirin-a-day concept
• limited efficacy in preventing platelet
  aggregation
• Abciximab (chimeric mAb) has been
  developed/approved as an effective treatment for
  the prevention of thrombosis and plaque formation
  in high risk patients.
• Targets the platelet receptors ?2,6?3/?V?3
  (integrins)
• Prevents platelet aggregation

32
Monoclonal Antibody Therapy Successes II

• Rheumatologic and Autoimmune Diseases
• TNF? is a proinflammatory cytokine that has been
  implicated as a causal factor in a variety of
  inflammatory diseases including Rheumatoid
  Arthritis.
• Infliximab is a chimeric mAb targeted to TNF?.
• Has displayed remarkable activity against
  Rheumatoid Arthritis and other inflammatory
  diseases (Crohns Disease).
• Approved in 1998. First for Crohns Disease now
  R.A.
• Cancer
• Most intensively studied area (25 years of
  research).
• Cancer poses the highest hurdles. Two major
  successes in recent years.
• Also being evaluated in modified versions
  involving the conjugation to radio-ionizing
  particles, immunotoxins, immunoliposomes.

33
Monoclonal Antibody Therapy Successes III-
Cancer Cont. -

• Rituximab (chimeric mAb)
• Targets CD20 a cell surface protein expressed
  exclusively on B cells.
• Approved for the treatment of B cell malignancies
  (NHL, CLL, ALL).
• Trastuzumab (humanized mAb)
• Her2 is a form of the EGF-Receptor that undergoes
  amplification in approximately 25 of breast
  cancers.
• 1st identified as a rat oncogene called neu
  (HER2/neu)
• Patients with amplified HER2 expression suffer a
  very poor prognosis.
• Trastuzumab (Herceptin) has displayed impressive
  activity against HER2 breast cancer both alone
  and in combination.
• Improved response rates and survival
• Approved in 1998
• Under further evaluation to optimize dosing and
  expand patients population.
• New versions under development (e.g., Herceptin
  conjugates with immunotherapy/radiation).

34
Protein Therapeutics - I

• The application of human proteins, or derivatives
  thereof, directly as drugs for the treatment of
  disease.
• Full-length proteins or peptide derivatives
• Most commonly are derived from naturally
  occurring secreted proteins that contain
  consensus peptide cleavage sites and signal
  sequences.
• These consensus sequences are being used to fish
  out and identify potentially novel protein
  therapeutics from the HGP.
• Therapeutic proteins are expressed in a suitable
  host (e.g., yeast) and purified to very large
  quantities for therapeutic applications (i.e.,
  tons).
• Notable successes have been achieved in the
  Pre-Genome era.
• Insulin and diabetes
• Growth hormone and hypopituitary dwarfism
• Interferon-? and hepatitis C
• An explosion of new drugs based on Protein
  Therapeutics is expected as a result of the HGP
  (and related genome projects) and because of
  advances in recombinant DNA technology.

35
Protein Therapeutics - II

• Challenges for Protein Therapeutic approaches
• Gene Identification
• Bioavailability (absorption), distribution and
  metabolism (PK)
• Acceptably convenient modes of administration
• Manufacturing
• Modified forms of proteins and peptides often
  need to be developed to make them useful as
  drugs.
• SAR involving amino acid substitutions and
  deletions (e.g., insulin)
• Conjugating to chemicals such as polyethylene
  glycol
• Pegylated proteins are protected from proteolysis
  and display longer plasma half-lives than
  unpegylated forms.
• Conjugating to other proteins or peptides
• Conjugation to human serum albumin using
  recombinant DNA technology provides a sustained
  plasma half-life.
• Formulations

36
(No Transcript)
37
Properties of Modified Insulins
HMR 1964
Glargine
Onset of Actions (Hrs)
1.11
0.71
Duration of Action (Hrs)
22.8
13.8
Peak Effect (Hrs)
Peakless
7.5
38
Protein Therapeutics - III- New Drugs on the
Horizon Based on Genomics -

• Osteoprotegrin (Amgen) An inhibitor of
  osteoclast activity for the treatment of
  osteoporosis (Phase 2).
• Pegasys (Roche) New form of ?-interferon for
  the treatment of viral infection (Phase 1).
• SD-01 (Amgen) Pegylated granulocyte-colony
  stimulating factor for lymphocyte/stem cell
  mobilization (Phase 1).
• Repifermin (SKB) Keratinocyte growth factor 2
  as a stimulator of wound healing (Phase 2).
• Xigris (Lilly) Protein C for the treatment of
  Sepsis (Phase 3).

39
Antisense Technology is Based on Simple
Watson-Crick Hybridization Mechanisms
Protein
Transcription
Antisense Hybridization
Antisense Suppression
Uracil
Adenine
Guanine
Cytocine
mRNA/OligonucleotideDuplex
DNADuplex
mRNA
40
Critical Factors to be Considered for Optimizing
Antisense Efficacy

• Medicinal Chemistry of Oligonucleotides
• Affinity Potency
• Toxicological considerations
• Metabolism
• Pharmacokinetics
• Selection of Optimized Antisense Sequences
• Small of ASOs actually bind to target RNA
• Extensive screening often required
• Choice of Terminating Mechanism (e.g., RNaseH)
• In-depth Understanding of ASO Pharmacokinetics
• Tissue/cellular distribution
• Pharmacokinetic/Pharmacodynamic relationships

41
Antisense Drug Discovery (Therapeutics)-
Shortening the Timelines between Concept and
Clinical Trials -

• Rapid identification of drug entity.
• 100 success rate in identifying an inhibitor.
• All targets are Drugable.
• Predictable pharmacokinetics (same from drug to
  drug).
• Predictable chemical class-related toxicology
  (same from drug to drug).
• Identical manufacturing procedures.

42
Antisense Drug Pipeline Isis Pharmaceuticals
Pre-Clinical
Product (form)
Target
Lead Indication
Phase 1
Phase 2
Phase 3
On Market
Vitravene (I)
Antiviral
CMV Retinitis
ISIS 3521 (P)
PKC-a
Cancer - NSCLC., others
ISIS 5132 (P)
C-Raf
Cancer - ovarian, others
ISIS 2503 (P)
H-ras
Cancer - pancreatic, other
ISIS 2302 (P)
ICAM-1
Crohn's Disease
'01
ISIS 2302 (T)
ICAM-1
Topical Psoriasis
ISIS 14803 (P)
Antiviral
Hepatitis C
ISIS 104838 (P, O)
TNF-a
RA, Crohn's
4Q00
ISIS 104838 (T)
TNF-a
Topical Psoriasis, et. al
1Q01
ISIS 107248 (P, O)
VLA-4
MS, Inflammatory
ISIS 107772 (P, O)
PTP-1B
Diabetes
ISIS 13650 (I)
C-Raf
Diabetic Retinopathy, AMD
I Intravitreal
P Parenteral
T Topical
O Oral
43
PKC Signaling
Extracellular
GF
GFR
FAS
GFR
PLC
P
Cytoplasm
DAXX
RAS
Grb2
SOS
P
P
DAG
PI3K
GTP
GDP
IP3
PKC
Mitochondria
PTEN
P
P
BAD
BAD
Bcl2
MAP Kinase
Anti-Apoptotic
Pro-Apoptotic
P04
AKT
Apoptosis
Transcription FactorActivation
GeneActivation
P
P
P
P
P
P
P
P
Nucleus
44
Isozyme-specific Reduction in PKC-a mRNA and
Protein Expression by ISIS 3521
ISIS3521
ISIS 3521
Saline
Control
No oligo
PKC?
PKC?
PKC?
PKC?
Protein
mRNA
45
Activity of ISIS 3521 AgainstCalu-1 (lung) Human
Tumor Xenograft
2.25
.9 NaCl
2
375 mM 3521
Treatment ( T/C) ISIS 3521 (25
mg/kg) 47 Taxol 42 Taxol ISIS 3521 21
1.75
1.5
1.25
Tumor Volume (cm3)
1
.75
.5
.25
0
Day 25
Day 32
Day 40
Day 46
46
ISIS 3521 a Potent Inhibitor of PKC-?
inNon-Small Cell Lung Cancer
Phase 2 Trial in combination with
carboplatin/taxol
Overall Survival And Time To Progression (TTP)
(N53, period ending May 2001)
Expectation for ISIS 3521
Chemotherapy Chemotherapy
Median TTP ? 5 months 6.3
months Survival ? 8 months 15.9 months
1.00
Survival
0.75
TTP
Survival Distribution Function
0.50
Overall Survival
0.25
Censored Observation
Time To Progression
TTP
Survival
Censored Observation
0.00
0
5
10
15
20
25
30
35
6.3
15.9
Months
Typical results with carboplatin/taxol only
47
Evidence Supporting PTP-1B as a Validated
Therapeutic Target for Type 2 Diabetes
a
a
ß
ß

• Abundant in insulinsensitive tissues.
• Non-specific PTP-1Binhibitors (e.g.,
  Vanadate)enhance insulin signalingin vitro/in
  vivo.
• Over-expression ofPTP-1B inhibits
  insulin-mediated IR/IRSphosphorylation/insulins
  ignaling.

PIP4,5
SHC
IRS-1
RAS
PI3K
PTEN
Raf
PTP-1B
PIP3,4,5
PDK1
MAPKK
AKT
MAPK
Metabolic ActionsAnti-Apoptosis
Cell Growth
48
Evidence Supporting PTP-1B as a Validated
Therapeutic Target for Type 2 Diabetes
ß
ß
a
a

• PTP-1B Knockout Mice
• Enhanced phosphorylationof IR/IRS in
  muscle/liver inresponse to insulin.
• Reduced fed blood glucoseand insulin levels.
• Improved performance ininsulin and
  glucosetolerance tests.
• Gain less weight on normal andhigh fat diets.
  Decreased fat cell mass.
• No adverse observations gt 2 years.

PIP4,5
SHC
IRS-1
RAS
PI3K
PTEN
Raf
PTP-1B
PIP3,4,5
PDK1
MAPKK
AKT
MAPK
Metabolic ActionsAnti-Apoptosis
Cell Growth
49
Phylogenetic Tree of Protein Tyrosine
Phosphatases- R. Hooft van Huijsduijnen / Gene
225 (1998) 1-8 -
50
Specific Inhibition of PTP-1B Expressionin HEPG2
Cells
120
PTP-1B ASO
PTP-1B
100
TC-PTPase
100
nM
80
PTEN
10
50
PTP-1B
mRNA ( no oligo)
60
40
TC-PTPase
20
PTEN
0
nM
10
50
100
100
PTP-1B ASO
Control ASO
51
Dose-Dependent Lowering of Blood Glucose Levels
in Diabetic Mice (db/db) Following PTP-1B
Antisense Drug
450
Saline
400
PTP-1B ASO50 mg/kg
350
PTP-1B ASO25 mg/kg
300
250
PTP-1B ASO10 mg/kg
Blood glucose concentration (mg/dL)
200
Control ASO50 mg/kg
150
Normal
100
50
0
0
1
2
3
4
Time after treatment initiation (weeks)
52
ISIS 113715 / L-888, 728 (PTP-1B Antisense)A
Novel Therapeutic Agent to Treat Type 2 Diabetes

• ISIS 113715 displays a very attractive
  pharmacology profile as an antidiabetic agent
• Normalized blood glucose in mouse and rat models
  of diabetes
• No hypoglycemia observed Prevention of diabetes
  onset
• Decreased hyperinsulinemia
• Improved insulin sensitivity and glucose
  homeostasis
• Reduction in weight gain on high fat diet
• Subcutenous dosing potentially as infrequent as
  once/month
• Suppression of PTP-1B protein levels and improved
  insulin sensitivity in non-human primates
• Mechanism of action is well understood
• Attractive safety profile
• Rodents
• Primates

53
Antisense Technology and the Future Remaining
Hurdles

• Oral bioavailability
• Targeting tissues/cell types that we cannot
  target today.
• Improve drug potency (10X)
• Evaluate chronic toxicities in large patient
  populations.
• Exploit new mechanisms of action (non-RNaseH).