Drug Discovery

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Drug Discovery

• Shu-Jen Chen, Ph.D.
• Molecular Medicine Research Center
• Chang Gung University
• Nov. 22, 2006

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Outline

• Why do we need new drugs?
• How do we get new drugs?
• The process
• The investment
• The risk
• The bottlenecks
• New Technology for drug discovery
• Target validation
• High-throughput chemistry
• High-throughput screening
• Computer-aided drug design
• Example of Target-based therapy Gleevec

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Why do we need new drugs?

• Current therapy is unsatisfactory
• Alzheimers disease, Parkinsons disease
• Cancer
• Multiple sclerosis
• Current therapy has sever side effects
• Antipsychotic, anti-inflammatory, asthma
• Drug resistance
• Methicillin-resistant Staphylococcus Aureus
  (MRSA)
• Vancomycin-resistant Enterococci (VRE)
• Multidrug-resistant Tuberculosis (MDR TB)
• Emerging new diseases
• Bovine Spongiform Encephalopathy (BSE)
• SARS

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Drug Discovery The Process
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Drug Discovery The Investment
16 years US900M (NT2.7B)
Discovery (2-10 years)
Preclinical Testing Laboratory and animal testing
Phase I 20-80 healthy volunteers used
to determine safety and dosage
Phase II 100-300 patient volunteers used to look
for efficacy and side effects
Phase III 1,000-5,000 patient volunteers used to
monitor adverse reactions to long-term use
FDA Review/ Approval
Additional Post-marketing Testing
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Drug Discovery The Risk
Compound No
Discovery
10000
500
30
Development
15
10
3
Market
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Drug Discovery The Bottlenecks
Genomic Sciences
High Throughput Screening
High Throughput Chemistry
High Throughput Chemistry
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Modern Technology for Drug Discovery
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Target Selection
Target identification to identify molecular
targets that are involved in disease
progression Target validation to prove that
manipulating the molecular target can provide
therapeutic benefit for patients Criteria
safety and long-term efficacy
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Biochemical Classes of Drug Targets
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Technologies for Target Selection

• Genomic Analysis
• Gene scan (gene mapping)
• Whole genome sequencing
• Positional cloning
• Transcriptional Analysis
• Differential display
• Microarray (gene chip)
• EST database analysis
• Quantitative PCR
• RNA in situ hybridization

• Proteomic Analysis
• 2D-PAGE
• Mass spectrum
• Functional Analysis
• Overexpression
• Gene Knockdown
• Gene knockout
• Transgenic mice system
• siRNA technology
• Antibodies

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Compound Library

• Synthetic chemicals
• Random library (100,000 1,000,000)
• Focused library (1,000)
• Structure-based design library (lt 1,000)
• Marketed drugs (lt 2,000)
• Natural products
• Purified natural chemical library (2,000-5,000)
• Natural product extracts (mixtures)

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Parallel Synthesis
1 core structure
3 x 3 9 analogs
3 analogs
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Solid Phase Synthesis
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High-throughput Screening
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Cell-based vs. Enzyme-based Assay
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High Density Microtiter Plates
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Automated Robotic System
Beckman ORCA
Beckman Core System
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Data Integration
Import assay results
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Docking compounds into proteins computationally
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Principle of molecular docking
Low free energy
Stable complex
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Ser144
Ser144
Cys145
Cys145
Gly143
Gly143
P1
P1
His41
His41
P3
P2
P3
P2
Asp48
Glu166
Asp48
Glu166
Met165
Met165
Met49
Pro168
Met49
Pro168
P4
P4
Ala191
Ala191
Activity 35 nM
Activity 2 uM
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Example of Targeted Therapy Gleevec for CML
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Leukemia

• Most common blood cancer
• Four major types
• Acute Myeloid Leukemia (AML),
• Chronic Lymphocytic Leukemia (CLL),
• Chronic Myeloid Leukemia (CML),
• Acute Lymphocytic Leukemia (ALL)

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Clinical Course of CML
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Epidemiology of CML

• Median age range 45-55 years
• Male-to-female ratio1.3 1
• 50 diagnosed by routine lab tests85 diagnosed
  during the chronic phase

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Treatment for CML

• Bone marrow transplant (BMT, 10 yr survival
  54-57)
• The only known and proven curative therapy
• Cure rate for young patients (30-40 y/o) is
  approximately 50-60
• The motality rate for BMT ranges from 15-25 for
  an ideal candidate, while an old patients (gt40
  y/o) has a mortality rate of 30-50
• Only 15-20 of CML patients are candidates for
  BMT (limited by the age and the lack of HLA
  mathced donors)
• Conventional chemotherapy (palliative only, 10 yr
  survival 12-25)
• IFN-a therapy alone (10 yr survival 17-49,
  discontinuation rate 31)
• IFN-a with Ara-C (discontinuation rate 50)

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Prevalence of the Philadelphia Chromosome in
Hematologic Malignancies
Leukemia of Ph Patients CML 95 ALL
(Adult) 1530 ALL (Pediatric) 5 AML 2
Faderl S et al. Oncology (Huntingt).
199913169-184.
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What is the Philadelphia chromosome?
A balanced translocation of Chr 22 and Chr 9
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Translocation of Bcr/Abl
Molecular Biology of the Cell, Albert et al, 2001
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Targeting Bcr/Abl for CML Treatment

• Abl is a tightly regulated tyrosine kinase
• v-Abl is a constitutively active tyrosine kinase
• v-Abl transforms NIH3T3 cells in high efficiency
• Bar/Abl fusion protein also display constitutive
  tyrosine kinase activity
• Bcr/Abl alone is sufficient to cause CML in
  experimental animals
• Altered adhesion to stromal cells and the
  extracellular matrix
• Constitutively active signal
• Decreased apoptosis

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Chemical optimization of Gleevec
phenylamino core structure
amide group To enhance activity
against tyrosine kinase
pyridyl group - to improve cellular penetration
N-methyl piperazine To increase water
solubility and bioavailability
flag methyl To reduce activity against Protein
kinase C
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Inhibition of Kinase Activity by Gleevec

• STI571 potently inhibited ABL tyrosine kinases
• STI571 also inhibited PDGF-R and c-KIT
• STI571 showed no inhition towards most Ser/Thr
  kinase

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In Vitro Selectivity of Gleevec

• STI571 at 1 uM effectively killed
  bcr/abl-overexpressing 32D cells
• STI571 at 10 uM showed no toxicity towards 32D
  cells and 32D cells overexpressing v-Src

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In Vivo Efficacy of Gleevec

• Model mice injected with 32D-Bcr/Abl (A) or
  32D-v-Src (B)
• Dosing d11-d18, ip

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Gleevec Phase I Trial Design

• Druker, et al (2001).
• Dose-escalating study of Gleevec (n83)
• Dose range from 25 mg-1000 mg/ day po
• Primary Endpoint?Safety, Tolerability of Gleevec
• Secondary Endpoints?Antileukemic Activity
• Hematologic Response gt50 ? in WBC count X2 wks
• Complete Hem Response ? WBC to lt10,000 Plt
  gt450,000
• Cytogenic Response of Ph cells (conversion to
  normal)

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Gleevec Phase I Trial Results

• Hematologic Response
• 98 of pts receiving gt300 mg/day had a complete
  hematologic response
• Cytogenic Response
• 54 of pts receiving gt300 mg/day had some
  cytogenic response
• 31 had major cytogenic responses
• 13 had complete cytogenic remissions

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Gleevec Phase I Study
150

• Time to normalization of WBC
• Dose 500 mg/day

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Gleevec Phase II Study

• Drucker et al (2001)
• Antileukemic activity and safety of Gleevec in
  Ph CML in blast crisis and Ph ALL
• Doses 300 mg-1000mg po qd
• Outcomes
• -Hematologic Responses
• -Marrow Responses
• -Cytogenic Responses

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Response to Gleevec in blast crisis and
accelerated phases of CML

• Gleevec was approved as 2nd line CML treatment
  for IFN failure patients
• The review process took only 72 days a record
  for anticancer drug

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Gleevec vs. IFN-a Ara-C in newly diagnosed
patients with CML

• Gleevec was approved as 1st line treatment for CML

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Gleevec 50 years from Discovery to Delivery
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Dosage and Cost

• Dosage
• 400 mg/day in Chronic Phase CML
• 600 mg/day in Accelerated/Blast Crisis
• Cost
• 2400/ month (400 mg/day)
• Not covered by insurance

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Advantage of Gleevec

• Gleevec as 1st line therapy for CML
• 6 years increased survival over interferon-alpha
  treatment
• 43,100 per life year saved
• Specifically targets an enzyme in cancer cells,
  not normal healthy cells
• Minimal side effects compared to other treatments
• Given orally instead of injections

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Side Effects of Gleevec

• Fluid retention
• Nausea and Vomiting
• Muscle Cramps
• Hemorrhage
• Diarrhea
• Rash
• Indigestion
• Headache
• Joint Pain

Discoutinuation rate - Gleevec 3.5 - IFN
31 - IFN Ara-C 50
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How Effective is Gleevec?

• Gleevec is more effective in chronic phase CML

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Resistance Issue

• Very effective
• Less toxic
• Resistance pose a problem

mutation
Gleevec
Bcr/Abl protein
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Mechamisms of Gleevec Resistance
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Why do we need new drugs?

• Current therapy is unsatisfactory
• Alzheimers disease, Parkinsons disease
• Cancer
• Multiple sclerosis
• Current therapy has sever side effects
• Antipsychotic, anti-inflammatory, asthma
• Drug resistance
• Methicillin-resistant Staphylococcus Aureus
  (MRSA)
• Vancomycin-resistant Enterococci (VRE)
• Multidrug-resistant Tuberculosis (MDR TB)
• Emerging new diseases
• Bovine Spongiform Encephalopathy (BSE)
• SARS

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Next Generation Kinase Inhibitor