Cell-Based Drug Screening and Testing.ppt

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 Cell-Based Drug Screening
        and Testing

           Ik-Hwan Kim

     School of Life Sciences and
           Biotechnology
           Contents
 Drug screening procedure
 Why cell-based drug screening
 Considerations in cell-based HTS
 Model disease : Cancer - NCI panel
Therapeutic Targets for
       Drugs
 Functional Genomics
 Structural Genomics
 Proteomics and Bioinformatics
 Conventional bioassays
Lead Compound Screening
 Chemical library and chemo-informatics
 Natural product extracts
 High throughput screening (HTS)
 Structure-based drug design
 Cell-based drug screening
Chance of successful drug
     development
    (Elli Lilly & Co.)
   Candidate Compounds 200.000
                 ↓
     Active Compounds 2,000
                 ↓
           True Hits 200
                 ↓
        Lead compounds 10
                 ↓
          Clinical Trials 1
                 ↓
         Market place 0.12
       Speed of HTS
(Screening 106 compounds)

       20 years (1992)
     → 200 weeks (1996)
     → 2 weeks (2001)

     • Assay vessel evolution (96, 384,
       1,536 and 3,456 wells)
     • Parallel processing
     • Innovation in hardware (Robotics)
     • Advances in assay technologies
What are Cell-Based Assays?
 Assays based on the use of live cells.
 Include a variety of assays that
  measure cell proliferation, viability,
  toxicity, motility, production of a
  measurable product, and morphology.
 Offer a more accurate representation
  of the real-life model
      Why Whole Cells for
       Drug Screening?
   Advantages
- Multiple (unknown) target
- Solubility/Transport issue
- Toxicity issue (Animal model: time-consuming)


   Disadvantage
- High throughput issues
- High signal:background ratio
- High SE of expt'l data
- Complexity of testing
- High cost
Therapeutics Index (TI)

      TI = IC50/EC50
In vitro and in vivo screening
 1) In vitro Primary Screening : hits 1-2% of
    initial compounds
 2) In vitro Secondary Screening : routinely
    functional assay based on whole cells
 3) In vivo Toxicity Testing : cost of being
    wrong is immense -
 4) In vivo ADME : Absorption, distribution,
    metabolism, Excretion
Problems of Animal-Based
        Methods
   1. Too slow
   2. Labour intensive,
   3. Too costly

   ⇒ In vitro cytotoxicity testing is being
     seriously evaluated
    Considerations for Cell-
    Based Assay Automation
 Environmental control. Control of
  temperature, pH, and humidity in order
  to maintain viability.
 Contamination protection. Bacteria,
  dust particle (false positive readings)
 Long incubations. Some experiments
  require long incubation times
Cell supply problem in cell-
        based HTS
    • Large quantity of wide range of cell lines
    • Maintenance of many live cell lines
    • May cause unacceptible delay on robots

    ⇒ 1) Use commercial pre-plated cells
       2) Cell culture robots (6 major
     company - consortium formed in 2001)
Cell viability assays suitable
            for HTS
    1. Dye-based assays
    2. Bioluminescent assays
    3. Advanced cytotoxicity testing
          - Apoptosis and necrosis
          - Flowcytometric analysis (?)
          - ATP/ADP ratio measurement
          - Oxygen biosensor system
  Prediction of Biochemical
Mechanism : NCI Cancer Model
NCI panal/Cell lines : 60 Human cancer cell
 lines

   Leukemia (K562...) : 10
   Small-cell lung cancer : 2
   Central nervous system : 8
   Colon cancer : 9
   Melanoma : 8
   Ovarian : 6
   Renal : 6
   Misc (P388, P388/ADR) :
   Prostate
   Breast (MCF7, MCF7/ADR...)
 Renamed IC50 values:

 GI50 : 50% Growth Inhibition
 TGI : Total Growth Inhibition
 LC50 : 50% Lethal Concentration
Index of Similarity = PCC

“COMPARE” - Statistical treatment
        0 < PCC < 1.0
*PCC (Pearson Correlation coefficient)
         Standard Agents
   Antimetabolites
    - Pyrimidine Biosynthetic Enzyme Inhibitors
    (UP785…)
    - Glutamine Antagonists (DON…)
    - Ribonucleotide reductase Inhibitors (IMPY…)
    - Antifolic agent (Methotrexate…)
    Topoisomerase II agents (Doxorubicin, m-Amsa)
    Antitubulin (Taxol…)
    Alkylating agents (methyl-CCNU…)
Applications of cell-based
drug assays

1. Neuron-like Cells (PC12,
   Neuroscreen1cells) : Neurite-outgrowth
2. Cell attachment and spreading assays :
   Cancer, Cardiovascular, Inflammatory, and
   Neurological diseases, wound healing
3. Receptor-expressed cells : Modification of
   receptor mediated signal transduction
Tissue-Based Drug
Screening (MatTek)

1. Human epidermis: Dermal
   irritancy and toxicology.
2. Epithelial tissue of the respiratory
   tract: Inhalation toxicity studies.
   Inhaled drug delivery studies.
3. Epidermal Keratinocytes resembling
   cornea: non-animal means to assess
   ocular irritation and other
   toxicological issues.
4. Buccal Tissue: Mucosal
   drug delivery
5. Vaginal epithelium:
   Vaginal anti-fungal
   products,
   contraceptives,
   lubricants.
6. Epidermal
   keratinocytes and
   melanocytes:
   evaluate cosmetic and
   pharmaceutical agents
   designed to modulate
   skin pigmentation.

				
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posted:5/16/2012
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