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BIOASSAY TECHNIQUES FOR DRUG DISCOVERY AND DEVELOPMENT

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Title: BIOASSAY TECHNIQUES FOR DRUG DISCOVERY AND DEVELOPMENT


1
BIOASSAY TECHNIQUES FOR DRUG DISCOVERY AND
DEVELOPMENT
Dr. Muhammad Iqbal Choudhary Distinguished
National Professor
International Center for Chemical and
Biological Sciences (H. E. J. Research
Institute of Chemistry Dr. Panjwani Center for
Molecular Medicine and Drug Research) University
of Karachi, Karachi-75270
2
CONTENT
  • Molecular basis of diseases
  • Stages in drug development
  • Why Bioassays?
  • Different types/classes of bioassays
  • Difference between bioassay and pharmacological
    screenings?
  • Various types of bioassays?
  • High-throughput bioassays-Definitions, advantages
    and disadvantages
  • Bioactivity directed isolation of natural
    products- Strategies
  • Bioassay-guided fractionation (BGF) and isolation

3
Drug Discovery-Past and Present
  • In the past, most drugs were either discovered
    by trial and error (traditional remedies) or by
    serendipitous discoveries.
  • Today efforts are made to understand the
    molecular basis of different diseases and then to
    use this knowledge to design and develop specific
    drugs.
  • In modern drug discovery process, bioassay
    screenings play an extremely important role.

4
What is Required to Develop a Modern Drug (NME)?
  • Decision Corporate decision to invest in
    specific therapeutic area, based on economic
    feasibility
  • Cost 1.4 billion- 1.8 billion
  • Duration 10-12 years of RD, and regulatory
    approval
  • People 600-800 scientists of multi-disciplinary
    expertise
  • Chemical Diversity Screening of 100,000- 200,000
    compounds
  • Global Approval Lots of paper works, based on
    often ill-planned studies, and malpractices

5
A Book Worth Reading
  • Bioassay Techniques for Drug Research
  • By
  • Atta-ur-Rahman, M. Iqbal Choudhary and William
    J. Thomsen
  • Harwood Academic Press, London
  • http//nadjeeb.wordpress.com/2009/05/9058230511.p
    df

6
Diseases- Molecular Basis
  • Overwhelming majority of diseases are caused
    by change in biochemistry and molecular genetics
    of human body (Molecular Pathology)
  • Over- and under-expression of catalytic proteins
    (enzymes)
  • Toxins produced by microorganisms
  • Viruses (wild DNA/molecular organisms) cause
    cancers, AIDS, influenza, Dengue fever, etc.
  • Mutation in DNA cause cancers
  • Malfunction of signaling pathways cause various
    disorders
  • Congenital diseases due to genetic malfunctions
  • Oxidation of biomolecules (proteins,
    carbohydrates, lipids, nucleic acid),
    degenerative diseases and ageing
  • Deficiency of essential elements, vitamin,
    nutrients, etc.

7
Courtesy of Prof. Dr. Azad Khan
I
8
Main Stages in Drug Discovery and Development
  • Selection of Disease Target/Designing of
    Bioassay
  • Discovery and Optimization of Lead Molecules
  • Preclinical Studies
  • Clinical Studies

9
Why we Need to Perform Bioassay?
  • To predict some type of therapeutic potential,
    either directly or by analogy, of test compounds.
  • Bioassay is a shorthand commonly used term for
    biological assay and is usually a type of in
    vitro experiments
  • Bioassays are typically conducted to measure the
    effects of a substance on a living organism or
    other living samples.

10
What is Bioassay?
  • Bioassay or biological assay/screening is any
    qualitative or quantitative analysis of a
    substances that uses a living system, such as an
    intact cell, as a component.

11
Essential Components of Bioassays/Assays
  • Stimulus (Test sample, drug candidate, potential
    agrochemical, etc)
  • Subject (Animal, Tissues, Cells, Sub-cellular
    orgenlles, Biochemicals, etc.)
  • Response (Response of the subject to various
    doses of stimulus)

12
Molecular Bank at the PCMD Over 12,500 compounds,
and 6,000 Plant Extracts
13
Bioassays/Assays
  • Whole animals
  • Isolated organs of vertebrates
  • Lower organisms e.g. fungi, bacteria, insects,
    molluscs, lower plants, etc.
  • Cultured cells such as cancer cells and tissues
    of human or animal organs
  • Isolated sub-cellular systems, such as enzymes,
    receptors, etc

14
Types of Bioassays?
  • In Silico Screenings
  • Non- physiological Assays
  • Biochemical or Mechanisms-Based Assays
  • In Vitro Assays
  • Assays on Sub-cellular Organelles
  • Cell based Bioassays
  • Ex-Vivo Assays
  • Tissue based Bioassays
  • NMR Based Drug Discovery
  • In Vivo Bioassays
  • Animal-based Assays/Preclinical Studies
  • Human trial/Clinical Trials

15
Predicting Drug Like Behavior- Lipinski Rule
of Five
  • Molecular weight about 500 a. m. u. (Optimum 350)
  • Number of hydrogen bond accepter 10 (Optimum 5)
  • Number of hydrogen bond donor 5 (Optimum 2)
  • Number of rotatable bonds 5 (Conformational
    Flexibility)
  • 1-Octanol/water partition coefficient between 2-4
    range

16
Broad Categories of Bioassays
  • Virtual Screenings
  • Primary Bioassays
  • Secondary Bioassays
  • Preclinical Trials
  • Clinical Trials

17
Virtual and In Silico Screenings
  • Ligand based or Target based
  • Target Selection
  • Data Mining (Chemical space of over 1060
    conceivable compounds)
  • Screening of Libraries of Compounds Virtually
  • Lead Optimization
  • Prediction of Structure-Activity Relationships
  • It Save, Time, Money and Efforts

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19
Primary Bioassay/Assays Screenings
  • Non- physiological Assays
  • Biochemical or Mechanism-Based Assays
  • Microorganism-based bioassays
  • Cell-based Bioassays
  • Tissue-based Bioassays
  • Many other In Vitro bioassays/assays

20
Examples of Primary Assays
  • Antioxidant Assays
  • Enzyme Inhibition Assays
  • Cytotoxicty Bioassays
  • Anti-cancer Bioassays (Cancer Cell Lines)
  • Brine Shrimp Lethality Bioassays
  • In Vitro Antiparasitic Bioassays
  • Anti-bacterial Bioassays
  • Antifungal Bioassays
  • Insecticidal Bioassays
  • Phytotoxicity Bioassays
  • Etc.

21
Salient Features of Primary Bioassay Screenings
  • Predictive Potential
  • General in nature
  • Tolerant of impurities
  • Unbiased
  • High-throughput
  • Reproducible
  • Fast
  • Cost-effective
  • Compatible with DMSO

22
Hit Rate of Primary Bioassay Screenings
  • A hit rate of 1 or less is generally considered
    a reasonable
  • False positive are acceptable
  • False negative are discouraged

23
Secondary Bioassays
  • Animal-based assays (In Vivo)
  • Toxicological Assessments in whole animals
  • ADME Studies
  • Behavioral Studies
  • Preclinical Studies

24
Importance of Standards in Bioassays/Assays
  • The results of the assay/bioassay need to
    validated by monitoring the effect of an
    available known compound (Standard).
  • Without judicious choice of standard and its
    reproducible results in an assay system, no
    screening can be claimed credible.

25
Importance of Reproducibility and Dose Dependency
  • Without reproducible results (within the margin
    of error or esd), an assay has any value. It is a
    share loss of time and efforts.
  • Dose dependency is the key to a successful
    outcome of study.
  • Without reproducibility and dose dependency, it
    can be magic, but not science

26
VINBLASTINE- A Novel Anticancer Drug from Flowers
of Sada Bahar
27
In Vitro Bioassays
  • In Vitro In experimental situation outside the
    organisms. Biological or chemical work done in
    the test tube( in vitro is Latin for in glass)
    rather than in living systems
  • Examples include antifungal, antibacterial,
    organ-based assays, cellular assays, etc

28
Examples of In Vitro Bioassays
  • Activity Assays
  • DPPH assay
  • Xanthine oxidase inhibition assays
  • Superoxide scavenging assay
  • Antiglycation assay
  • Bioassays (cell-based)
  • DNA Level
  • Protein Level
  • RNA Level
  • Immunology assay
  • Toxicity Assays
  • MTT assay
  • Cancer cell line assays

29
In Vivo Screenings or Pharmacological Screenings
  • In Vivo Test performed in a living system such
    as antidiabetic assays, CNS assays,
    antihypertensive assays, etc.

30
Examples of In Vivo Bioassays
  • Animal Toxicity
  • Acute toxicity
  • Chronic toxicity
  • Animals Study
  • Animal model with induced disease
  • Animal model with induced injury
  • Pre-Clinical Trials
  • Clinical Trials

31
High-throughput Assays
  • The process of finding a new drug against a
    chosen target for a particular disease usually
    involves high-throughput screening (HTS), wherein
    large libraries of chemicals are tested for their
    ability to modify the target.

32
HIGH-THROUGHPUT BIOLOGICAL SCREENINGS
  • 96-384 Well plates (medium throughput) and more
    (high-throughput).
  • Development of straight-forward in-vitro
    biological assays (enzyme-based, cellular and
    microbiological assays) into automated
    high-throughput screens (HTS).
  • Rapid assays of thousands or hundreds of
    thousands of compounds (upto 200,000 samples per
    day).
  • Specifically suitable for the isolation of
    bioactive constituents from complex plant
    extracts or complex combinatorial library.

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High-throughput Screening Strategy for Enzyme
Inhibition Assays
Inhibition (E-S)/E ? 100 E Activity of
enzyme without test material S Activity of
enzyme with test material
Enzyme Buffer Potential inhibitor
Substrate
Incubation
Measurement of absorbance
96-well plate
12
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Some Examples of Assays at the ICCBS
39
Examples of Primary Assays
  • Antioxidant Assays
  • Enzyme Inhibition Assays
  • Cytotoxicty Bioassays
  • Anti-cancer Bioassays (Cancer Cell Lines)
  • Brine Shrimp Lethality Bioassays
  • In Vitro Antiparasitic Bioassays
  • Anti-bacterial Bioassays
  • Antifungal Bioassays
  • Insecticidal Bioassays
  • Phytotoxicity Bioassays
  • Etc.

40
Examples of In Vivo Assays
  • Metabolic Disorders (Diabetes, IGT, etc)
  • Cardiovascular
  • CNS Assays (Anti-depressant. Anti-anxiety,
    Anti-epilepsy, memory, etc)
  • Anti cancer
  • Drug Metabolism
  • Anti-parasitic
  • Anti-obesity
  • Toxicity

41
Enzyme Inhibition- Key Tool in Drug Development
A wide range of diseases are enzyme related. More
than 30 of the drugs in clinical use are enzyme
inhibitors. Many pesticides and insecticides
(chemical weapons!) also work as enzyme
inhibitors in the target organisms. Plants and
other living sources, as well as medicinal
chemistry can provide novel and potent enzyme
inhibitors.
42
Medium-throughput Screening Strategy for Enzyme
Inhibition Assays
Inhibition (E-S)/E ? 100 E Activity of
enzyme without test material S Activity of
enzyme with test material
Enzyme Buffer Potential inhibitor
Substrate
Incubation
Measurement of absorbance
96-well plate
12
43
Example Urease Inhibition
  • Urease catalyzes the hydrolysis of urea into
    carbon dioxide and ammonia.
  • The reaction occurs as follows
  • (NH2)2CO H2O ?CO2 2NH3
  • Ammonia in water forms ammonium hydroxide, a
    base.
  • Urease inhibition is a successful approach
    towards the treatment of diseases caused by
    ureolytic bacteria.

43
44
Inhibition of Urease- Inhibitors Type
  • Substrate Like Inhibitors Inhibitors which bind
    in a substrate or active-site directed mode.
  • Mechanism Based Inhibitors Inhibitors which bind
    in a non-substrate like manner or in
    mechanism-based directed mode

44
45
Urea Derivatives- Novel Urease Inhibitors
IC50 1.250.021 µM
Substrate like inhibition mechanism -structurally
similar to the natural substrate urea.
Standard Thiourea IC50 (Jack bean) 210.11 µM
Letters in Drug Design Discovery, Volume 5, 
Number 6, September 2008, pp. 401-405(5)
45
46
Substrate Like Novel Urease Inhibitors
Thioureas
1,2,4-Triazole-3-thiones
IC50 15.030.02 µM
IC50 16.70.178 µM
Oxadiazoles
IC50 16.10.12 µM
Dihydropyrimidines
Triazoles
IC50 5.360.027 µM
IC50 10.660.16 µM
Standard (Thiourea) IC50 21 0.11 µM
47
Glycation
Occurs in everyone, but at a faster rate in
diabetics AGEs formation effect the
molecular functioning of the body and cause
various diseases Activate RAGEs (Receptors of
AGEs) which contribute in triggering a number of
disease-causing inflammatory response
48
Prevention of Non-enzymatic Glycation
  • Inhibition of AGEs formation can lead to the
    prevention of diabetic complications by
    suppressing or delaying the formation of AGEs .
  • Various inhibitors have been discovered, such as
    Aminogunadine, Aspirin, Rutin, Antioxidants, AGE
    breakers, etc.
  • Aminoguanidine (AG), a potent AGEs inhibitor also
    underwent the clinical trials.Idealy AGEs
    inhbitors should be able to reverse the process
    of glycation, and repair the damage.

49
In Vitro Assay for Inhibition of Protein Glycation
Inhibitor (1 mM) HSA (10mg/mL)
Fructose (500 mM) Sodium Phosphate Buffer
Activity was monitored at Excitation 330 nm
Emission 440 nm.
Incubation 7 days at 37 C
50
Anti-glycation Activity of Some Natural Compounds
(Flavonoid glycoside)
Plant Name Tagetus patula
Rutin IC50 294.50 mM
51
Benzimidazoles

3-(6-Nitro-1H-benzimidazol-2-yl)-1,2-benzenediol
2- (6-Nitro-1H-benzimidazol-2-yl)-1,4-benzenediol
IC50 17.7 0.001 µM
IC50 48.7 0.006 µM
Rutin IC50 70 0.5 µM
52
Oxidation and Human Health
  • One of the paradoxes of life on this planet is
    that the molecule that sustain aerobic life,
    oxygen, is not only fundamentally essential for
    energy metabolism and respiration, but implicated
    in many diseases and degenerative conditions.
  • Marx, Science, 235, 529-531 (1985).

53
Methods Used to Determine Antioxidant Potential
DPPH Radical Scavenging Assay
  • For quantitative determination of electron
    donation.
  • The molecule of 1, 1-diphenyl-2-picrylhydrazyl
    (DPPH) is a long-lived organic nitrogen radical.

54
Principle
  • The delocalization gives rise to the deep violet
    color.
  • Characteristic absorption band in ethanol
    solution at
  • 515 nm.
  • When a solution of DPPH is mixed with that of a
    substance (RH) which can donate a hydrogen atom,
    then pale yellow reduced form of DPPH is formed.

55
Protocol
Sample in DMSO
56
Superoxide Anion Scavenging Assay
The assay involves a non-enzymatic generation of
superoxide anion radicals. The superoxide anion
scavenging activity was determined by measuring
the reduction in rate of formation of blue
colored formazan dye which absorbs at 560 nm. A
sample with antioxidant potential scavenge the
super oxide anion radicals and eventually reduces
the rate of formation of formazan dye, which can
be monitored by means of decrease in the
absorbance.
57
Superoxide Anion Scavenging Assay
58
Superoxide Anion Scavenging Assay
59
Flavones
Studied in DPPH BHA 44.02.70 PG 30.00.27
IC50 ?M 40.261.04
Source Whole plant of Iris tenuifolia Pall.
IC50 ?M 159.1534.49
IC50 ?M gt500
Source Whole plant of Iris unguicularis
Isolated by Dr. Sumaira Hareem
60
Studied at 500 ?M in DPPH and SO BHA 44.02.70
/97.03.0 PG 30.00.27 /104.02.4
DPPH Inactive Superoxide anion 40.35 ?M 1.87
DPPH Inactive Superoxide anion 97.99 ?M 2.65
DPPH Inactive Superoxide anion 30.98 ?M 1.65
61
Xanthone glycoside
Studied at 500 ?M in DPPH BHA 44.02.70 PG
30.00.27
Source Iris unguicularis Isolated by Dr.
Sumaira Hareem
Studied in DPPH BHA 44.02.70 PG 30.00.27
IC50 ?M 22.45 0.35
62
NMR-BASED SCREENING IN DRUG DISCOVERY
63
NMR-A Versatile Tool in Drug Discovery
64
ON-LINE ISOLATION AND BIOASSAY SCREENING
UV/VIS DETECTOR (Photodiode Array Detector)
CHROMATOGRAPHIC METHODS
Sample
FRACTION COLLECTOR
ON-LINE SPECTROMETERS
-NMR -MASS -IR -ICP
SPECTRAL AND STRUCTURAL DATABASES Dictionary of
Natural Products, Bioactive Natural Products
Database, DEREP, NAPRALERT, MARINLIT, Marine
Natural Products Database, STN Files
96-well plates or 384-well microplate
SPLITER
BIOASSAYS
65
Fragment Based Drug Discovery
Thrombin Inhibitor
HIV Protease Inhibitor
66
Fragment Based Drug Discovery
C. Acetylcholinesterase Inhibitor
67
Substrate Binding Specificity
  • Geometric Complementarity
  • Electronic (electrostatic) Complementarity
  • Induced fit vs. Lock Key
  • Stereospecific (enzymes and substrates are chiral)

68
NMR for Drug Research
1. Detect the weakest ligandtarget
interactions even with millimolar binding
constants. 2. Enables a determination of
binding constants. 4. Allows direct screening
and deconvolution of mixtures from natural
sources or combinatorial chemistry. 5.
Provide structural information for both target
and ligand with atomic resolution.
69
NMR for Drug Research
  • Fragment based discovery
  • Target identification
  • Lead optimization

70
NMR for Drug Research
  • Promising new method in drug discovery
  • Unmatched screening sensitivity.
  • Abundance of information about the structure and
    nature of molecular interaction and recognition.

71
Basic Development of NMR Spectroscopy for Drug
Research
  • Cryoprobe technology which increase
    signal-to-noise ratio and lower accessible
    binging affinities.
  • Flow probe alleviating the need for NMR tubes and
    time-consuming handling.
  • Micro-coil tubes (micro- and nano-probes) reduce
    the required sample volumes and also superior Rf
    field homogeneity. Thus facilitating difference
    based NMR screening methods.

72
Experiments Commonly Used in NMR-Based Drug Discovery Experiments Commonly Used in NMR-Based Drug Discovery Experiments Commonly Used in NMR-Based Drug Discovery
Acronyms Full Name Brief Definition
HSQC/HMQC Heteronuclear Single Quantum Correlation/ Heteronuclear Multiple Quantum Correlation 2D experiments which correlate proton and heteronuclear resonances. Very useful for protein binding studies and central to the chemical shift perturbation method.
TROSY Transverse Relaxation-Optimized Spectroscopy Technique that, by taking advantage of the interference of different relaxation mechanisms, allows for a significant increase in the molecular weight limit of biomolecular NMR.
SEA-TROSY Solvent-Exposed Amides Transverse Relaxation-Optimized Spectroscopy TROSY-based experiment that, by detecting only solvent-exposed amides, greatly simplifies the spectrum of high molecular weight proteins. This facilitates the use of chemical shift perturbation methods for screening.
INEPT Insensitive Nuclei Enhanced by Polarization Transfer Technique which uses heteronuclear coupling constants to transfer magnetization to insensitive nuclei, allowing their detection.
CRIPT Cross Relaxation-Induced Polarization Transfer Alternative to the INEPT technique where magnetization is transferred using cross-correlated relaxation.
73
Experiments Commonly Used in NMR-Based Drug Discovery Experiments Commonly Used in NMR-Based Drug Discovery Experiments Commonly Used in NMR-Based Drug Discovery
Acronyms Full Name Brief Definition
NOE/NOESY Nuclear Overhauser Effect/NOE Spectroscopy Effect that can be used to measure approximate through-space proton to proton distances. NOEs are the main NMR parameter used for conformational analysis and protein structure determination by NMR. NOEs are in general measured using a 2-D NMR experiment termed NOESY.
STD Saturation Transfer Difference Technique that allows the identification of ligands from a mixture of low molecular weight compounds by transferring saturation from the macromolecular target to the ligands.
Water-LOGSY Water-ligand observed via gradient spectroscopy Technique which uses water molecules to mediate the transfer of magnetization from the macromolecular target to the ligand.
SAR by NMR Structure Activity Relationships by NMR Structure-based NMR approach for the discovery of high affinity protein ligands based on chemical shift perturbation.
NMR-DOC NMR docking of compounds Structure-based NMR approach well-suited for very high molecular weight proteins which relies on selective isotope enrichment and requires no previous knowledge of the chemical shift assignments of the protein.
74
FRAGMENT-BASED DRUG DISCOVERY
  • Target- or Receptor-Based Screening- Does ligand
    interact with the target by following the changes
    in the chemical shifts of target protons?. It
    observe and compare the chemical shifts of
    targets in the absence and presence of ligand
  • Ligand-Based-Screening- Does ligand is
    interacting with the target by following the
    changes in the NMR parameters of ligand after the
    addition of the target

75
Receptor Based Screening by Chemical Shift Mapping
  • Identification of high affinity ligands by
    mapping the chemical shifts changes in the
    receptor spectrum (1H-15N- HSQC)
  • Require more quantities of receptor (proteins)

76
RECEPTOR-BASED SCREENING FOR DRUG DISCOVERY
77
Receptor Based HSQC/HMQC
  • 2D 1H, 15N or 1H, 13C-HSQC are used in the
    absence and presence of ligand.
  • The affinity constant between the ligand and the
    target can be accurately measured by determining
    the chemical shift changes as a function of
    ligand concentration.
  • 1H, 15N-HSQC experiment use to monitor changes
    in the amide protons and nitrogen nuclei of the
    backbone and Asn and Gln side chains (it
    requires the protein sample to be enriched in
    15N).
  • 1H, 13C-HSQC experiment gives information about
    the chemical shift changes in all side chains.
  • Drug-discovery programs usually deals with very
    large proteins. Using traditional method very
    long correlation time of protein (MW gt30 kDa)
    causes their NMR resonances to be too wide to be
    detected.

78
2D 1H15N-HSQC Experiment (Chemical shift
perturbation method)
1H (ppm)
The black contours correspond to FKBP (family of
enzymes that function as protein folding
cheprons), the macromolecular target, whereas the
red contours. correspond to the complex formed by
FKBP and phenylimidazole.
79
SAR by NMR
  • The first step of the method is the screening of
    libraries of low molecular weight compounds
    (fragments) using the 1H,15N-HSQC spectrum.
  • Once a hit is identified, the binding site can be
    discovered by deconvolution of the mixture,
    identification of the actual binding compound and
    mapping the chemical shift changes on the surface
    of the target .
  • This information is used to guide the
    combinatorial search for modified ligands of
    higher affinity.

80
SAR by NMR
  • Various step are involved in the screening of
    different libraries in search of compounds that
    will cause chemical shift perturbation in a
    different second site at the surface of the
    protein.

81
SAR by NMR
  • This step requires the screening to be carried
    out on a protein solution in the presence of
    saturating amounts of the first ligand so that
    the first binding site is fully occupied. When a
    hit for the second binding site is obtained the
    chemical shift changes are again mapped on the
    surface of the protein in order to ascertain the
    relative positions of the two binding sites.
  • After optimization of the affinity of the second
    hit, the two compounds (fragments?) can be
    covalently linked, yielding a lead compound of
    high affinity due to the chelating effect.

82
Structure-Activity-Relationship (SAR) by NMR
  • Identification of ligands with high binding
    affinity from library of compounds by using 2D
    1H-15N- HSQC
  • Optimization of ligands by chemical modification
  • Identification of ligand (optimized) binding by
    again recoding 2D 1H-15N- HSQC
  • Re-optimization of ligand by chemical
    modifications
  • Lining two ligands with appropriate linkers and
    checking the affinity again

83
SAR by NMR
84
SAR by NMR
Use of the SAR by NMR approach for the discovery
of inhibitors of Stromelysins (matrix
metaloproteineases).
85
Pre-clinical Trials
  • Involve in vivo (test tube) and in vivo (animal)
    experiments using wide-ranging doses of the study
    drug to obtain preliminary efficacy, toxicity and
    pharmacokinetics information.
  • Assist pharmaceutical companies to decide whether
    a drug candidate has scientific merit for further
    development as an investigational new drug.

86
Clinical Trials
  • Human Trial/Clinical Trials
  • Phase I (Safety 20-80 Volunteers)
  • Phase II (Efficacy/Safety 100-300 patients)
  • Phase III (Efficacy/Safety 300-3000 patients)
  • Phase IV (Post Approval/Marketing Studies)
  • Randomized, Double-blind, Placebo

87
VARIOUS STAGES IN DRUG DEVELOPMENT
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BIOASSAY-GUIDED FRACTIONATION (BGF)
  • Bioassay-guided fractionation (BGF) of Isolation
    is the process in which natural product extract
    or mixtures of synthetic products is
    chromatographically fractionated and
    re-fractionated until a pure biologically active
    constituent(s) is isolated.
  • At every stage of chromatographic separation,
    every fraction is subjected to a specific
    bioassay to identify the most active fraction(s).
  • Only those fraction(s) which are active are
    further processed.

91
  • Thank You Very Much
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