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Drug Discovery &
Development
Dr. Prashant Shukla
Junior Resident
Dept of Pharmacology
1/29/2015 1
Definitions: Drug: A chemical substance of known
structure, other than a nutrient or an
essential dietary ingredient, which, when
administered to a living organism,
produces a biological effect.
Discovery phase: Identification of a new
chemical entity as a potential therapeutic
agent.
Development phase: Compound is tested
for safety and efficacy for one or more
clinical indications, and in suitable
formulations and dosage form.1/29/2015 2
An important change:
The term “ drug discovery” is classical and
conventional. It should be replaced with
the term “ drug invention”.
In the past, drugs were discovered as
natural products and used as such.
Now, drugs are sculpted and brought into
being by pharmacologists in air
conditioned labs.
The term invention emphasizes this
process. 1/29/2015 3
New Drug definition (CDSCO)
According to Rule 122 E:
1/29/2015 4
(a) A new substance of chemical, biological or
biotechnological origin; in bulk or prepared
dosage form; used for prevention, diagnosis,
or
treatment of disease in man or animal; which,
except during local clinical trials, has not been
used in the country to any significant extent;
and which, except during local clinical trials,
has not been recognized in the country as
effective and safe for the proposed claim.
New Drug definition (CDSCO)
1/29/2015 5
(b) A drug already approved by the
licensing authority, which is now proposed
to be marketed with modified or new
indications, dosage forms (including SR
dosage form) and route of administration.
New Drug definition (CDSCO)
1/29/2015 6
(c) A fixed dose combination of two or more
drugs, individually approved earlier, and
now proposed to be combined for the first
time in a fixed ratio, or if the ratio of
ingredients in an already marketed
combination is proposed to be changed,
with certain claims, viz. indications, dosage
form (including SR dosage form) and route
of administration.
Stages of the New drug
synthesis: Drug discovery: Candidate molecules are
chosen on the basis of their pharmacological properties.
Preclinical development: Non-human studies (e.g. toxicity testing, pharmacokinetic analysis and formulation) are performed.
Clinical development: The selected compound is tested for efficacy, side effects and potential dangers in volunteers and patients.
1/29/2015 7
*
1/29/2015 8
1/29/2015 9
Drug discovery
Usual time duration: 2-5 years
Usual no. of projects: 100
It consists of following components:
1. Target selection and validation
2. Lead-finding or Lead generation
3. Lead optimisation
4. Pharmacological profiling1/29/2015 10
Reverse Pharmacology AKA target based drug discovery (TDD)
A hypothesis is first made that modulation of the activity of a specific protein target will have beneficial therapeutic effects.
The reverse pharmacology approach takes about 2 years to obtain a new drug candidate.
It is much faster and more efficient than the classical approach, which takes about
1/29/2015 11
Comparison:
1/29/2015 12
Properties of ideal drug target:
1/29/2015 13
Various targets of drug action
1/29/2015 14
The majority of drug targets are
a) G-protein coupled receptors (est total
5000)
b) Nuclear receptors (est total
>150)
c) Ion channels (est total 1000)
d) Enzymes (est total
uncertain)
Currently we are exploiting only 120 distinct drug
targets.
The potential drug targets that remain to be
Target identification strategies Gene Expression profiling: Genomics
Focussed Proteomics
Metabolic pathways analysis: Molecular
Biology
Phenotype analysis
Genetic association1/29/2015 15
Target identification
strategies... Inverse Docking: It is a computational
docking program in which a specific
small molecule of interest is tested
against a library of receptor structures.
Bio informatics: It derives knowledge
from computational analysis of
biological data. It includes information
stored in genetic code, patients
statistics and scientific literature.1/29/2015 16
In the earlier times, the complex
biological responses of chemicals were
first seen. And then the drug targets
were explored for those chemicals.
Now target identification has become the
first step of drug discovery.
Limitation: Drugs which do not act
through receptors- Antacids, Osmotic
diuretics, Alkylating agents, Psoralens
and Activated charcoal can not be
recognised.1/29/2015 17
Target Validation
To identify the most useful target among the various identified targets, Target Validation is done.
Identified targets are analysed and compared for
1. Ability to regulate biological and chemical process/ molecules in the body.
2. Association with a specific disease.
It is done using genetic knockout, knockdown and suitable disease
1/29/2015 18
Lead finding/ Lead generation
Approaches to new drug molecule
1. NEWER TECHNIQUES
Molecular modeling
Combinatorial chemistry
Biotechnology
Genetic medicine
Immunopharmacology
1/29/2015 19
BIOLOGICS or
BIOLOGICAL
COMPOUNDS
Molecular modelling AKA Rational drug designing.
Aided by three dimensional computer
graphics.
Allows design of structure based on new &
known molecules.
Highly selective targeted compounds are
created by enhancing desired properties of
known molecules. 1/29/2015 20
Combinatorial
chemistry It is systematic and repetitive covalent
connection of set of different building
blocks of varying structures to each
other to yield a large array of diverse
molecular entities.
This process is
1. Faster, more efficient and cheaper.
2. Millions of compounds can be
synthesized : Chemical Compound
Library.1/29/2015 21
Limitations of Combinatorial
chemistry:
1. Building and maintaining huge
compound libraries is a costly
business.
2. Even the largest compound collection
represents only a fraction of the
number of “drug like” molecules that
exists in theory- estimated to be about
1060 .1/29/2015 22
Biotechnology
Therapeutic agents produced by biotechnology rather than conventional synthetic chemistry are called Biopharmaceuticals.
Involve the use of recombinant DNA technology /genetic engineering
1. to clone & express human genes.
2. to produce large amount of hormones like insulin.
1/29/2015 23
Genetic medicine Transfer of Genetic material 1. A single gene which is typical for gene
therapy.2. Fragments of coding sequences (as in
RNA modification therapy- MC being anti sense oligonucleotide strategy).
3. Entire genome (as in the case of SSC and ESC therapy).
Vectors are Viruses and Liposome-plasmid complex.
Diseases addressed: Hereditary diseases like SCID, Haemophillia, etc.
1/29/2015 24
Immunopharmacology
Deals with finding the Biological immune modifiers or Immuno-modulating agents that cause selective up-regulation or down-regulation of specific immune responses.
Examples include:
1. Rituximab- Anti CD 20 monoclonal antibody for RA.
2. Adalimumab- Anti TNF-α inhibitor antibody for RA. 1/29/2015 25
Lead finding/ Lead generationApproaches to new drug molecule:
2. OLDER TECHNIQUES
Animal models as human disease.
Natural Products like plants , animals & micro- Organisms:
1. Random screening approach
2. Ethnobiological approach
Traditional Medicines.
Modification of structure of known drugs to develop “Me-too/ derivative medications/ follow-up drugs” drugs.
1/29/2015 26
Source of leads: Plants
1/29/2015 27
Papaver somniferum: Morphine
Atropa belladona: Atropine
Rauwolfia serpentina: Reserpine
Digitalis lanata: Digoxin
Strychnos toxifera: d- TC
Pilocarpus microphyllus: Pilocarpine
Salix alba (Willow bark): Aspirin
Bark of Yew tree: Paclitaxel
Cinchona tree: Quinine
Source of leads: Animals
Kraits: α- Bungarotoxin
Marine invertebrates: Arabinose
nucleosides
Cone snail toxin: Ziconotide (Prialt)
Marine invertebrates: Bryostatin-like
compounds
Dinoflagellates: Saxitoxin1/29/2015 28
Source of leads: Micro-
organisms Streptomyces notatum: Penicillin
Streptomyces venezuelae: Chloramphenicol
Penicilium griseofulvum: Griseofulvin
Streptomyces griseus: Streptomycin
Streptomyces gradiae: Neomycin
1/29/2015 29
SCREENING The usual approach is to clone the
target protein- the human form. This is
because the sequence variation among
species is associated with
pharmacological differences and it is
essential to optimise for activity in
humans.
An assay system is then developed to
measure the functional activity of the
target protein. 1/29/2015 30
Desired characteristics of the
assay:1. Should run automatically (if possible,
with an optical read out e.g. Fluorescence or optical absorbance).
2. Should be in a miniaturised multiwellplate format- for reasons of speed and economy.
Robotically controlled assay has become the standard starting point for most drug discovery projects.
e.g. High through put screening1/29/2015 31
Virtual screening (VS)
It is based on the computationally
inferred or simulated real screening.
Advantages compared to laboratory
experiments are:
1.low costs.
2.Investigate compounds that have not
been synthesized yet.
1/29/2015 32
Virtual screening (VS)...
3. VS can be used to reduce the initial
number of compounds before using
expensive HTS methods.
4.The number of possible virtual
molecules available for VS is much
higher than those available for HTS.
Disadvantage is that it can not substitute
the real screening.
1/29/2015 33
Two types of approaches used in
virtual screening
I. Target based virtual screening
(TBVS), or Receptor based virtual
screening
II. Ligand based virtual screening
(LBVS), or Similarity based virtual
screening.
1/29/2015 34
Target based virtual screening
(TBVS) Exploits the molecular recognition between
the ligand and a target protein information about the target.
Selection of chemical that has high affinity for the target’s active site.
Structural information can be determined by Nuclear Magnetic Resonance(NMR) or X-ray diffraction.
1/29/2015 35
Target based virtual screening
(TBVS) TBVS relies on 3D structures of protein
targets and on 3D databases of chemicals.
TBVS allows the identification of structurally novel ligands that may present interaction modes similar to the already known ligands.
Even new interaction pattern identification with different parts of the target’s active sites.
This methodology uses virtual filtering of all available ligands in a suitable database.
1/29/2015 36
Ligand based virtual screening
(LBVS) There is no structural information about
the target.
The screening focuses on physical and chemical based searches among the ligands.
Through pharmacophore pattern matching.
On similarity searching using descriptors that may be 1D, 2D or 3D.1/29/2015 37
Chris Lipinski’s rule of five
Linear descriptors (1D) are used to identify chemicals which do not violate any rules for solubility and permeability:
1. H-bond donors <5.
2. Molecular weight <500.
3. Partitioning coefficient (Log P) <5.
4. H-bond acceptors <10 (=5×2).
The “rule of five”name came from the cutoffs all being multiples of five. But there are only four rules.
1/29/2015 38
Ligand based virtual screening
(LBVS) The complete structure of the ligands can
be considered in the quantitative structure-activity relationships (QSAR) methods.
QSAR methods can make accurate prediction of the relative conformation and alignment of the ligands.
LBVS are more limited than TBVS since it is biased by the properties of the already known ligands for a given target.
1/29/2015 39
High through put screening
(HTS) The “Real Screening”.
It is the process of testing a large number of diverse chemical structures against disease targets to identify “hits”.
Compared to traditional screening methods, HTS is characterised by:
1. Simplicity
2. Rapidness
3. High information harvest
4. Based on ligand-target interaction principle 1/29/2015 40
High through put screening...
Various technologies used for HTS are:
1. Fluorescence
2. Nuclear Magnetic Resonance (NMR)
3. Affinity chromatography
4. Surface plasmon resonance
5. DNA microarray
HTS can analyse around 10,000-
100,000 samples/day.
1/29/2015 41
End results of screens:
Hit: A molecule with confirmed
concentration-dependent activity in a
screen, and known chemical structure.
Progressible hit: A representative of a
compound series with activity via
acceptable mechanism of action and
some limited structure-activity relationship
information.1/29/2015 42
Lead Optimisation
The aim of this stage is:
1. Increase the potency of the
compound on its target.
2. Increase its selectivity.
3. Increase its metabolic stability.
Usually one project out of five passes this
stage.
1/29/2015 43
Lead Optimisation...
Various steps:
1. Identification of the Pharmacophore
(relevant groups on a molecule that
interact with a receptor and are
responsible for the biological activity).
2. Functional group modification:
Modification of the group may enable
or disable certain biological effects.
1/29/2015 44
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3. Structure-Activity relationship:
Some of these features are important for
the activity and the others are not.
(1) NH2 and sulfonyl (R) should be para.
(2) NH2 should be unsubstituted.
(3) Benzene ring should not be replaced
by other ring systems.
4. Structure modification to increase potency and therapeutic index:
A. Homologation: a homologous series is a group of compounds that differ by a constant unit, usually CH2.
B. Chain branchingC. Ring-chain transformationAffects (1) lipophilicity,
(2) interaction with the enzyme or receptor. It could increase or decrease drug potency and therapeutic index.
D. Bioisosterism.
1/29/2015 46
5. Quantitative structure-activity
relationships (QSAR-rational drug
design)
Based on the fact “the biological properties
of compounds are a function of its
physico-chemical parameters”.
Fundamental physicochemical parameters
a) Electronic effects: Hammett equation
b) Lipophilicity effects: Hansch equation
c) Steric effects: Taft equation1/29/2015 47
6. Molecular graphics-based drug design: To find a structure match, a computer technology called DOCKING is used. It is the computer-assisted movement of a terminal-displayed molecule into its receptor.
Docking algorithms deal with ligandconformation prediction and orientation within the target active site. It predicts the various forces acting between target and ligand.
Scoring function is a mathematical function to rank protein-ligand complexes according to their predicted binding affinity.1/29/2015 48
The main problem is that lead
optimisation often seems impossible
despite much ingenious and back-
breaking chemistry. It is because lead
compounds, like anti social teenagers,
refuse to give up their bad habits.
In other cases, the compounds
although they produce the desired
effects on the target molecule and have
no other obvious defects, fail to produce
the desired effects in animal models of
disease. This implies that the target is
not a good one.
1/29/2015 49
Out of the above steps, Target identification and Lead Finding is often carried out in academic research laboratories.
Screening for biologic activity and chemical modification of a known active molecule are usually carried out in industries due to their high costs.
Translational research/ medicine or Experimental medicine: The process of moving from the basic science laboratory to the clinic. It involves the pre clinical and clinical steps of drug discovery.
1/29/2015 50
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Pre clinical development
Usual time duration: 1.5 years
Usual no. of Compounds: 20
The aims of pre clinical testing are:
1. Pharmacokinetics
2. Short term toxicology
3. Formulation
4. Synthesis scale up
1/29/2015 52
Work falls in four categories:
1. Safety Pharmacology:
Pharmacological testing to check that
the drug does not produce any
hazardous side effects.*
2. Preliminary toxicological testing to
eliminate genotoxicity and to determine
the maximum non-toxic dose of the
drug (usually when given daily for 28
days, and tested in two species). 1/29/2015 53
Work falls in four categories:3. Animal studies: Pharmacokinetic testing i.e.
studies on absorption, metabolism, distribution and elimination in laboratory animals like Mice, chicken, monkeys, and guinea pigs.
4. Chemical and pharmaceutical development:
a) Feasibility of large- scale synthesis and purification
b) Stability of the compound under various conditions
c) To develop a formulation suitable for clinical studies.
1/29/2015 54
Animal studies: components Toxicology studies: Extended
programme in animals studies. These
could be acute, sub acute or chronic
toxicity studies.
1. Acute: 24-48 hours
2. Sub Acute: Few weeks
3. Chronic: For months
1/29/2015 55
Animal studies: components... Mutagenicity studies: comprises of in
vitro tests where either unicellular organisms or tissue cultures are exposed to the drug.
Following battery of tests is used:
1. Ames test in S. Typhimurium.
2. Cytogenetic assay in mammalian cells.
3. Micronucleus assay in rodent hematopoietic cells.
First two tests should be completed before Phase I study commences.
But the battery should be complete in all 1/29/2015 56
Animal studies: components... Carcinogenicity studies:
Two animal species with low incidence of spontaneous tumours are used. 3 doses- high, low and intermediate are employed.
The study usually lasts for most of the animal’s life.
Detailed autopsy and histological examination are performed at the end of the study.
1/29/2015 57
Animal studies: components... Reproductive studies: Extensive studies of
a potential drug in the pregnant animals is mandatory.
PK variations and plasma conc. of the drug in mother and foetus are recorded.
Aims: Teratogenic potential
Effect on gametes, uterine growth, parturition, post natal
development and lactation.
1/29/2015 58
Animal studies: End
parameters1. Therapeutic dose =
Lethal dose(LD 50)/ Effective dose (ED
50)
2.Maximum Tolerated dose (MTD)
3.Minimal lethal dose (LD10)
4.No adverse effect level (NOAEL) dose: It
is the largest amount of drug which
causes no detectable adverse effect
with regards to morphology,
physiology, growth, reproduction and
life span of the organism exposed to
drug.1/29/2015 59
Animal studies: End
parameters...NOAEL is calculated in at least three
species with one being a rodent.
NOAEL is extrapolated to humans.
5. Human equivalent dose (HED)= Animal
NOAEL x (Wanimal/ Whuman)1-b
where W is the weight in Kg and b is a
correction factor (equal to 0.67) used to
convert mg/kg to mg/m2
1/29/2015 60
Animal studies: End
parameters...HED (mg/kg) = Animal Dose (mg/kg) x
[Animal Km / Human Km(37)]*
6. The dose to be used for initial human
studies is called “Maximum
recommended starting dose (MSRD)”
calculated as
MRSD = HED/ Safety factor (Sf)
Sf allows the interspecies variability in
drug disposition. Default Sf is 10. 1/29/2015 61
Good laboratory practices
(GLP)It is defined by the OECD (Organization
for Economic Co-operation and
Development) principles as
“a quality system concerned with the
organizational process and the
conditions under which non-clinical
health and environment safety studies
are planned, performed, monitored,
recorded, archived and reported.”
1/29/2015 62
Good laboratory practices (GLP)...
1/29/2015 63
Limitations of pre clinical
testing1.Toxicity testing is time consuming and
expensive. 2-6 years may be required
to collect and analyse data on toxicity
for testing in humans.
2. Large no. of animals may be needed
to obtain valid pre clinical data. It
raises ethical issues.
1/29/2015 64
Limitations of pre clinical
testing...3.Extrapolations of therapeutic index and
toxicity data from humans are
reasonably predictive for many but not
for all toxicities.
4. For statistical reasons, rare adverse
effects are unlikely to be detected in
preclinical testing.
1/29/2015 65
Alternatives to animals:
1/29/2015 66
Micro-organisms
Cell components
Cell cultures
Tissue cultures
Tissues slices
Isolated & perfused organs
Computer modelling
1. In silico ADMET
2. Physiologically Based PK simulations
5 R’s1. Reduce the no. of animals used
to a minimum.
2. Refine the way that experiments are carried out so that the effect on the animal is minimized and animal welfare is improved.
3. Replace animal experiments with alternative (non-animal) techniques wherever possible.
4. Rehabilitation When death is not the end point.
5. Reuse Whenever and wherever possible.
1/29/2015 67
What after preclinical phase??
Once the preclinical trials are over, sponsors are required to submit the “Investigational New Drug” application.
It contains information regarding:
1. Preclinical data: PK, PD & Toxicological
2. Manufacturing data: Composition, Manufacturing process, Stability & Shelf life.
3. Protocol of clinical trials1/29/2015 68
In Silico drug design or CADD
1/29/2015 69
Target Ident.
Target Validation
Lead Ident.
Lead OptPreclinical
ToxClinical trials
Bioinformatics
Inverse docking
Protein pred.
Target
druggabilty
Tool compound
design
Lib. Design
Docking scoring
De novo design
QSAR
3 D- QSAR
Struc. based
opt.
In silico ADMET
PBPK
simulations
No options
Drug Discovery: Past & Present
Criterion Last century Present era
Drug
development
Predominantly
compound centred
Predominantly
target centred
Source of Leads Natural
Compounds
Natural +
Synthetic
compounds
Time taken Many years Few years
First step Finding
appropriate
biological
response
Finding
appropriate drug
target
Safety Lesser safe More safe
Screening Animal disease
models
Virtual Screening/
HTS1/29/2015 70
Criterion Last century Present era
Animal use Frequent Relatively less
Ethical
Considerations
Less stringent More Stringent
No. of
Compounds
explored
Less More
No. of Targets
explored
Less More
Change in term Drug discovery Drug Invention
1/29/2015 71
Drug Discovery: Past & Present
Future Prospects
1/29/2015 72
Summary:
1/29/2015 73
1/29/2015 74
Target
identification
Target
validation Lead finding
OLDER TECHNIQUESNEWER TECHNIQUES
Lead
Optimisation
Preclinical
Development
Clinical
Development
VS
HTS
HITSLEAD COMPOUND
Thank you for your patience!
1/29/2015 75
