Drug discovery and Devlp- Durgashree Diwakar

BY- DURGASHREE.M.D
M.PHARM, PHARMACOGNOSY
KLE COLLEGE OF PHARMACY
BENGALURU
1

Introduction
History of Herbs as Source of Drugs and Drug
Discovery
The lead structure selection process
Structure development
Product Discovery Process.
Drug registration.
Selection and Optimization of the lead compound.
Phases of a clinical trial.
Protocol design of lead molecule.
2

 Drug discovery is the process through which potential
new medicines are identified.
 It involves a wide range of scientific disciplines,
including biology, chemistry and pharmacology. [3]
3

 Natural products and their structural
analogues have historically made a
major contribution to pharmacotherapy,
especially for cancer and infectious
diseases.
 Natural products also have challenges
for drug discovery, such as technical
barriers to screening, isolation,
characterization and optimization. [4]
4

 Collection, identification, and authentication of the plant.
 Extraction of non-polar to polar solvent and preparation of
extract for phytochemical analysis.
 Evaluation of plant extract by the different biological test
methods.
 Chromatographic analysis by activity–guided fractionation
of the extract.
 Structure elucidation using spectroscopic techniques.
 Testing of each bio active compound in all invitro and in-
vivo phytopharmacological test methods.
 Clinical trials (Phase I-III). [5]
5

 A drug is a chemical substance, typically of known
structure, which is administered to a living organism,
produces a biological effect.
 A pharmaceutical drug, also called a medication or
medicine, is a chemical substance used to treat, cure,
prevent, or diagnose a disease or to promote well-being.
7

 Sources of drugs may be natural, synthetic, and
biosynthetic.
 Drugs of plant, animal, microbiological, marine, mineral,
geographical origins constitute the natural sources. [1]
8

 Historically, natural products have been used since ancient
times and in folklore for the treatment of many diseases
and illnesses.
 Natural products (secondary metabolites) have been the
most successful source of potential drug leads.
 Natural products provide unique structural diversity.
9

 The Ebers Papyrus (2900 B.C.) is an Egyptian
pharmaceutical record, which documents over
700 plant-based drugs ranging from gargles,
pills, infusions, to ointments.
 The Chinese Materia Medica (1100 B.C.) (Wu
Shi Er Bing Fang, contains 52 prescriptions),
 Shennong Herbal (~100 B.C., 365 drugs) and
 The Tang Herbal (659 A.D., 850 drugs)
 Are documented records of the uses of natural
products .
10

 The Greek physician, Dioscorides,
(100 A.D.), recorded the
collection, storage and the uses of
medicinal herbs.
 The Greek philosopher and
natural scientist, Theophrastus
(~300 B.C.) dealt with medicinal
herbs.
11

 The dominant source of knowledge of natural product uses
from medicinal plants is a result of man experimenting by
trial and error for hundreds of centuries.
 For Example:
 The plant, Alhagi maurorum Medik (Camels thorn)
secretes a sweet, gummy material from the stems and
leaves during hot days.
 This gummy sap is called “manna”.
12

 It has been documented and claimed by the Ayurvedic
people as useful in the treatment of
-anorexia,
-constipation,
-dermatosis,
-fever,
-leprosy, and obesity.
 It was also used by the Israelis who boiled the roots and
drank the extract as it stopped bloody diarrhea.
 The Konkani people smoked the plant for the treatment of
asthma.
 The Romans used the plant for nasal polyps.
13

 Traditional medicinal practices have
formed the basis of most of the
medicines.
 Like the synthesis of acetyl salicyclic
acid (aspirin) derived from the
natural product, salicin isolated from
the bark of the willow tree Salix
alba L.
 Several alkaloids including Morphine,
isolated from Papaver somniferum
(opium poppy), which is used as pain
killer, and so on..
14

 Macro and Micro Fungi is a part of
human life for thousands of years.
 They were used as food (mushrooms),
in preparation of alcoholic beverages
(yeast), medication for cultural
purposes.
 Natural Products discovered from
fungus is Penicillin from fungus
Penicillium notatum, discovered by
Fleming in 1929.
15

 Plants have proven to be novel source of bioactive natural
products .
 Example: Paclitaxel (Taxol®), isolated from the bark
of Taxus brevifolia (Pacific Yew).
 The marine environment has a clear track record in
offering novel structural entities.
 Exploration of the marine environment and organisms
(algae, sponges, ascidians, tunicates and bryozoans)
became possible due to modern technology,
-SCUBA (1970s)
-manned submersibles (1980s)
-remotely operated vehicles (ROVs)
(1990s).
16

 These progressive advancements in the past 40 years of
exploration of the marine environment have resulted in the
isolation of thousands of structurally unique bioactive
marine natural products.
 Algae (macroalgae, seaweed) are represented by at least
30,000 species worldwide supplying oxygen to the
biosphere, food for fish and man, medicine.
 It is prolific source of structurally unique natural products.
 The brown alga, Dictyota dichotoma afforded diterpenes,
4-acetoxydictylolactone , and dictyolides A , B which
display antitumor activities. [2]
17

YEAR DRUG INDICATION DISCOVERED- COMPANY
1826 Morphine Analgesic Friedrich
Serturner
E. Merck
1899 Acetyl salicylic acid Analgesic Felix Hoffmen Bayer
1941 Penicillin Antibacterial Merck
1983 Cyclosporin A Immunosuppress
ant
Sandoz
1987 Artemisinin Antimalarial Tu Youyou Baiyunshan
1987 Lovastatin Anti-
hyperlipidemic
Merck
1990 Acarbose Anti-diabetic
(type 2)
Bayer
1993 Paclitaxel Anticancer BMS
18
[5]

 Lead selection refers to the process by which the early hits
are interrogated in a vigorous, multi-step screening process
to select lead molecules that meet pre-established criteria
for progressing to the next stage.
 Lead Structure: a structure that has some activity against
the chosen target, but is not yet good enough to be the
drug itself.
19

 Drug development is the process of bringing a new
pharmaceutical drug to the market once a lead compound
has been identified through the process of drug discovery.
 Drug discovery is the process by which new candidate
medications are discovered.
 The lead compound is the compound under discovery
stages.
 It is the chemical compound that occurs prior to preclinical
and clinical development and has pharmacological or
biological activity likely to be therapeutically useful.
20

 Organic compounds are identified which interact with the
target protein and modulate its activity by using random
(screening) or rational (design) approaches.
 Natural product and synthetic compound libraries with
millions of compounds are screened using a test assay.
21

 Random screening: all compounds including synthetic
chemicals, natural products of plant, marine, and
microbial origin from a given series are tested.
 Non-random method: In this method, only such
compounds having similar structural skeletons with that of
lead are tested.
 Molecules are chemically modified and subsequently
characterized in order to obtain compounds with suitable
properties to become a drug.
 Once compounds with desirable in vitro profiles have been
identified, these are characterized using in vivo models. [6]
22

 Drug discovery
process is of 4 steps.
24

1.Target Identification
 Target identification and characterization begin with
identifying the function of a possible therapeutic target
and its role in the disease.
 A good target should be efficacious, safe, meet clinical, and
commercial requirements.
 A recent analysis shows that current drug therapy is based
on less than 500 molecular targets.
 This includes 45% of GPCR, 28% enzymes, 11% hormones,
and factors, 5% ion channels, and 2% nuclear receptors.
 Thus, the number of drug targets that can be exploited in
the future is at least 10 times the number of targets.
25

 Besides classical methods of cellular and molecular
biology, new techniques have becoming more important.
 These methods aim to-
- discovering new genes and protein
- Quantifying and analyzing gene and protein
expression between diseased and normal cells.
 Method frequently used are genomics and proteomics.
26

Genomics:
 The term genomics was coined in the mid-1980s.
 This new discipline has evolved from two independent
advances.
i) Automation: Resulting in a significant increase in the
number of experiments that could be conducted in the
given time and thereby generating a vast amount of
scientific data.
ii) Informatics: The ability to transform raw data into
meaningful information and knowledge by applying
computerized techniques.
Without Informatics data would remain raw material.
28

 Clearly the identification of new and clinically relevant
biological targets has benefited from the genomic
approach.
 The Huge genomic initiatives which started in the early
1990s have led to an enormous amount of DNA sequence
information.
29

Proteomics:
 Target Identification with proteomics is performed by
comparing the protein expression levels in normal and
diseased tissues.
 Two-dimensional polyacrylamide gel electrophoresis (2D-
PAGE) is used to separate the proteins.
 Differential protein expression between normal and
diseased samples probably indicates the protein of interest,
which is involved in the pathophysiology and could
therefore be a potential drug target.
 This assumption has to be confirmed during the target
validation process.
30

2. Target validation
 The validation of a drug target involves
demonstrating the relevance of the target
protein in a disease process and ideally
requires both gain and loss of function
studies.
 This is done primarily with highly specialized
knock-out and knock-in animal models that
are capable of mimicking the target disease
state.
 The development of in-vivo models is always
costly and time-consuming.
31

 Methods used for target identification at the invitro level
are RNA and protein expression analysis and cell based
assays.
 Target validation is one of the bottlenecks in the drug
discovery process, since this phase is less adaptable to
automation.
 The careful validation of target proteins, not only with
respect to disease relevance, but also “druggability”, will
reduce the failure rate and reduce the overall efficiency of
the drug discovery process.
32

3. Lead identification
 In the lead generation phase, compounds are identified
which interact with the target protein and modulate its
activity.
 Such compounds are mainly identified using random
(screening) or rational (design) approaches.
33

 High-throughput screening is used to test large number of
compounds for their ability to affect the activity of target
proteins.
 Today entire in-house compound libraries with millions of
compounds can be screened with a throughput of 10,000 to
100,000 compounds per day.
34

 Combinatorial chemistry and parallel synthesis are
employed to generate such huge number of compounds.
35

 A different approach in lead identification is in-silico or
virtual screening.
 With this computer method, the 3D structure of
compounds from virtual or physical libraries are docked
into binding sites of the target protein with predicted
structure.
36

 Another screening approach, namely NMR-based
screening fills the gap between HTS and virtual screening.
 This method combines the random screening approach
with the rational screening-based approach to lead
discovery.
 Small organic molecule that binds to proximal subsites of
protein are identified through screening and linked
together in a rational approach, to produce high-affinity
ligand.
37

4. Lead optimization
 In this phase, small organic molecules are chemically
modified and pharmacologically characterized to obtain
compounds with suitable pharm. dynamic and kinetic
properties to become a drug.
 Lead optimization is mainly a cross-talk between the
traditional disciplines in drug discovery: medicinal
chemistry and in vitro/ in vivo pharmacology.
38

 Leads are characterized with respect to
pharmacodynamics, physiochemical, pharmacokinetic
properties, and toxicological aspects.
 In parallel to compound characterization with respect to
potency and selectivity, in vitro assays for the prediction of
pharmacokinetic properties should be performed.
Examples of in vitro systems: Caco-2 or Madin-Dardy
canine kidney cells, human hepatic microsomes, etc…
39

 Once compounds with desirable in vitro profiles have been
identified, these are characterized using in vivo models.
 These tests usually require larger quantities of compounds
and are conducted with selected molecules only.
40

 Ideas on how to modify a lead compound with respect to
pharmacodynamic properties can originate from molecular
modeling, quantitative structure-activity relationship
(QSAR)-studies, and structural biology.
 These rational approaches are valuable tools in the lead
optimization phase since they connect biology with
chemistry and allow a thorough understanding of the
relationship between chemical structure and biological
function.
41

 Ideas on how to modify compounds with respect to
physicochemical and pharmacokinetic properties, in silico
methods play an important role probably the most widely
used ADME- model is Lipinski’s rule-five.
 It is vital to conceive lead optimization as a simultaneous
multi-dimensional optimization process rather than a
sequential one. [5]
42

1. ARTEMESIN
 People are at risk from Malaria.
 Therapies now include the use of anti-malarial compounds
derived from Artemisia annua, a member of Asteraceae.
44

 The medicinal properties of this plant have been part of
traditional Chinese medicine for at least 1000 years.
 The WHO now recommends the use of artemisinin-based
combination therapy (ACT) in the regions where the
tropical malaria parasite has developed multi-drug
resistant to the more common anti malarial drug.
45

 Artemisia annua plant is the only source of
antimalarial drug artemisinin.
 Biological sources are leaves, flower heads of Artemisia
annua.
 The active constituents are artemisinin, dihydroartemisinin,
Artemisin, Artemisic acid.
 It is effective against malaria and cerebral malaria.
 Chemically artemisinin is a sesquiterpene lactone
containing an unusual peroxide bridge.
 For extraction 2 methods are followed: Soxhlet, Microwave-
assisted extraction.
46

 Plant material was dried mashed into powder.
 The solvent methanol, hexane, and ethyl acetate are used.
 For extraction 250g of powder herb macerated using
methanol as a solvent, Magnetic stirrer 700rpm for one
hour.
 This process is continued till colorless.
 The extract was evaporated 100ml.
 Partition the extract using 50ml hexane.
 Partitioning is done many times until the hexane layer
becomes colorless.
47

 Two layers got from this process:
1. Hexane Extract (non-polar fraction).
2. Methanol Extract (polar fraction)
 Each extract was concentrated using a rotavapour at a
temperature of 40 degrees Celsius.
48

 Estimation by TLC method:
 Mobile phase- Ethyl acetate: Hexane (3:97).
 Stationary phase- 60F254 Silica gel
 Detecting reagent- Anisaldehyde- sulphuric acid
reagent.
 Spot volume- 10 microliters of test and standard
sample.
 Spot color- pink color at room temperature 25+or -2
degree Celsius, Relative humidity- 45+or-2%.
49

 HPLC method -
 Column- C18
 Detector- UV 260nm
 Mobile phase- Phosphate buffer: Methanol (6:4)
[pH 7.9]
 Flow rate 1 ml/min.
 Caliberation curve was plotted with different
concentrations.
50

2. ANDROGRAPHOLIDE
 Andrographis paniculata or Kalmegh is one of the most
widely used plants in ayurvedic formulations.
 It is used in activities like anti-malarial activity, anticancer
activity, anti-diabetic activity, Liver protection.
51

 Extraction: Plant material was dried under shade,
powdered using a blender, and stored in airtight bottles.
 Whole dried crushed Andrographis paniculata was
extracted, twice with a 1:1 mixture of dichloromethane and
methanol by cold maceration.
 Time period of extraction is 8 hours.
 Filter the extract and concentrate.
 The filtrate was purified and cold crystallization is carried
out.
52

 Identification Test: To confirm that the above-obtained
diterpene lactones obtained from Andrographis
paniculatais Andrographolide the above filtrate was
evaporated under reduced pressure until fully dried.
 About 5mg of filtrate powder was dissoloved in 5ml of
warm ethanol and 1mg of standard Andrographolide was
dissolved in 0.5 ml of warm ethanol separately.
53

 A pre coated plate of silica gel 60 F254 aluminum sheets
(10*10 cm) was used and
 The mobile phase used was- Chloroform: Methanol: Ethyl
acetate (8:1.5:1).
 5 microliters were used for each spot.
 TLC of the isolated samples was firstly detected by UV
radiation and then confirmed by spraying with 2% wv
solution of 3,5-dinotrobenzoic acid in ethanol and an
excess of 5.7% wv of potassium hydroxide in ethanol.
 Results completely match with standard Andrographolide
which clearly indicates that obtained diterpene lactones is
Andrographolide.
54

 HPTLC and HPLC both are very effective methods and can
be used for phytochemical profiling of whole plant, leaves,
and other parts of Andrographis paniculata and
quantification of Andrographolide.
55

 UV Study:
 The Concentration of purified Andrographolide and
standard Andrographolide concentration 25microgram/ml
in ethanol.
 The maximum wavelength was found to be 223 nm, which
matches with standard value of Andrographolide. [6]
56

 Clinical Trials are experiments and observations done in
clinical research.
 These are scientific studies conducted to find better ways
to prevent screens for diagnosing or treating disease.
 These clinical trials also show which medical approach
work better for a certain group of peoples having identified
illness condition.
 These studies follow strict scientific standards which
protect patients and produce reliable clinical trial results.
57

 Clinical Trial may be defined as any investigation in
human subjects intended to discover or verify the
clinical pharmacological and other pharmacodynamic
effects of an investigational product
 And to identify any adverse reaction and to study
absorption, distribution, metabolism, and excretion of
an investigational product.
58

1. Clinical Phase zero:
 These studies aim to find out if a drug behaves in the way
researchers expect it.
 It involves a small number of people 10 to 20 and they have
a small number of doses.
 Testing a low dose of the treatment to check it isn’t
harmful. It is not randomized.
 Phase 0 study is a micro-dosing study.
59

 Micro-dose is less than 1/100 of the dose of a test
substance calculated to produce a pharmacological
effect with a max dose less than or equal to 100
micrograms.
 In clinical phase 0 there are fewer chances of adverse
effect, short duration of the study, less number of
volunteers only 10 to 15 are required.
 It reduces the cost of research.
60

2. Clinical Phase 1:
 It is the first stage of testing in human volunteers.
 It is designed to access the safety, tolerability, pK of drugs.
 20 to 50 healthy volunteers are taking part. The duration of
the study is from 6 months to 12 months.
 The aim of the phase 1 trial is to determine the maximum
tolerated dose of the drug under investigation.
61

 Phase 1 study includes Single ascending dose studies and
Multiple ascending dose studies.
 Investigation of difference in absorption caused by food.
 Finding of Clinical Phase 1 is about side effects, what
happens to the treatment in the body. It is not randomized.
 The first few patients are given a very small dose under
investigation, if the results are satisfactory then the dose is
gradually increased with each group. The researchers
monitor the side effects.
62

63
 It is Therapeutic Explanatory Trial.
 20 to 300 healthy volunteers are taking part.
 Phase II confirm the effectiveness, monitor side effect,
and further evaluate safety.
 Phase II trials are usually larger than phase I.
 These trials find out the side effects and look at how
well the treatment works. Sometimes it is randomized.

 Randomized trial means researchers put the people taking
part into treatment groups at random.
 Duration of phase II is from 6 months to several years.
 Phase II study types are
-Phase IIA: designed to access dosing requirements.
It is a single-blind comparison with standard drug
-Phase IIB: designed to study efficacy.
It is double-blind compared with placebo or standard
drug.
64

 It is a Therapeutic Confirmatory trial.
 20 and 300 healthy volunteers are taking part because
differences in success rates may be small so the trial
needs many patients to be able to show the difference.
 Duration of Phase III is up to 5 years.
 These trials compare a new treatment with the best
currently available treatment.
 Phase III trials find out which treatment works better
for the particular type of disease.
65

 It involves a detailed study of side effects.
 The trials are randomized.
 The subtypes of Phase III are
Phase IIIA: For generation of sufficient and significant
data.
Phase IIIB: Allows patient to continue the treatment, label
expansion, additional safety data.
 Phase III is the end of clinical trial activities.
66

 Clinical phase 4 is the Post Marketing Surveillance.
 It did not include fixed duration and number of patients.
 Clinical Phase IV is the trials done after the drug has been
shown to work and has been licensed.
 It aims to find out more about the side effects and safety of
drugs. What are the risk and long-term benefits drugs
have?
67

 Helps to detect rare ADRs, Drug interactions and also to
explore new uses for drugs.
 Clinical Phase 4 reports to be submitted by the
manufacturer every 6 months for 2 years and then annually
for the next 2 years marketing approval.
 The ADR can be reported to a formal reporting system.
 Harmful effects discovered may result in the withdrawal of
the drug from the market.
 Clinical Phase IV confirms the efficiency and safety profile
in large populations during drug practice. [6]
68

1. Drugs: Their Natural , Synthetic, and Biosynthetic sources – Springer
link
2. A Historical Overview of Natural Products in Drug Discovery.
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901206/)
3. Drug Discovery (https://www.nature.com/subjects/drug-discovery)
4. Natural products in drug discovery: advances and opportunities.
(https://www.nature.com/articles/s41573-020-00114-z)
5. Modern methods of drug discovery- Alexander Hillisch and Rolf
Hilgenfeld.
6. A Textbook of Phyto Chemistry- By Rageeb, Namitha, Tanvir and
Bharat.
70

Drug discovery and Devlp- Durgashree Diwakar

Recomendados

Recomendados

Mais conteúdo relacionado

Mais procurados

Mais procurados (20)

Semelhante a Drug discovery and Devlp- Durgashree Diwakar

Semelhante a Drug discovery and Devlp- Durgashree Diwakar (20)

Mais de Durgashree Diwakar

Mais de Durgashree Diwakar (8)

Último

Último (20)

Drug discovery and Devlp- Durgashree Diwakar