PROCESS OF

DRUG DEVELOPMENT
 OBJECTIVES & OUTCOMES

Objectives:
 Describe general steps in drug discovery and development

Outcomes: At the end of this lesson, students are able:

 To present general steps in drug discovery and development
 INTRODUCTION

 Drug discovery and development - the past

 Before the twentieth century
• Herbs and potions
• Isolate, purify and determine the structure of the active principles.
 After the twentieth century
+ Trial and error
+ Synthesize numerous analogs in an attempt to improve the
pharmacological profile of the natural products (lead compound)
+ Overall pattern for drug discovery and drug development evolved
(rarely understood mechanism of action)
 Drug discovery and development - the present

Target oriented

+ An understanding of the structure and mechanism of the target

is crucial to drug discovery and development
+ Rapid advances in the biological sciences have resulted in a
much better understanding of how the body functions at the
cellular and molecular levels
- It takes average 15 years to be approved as a marketable drug and
costs > $800 million
 Drug discovery and development process

- Choose a disease
- Choose a drug target
- Identify a bioassay
- Find a lead compound
- Identify structure-activity relationships
- Optimize the lead compound
- Patent the drug candidates
- Complete the preclinical and clinical studies
 CONTENTS

PART 1. DRUG DISCOVERY: PART 2. GETTING THE DRUG TO

FINDING A LEAD THE MARKET

1. Choosing a Disease 1. Preclinical and clinical trials

2. Choosing a Drug Target 2. Patenting and regulatory affairs
3. Identifying a Bioassay 3. Chemical and process development
4. Finding a Lead Compound
5. Isolation and Purification
6. Structure Determination
 PART 1.
DRUG DISCOVERY: FINDING A LEAD
 Finding a
Choosing a Choosing a Identifying a Isolation and Structure
Lead
Disease Drug Target Bioassay Purification Determination
Compound
 1

Finding a
Choosing a Choosing a Identifying a Isolation and Structure
Lead
Disease Drug Target Bioassay Purification Determination
Compound
 1. Choosing a Disease
• Criteria
– Consider economic and medical factors
– Need for new and/or improved drugs
– The first drug for a disease in the world
Sildenafil, simvastatin, cyclosporin, etc.

• Great attention diseases:

Cancer, cardiovascular, diabetes, obesity, depression, migraine, etc.
 2

Finding a
Choosing a Choosing a Identifying a Isolation and Structure
Lead
Disease Drug Target Bioassay Purification Determination
Compound
 2. Choosing a Drug Target
2.1. Drug targets
An understanding of which
biomacromolecules are involved in a
particular disease state is clearly important.

 Possible drug targets:

Receptor, enzyme, etc. which is involved in a particular disease
  Example:

- Serotonin receptor agonists for

migraine
- Dopamine receptor antagonists as
antidepressants

- Angiotensin converting enzyme

inhibitors for hypertension
2.2. Discovering a Drug Target

 In the past, the discovery of drug targets depended on finding the drug
first.
Use to find molecular target after discovering a drug which was usually natural
product (ex: morphine: analgesic agent derived from plants )

Analgesic effect

The detection of drug targets was very much a hit and miss affair.
 Finding body’s own chemical messenger (natural ligand) led to the
discovery of molecular target.

Ex. A variety of peptides and proteins have been discovered which act as the
body's own analgesics (enkephalins and endorphins).

Enkephalin
 Mapping human genome
– Completion of human genome project revealed a huge number of
new receptors and enzymes which are potential drug targets
• Function and natural ligands for most of these receptors and
enzymes are not known
 How to find function of these receptors and enzymes?
• Transgenic animal

 How to find ligands for these receptors or enzymes?

• Random search (combinatorial chemistry)
• Computer-aided molecular modeling
 2.3. Target specificity between species

- The more selective a drug is for its target, the less chance there is that
it will interact with different targets and have undesirable side effects.

• The best targets to choose are those that are unique to the microbe and
are not present in humans.

Ex. Penicillin (Antibiotics ) has few side-effects.

(it targets transpeptidase which mammalian cells
do not have).
• If the enzymes are present in both microbe and human: need to
be different enough for selectivity.

Ex: - Antifungal agents (Fluconazole)

Inhibits a fungal demethylase which is

significantly different from the mammalian
enzyme
2.4. Target specificity and selectivity within the body
– Enzyme isozymes
H3C
COOH

O CH3

O N
N CF3
H2NSO2

Aspirin for COX (1 and 2)

Celecoxib for COX-2
- Receptor subtypes

Antihistaminic agents for H1 Antiulcer agents for H2

2.5. Targeting drugs to specific organs and tissues
 β-Adrenergic receptors

(Noradrenaline) (Salbutamol)

Heart: β1 - Adrenergic receptor Lung: β2 - Adrenergic receptor

 Dopamine receptors
Parkinson’s disease: Dopamine transmission is deficient in certain regions
of the brain but elsewhere in the body functions normally.
⇛ Brain specific dopamine agonist for Parkinson’s disease
 Pitfalls

• Human body is a highly sophisticated system

– Generally many chemical messengers, receptors, and enzymes
are involved for any given function.
Ex:
– Blood pressure control
• Angiotensin converting enzyme, angiotensin II receptor, β1-
adrenoreceptors, calcium ion channels, potassium ion
channels, etc.
– Therapy for asthma
• Bronchodilator (β2-agonist) and anti-inflammatory agent (corticosteroid)
 3

Finding a
Choosing a Choosing a Identifying a Isolation and Structure
Lead
Disease Drug Target Bioassay Purification Determination
Compound
3. Identifying a Bioassay

3.1.Choice of bioassay
– Choosing the right bioassay or test system is crucial to the success of a
drug development
– Requirements for the test system:
• Relevant to the disease
• Simple
• Fast

-Can test:
• In vitro (i.e. on isolated cells, tissues, enzymes or receptors).
• In vivo (on animal)
3.2. In Vitro Tests
• No live animal is involved: cells or enzymes
 Enzyme inhibitors: tested on the pure enzyme in solution.
 Can determine an enzyme inhibitor:
+ competitive
+ or non-competitive
+ IC50 values (half maximal inhibitory concentration).
 Receptor agonists and antagonists: tested on isolated tissues or cells
which express the target receptor on their surface.

- Tissues can be used to test drugs for physiological effects:

Bronchodilator activity can be tested by observing how well
compounds inhibit contraction of isolated tracheal smooth muscle.
- Antibacterial drugs are tested
in vitro by measuring how
effectively they inhibit or kill
bacterial cells in culture.
3.3. In Vivo Tests

• Animal testing
– Test the activity of a drug candidates against the animal which has
observable symptoms
• Usually observable symptoms are induced

• Transgenic animals
Animals with altered genetic code
Transgenic mouse can have human receptor or
enzyme
Alter the mouse gene so the mouse became
susceptible to a particular disease
• Problems associated with animal testing:
– Slow and causes animal suffering
– Problems of pharmacokinetics
– Induced observable symptoms might be caused by a different physiological
mechanism than the one intended
– Different results may be obtained in different animal species
 Ex: Penicillin methyl ester prodrugs:

In the mouse or rat In rabbit, dog or man

hydrolyzed ⇛ active penicillins not hydrolyzed

 4

Finding a
Choosing a Choosing a Identifying a Isolation and Structure
Lead
Disease Drug Target Bioassay Purification Determination
Compound
4. Finding a Lead Compound
• Lead compound
– Compound with the desired pharmacological effects
• May have weak activity with undesirable side-effects
– Provides a start for the drug development process.
 Methods of finding lead compounds

– Screening of natural products

– Medical folklore
– Screening of synthetic small molecular weight compounds
– Existing drugs
– Starting from the natural ligand or modulator
– Combinatorial synthesis
– Computer-aided design
– Serendipity and the prepared mind
– Computerized searching of structural databanks
– Designing lead compounds by NMR
 4.1.Screening of Natural Products
• Natural products
– Provided biologically active compounds for centuries
– Most of the biologically active natural products are secondary metabolites
– Often they have extremely novel chemical structures
• Difficult to produce large quantities

H3C
CH3
H
O O OH
O O
H3C
H3C
O
O NH O CH3 O
CH3
H3C
H H
O
H
O O
O O O CH3
OH OH
O O
H3C

Taxol Artemisinin
 Plant Kingdom

Animal Sources Microbiological World

Marine World
4.1.1.The Plant Kingdom

– Plants provide a bank of rich, complex and highly varied structure which
are unlikely to be synthesized in laboratories
• Useful drugs by themselves
• Served as basis for synthetic drugs
– The vast majority of plants have not been studied
– Conserve the rainforest, plants and trees
• Products from plants and trees
H
HO H2C
CH3
O H
N HO N
O H
H
OCH3
NCH3
H3CO
O
HO
Morphine
N
O
Cocaine Quinine
 Nicotiana

Amanita muscaria

O
H 3C CH3 N
N CH3
CH3
CH3 N
HO
Muscarine

Nicotine
 O
H3C
O

O O OH
CH3 O
H3C
CH3 H OH
O NH O CH3 CH3
H3C
H OH
CH3
O O
O H
O H O
OH OH O O
OCH3
CH2OH
O O
O Digitalin H3C
OH Taxol
OH
OH
OH

Taxus brevifolia
4.1.2. The Microbiological World
• Antimicrobial agents
– Started after the discovery of penicillin
NH2 O
O H H
H H S
S CH3 HOOC N
N H
H
N N O CH3
CH3
O O
COOH
Penicillin G COOH O
Cephalosporin C

Penicillium

Acremonium
 Streptomyces Streptomyces venezuelae

H 3C CH3
HO CH3 N
H
OH

CONH2
OH
OH O OH O

Tetracycline OH

OH

HN CHCl2
O2N

O
Chloramphenicol
 4.1.3. The Marine World

• Biologically interesting compounds from marine sponges and corals

H H

H3C Curacin A
N OCH3
S
CH2
CH3
HO
H3C
CH2
Brevetoxin B H O O
OHC
H H
CH3 CH3 H O
CH3
H CH3 H H O O H
O O
H
O O
O O O H H H CH3
H H H CH3
 4.1.4. Animal Sources

• From frog skin

– Epibatidine
• Analgesic

Cl H 3C

H NH
N N H 3C O

HO O CH3
CH3 CH3
Epibatidine N

O O

HO
H Batrachotoxin
 4.1.5. Venoms and Toxins

• Tools to study receptors, ion channels and enzymes or serve as lead

compounds due to specific binding
– a-Bungarotoxin from cobra
– Mol wt.: 8000, 74AA CH3
• Teprotide from brazilian viper HS N
– E-W-P-R-P-Q-I-P-P
O COOH
– Led to captopril
Captopril
 4.2. Medical Folklore CH3
N

• Rhubarb root: laxative (danthron)

• Artemisinin: antimalaria CH2OH
• Opium puppy: morphine O
Atropine
• Solanaceae: atropine O
• Snakeroot plant: reserpine

OH O OH
N
H3CO N
H H H

O
H
H3CO OCH3
O

O O OCH3
OCH3
Danthron Reserpine OCH3
 4.3. Screening Synthetic Banks

• Statistically only one compound out of 5000 – 10000 compounds make to

the pharmaceutical market
– Hugh number of synthetic compounds are left behind
– Limited usefulness due to lack of diversity

O NH2
O NHNH2

N
N
Isoniazid
Quinoline-3-carboxamides
F
 4.4. Existing Drugs
4.4.1. ‘Me too’ and “me better” drugs

– Use competitor’s drug as a lead compound and improve therapeutic

properties ⇛ avoids patent restrictions, retains activity, and ideally has
improved therapeutic properties.

CH3
Cilazapril
HS N Enalapril

CH3 N
O COOH
N N
Captopril EtO2C N EtO2C N
H H
CO2H O
O COOH

(Merck) (Hoffmann-La Roche)

Captopril and 'me too' drugs.

4.4.2. Enhancing a side-effect
– From antibiotics to antidiabetics and diuretics
O
O O
S
N N CH3
(antidiabetic)
H H

H 3C
Tolbutamide
O O
S
NH2 O O O O
S S
H 2N NH
H 2N
Sulfanilamide Cl N (diuretic)
(antibiotics)
Chlorothiazide
 4.5. Starting From Natural Ligand

• Natural ligands for receptors

– Agonists
• Adrenergic β-agonists from adrenaline and noradrenaline
OH
OH
H H
N CH3 HO N
HO
CH3 CH3
CH3 HO
HO Dobutamine
Salbutamol
 – Antagonists
• Histamine receptor (H2) antagonist from histamine
CH3
NH2 H H
HN N N
HN S CH3
N
N N
Histamine CN
Cimetidine

• Adrenergic receptor antagonist from epinephrine

OH
H OH
HO N H
CH3 N CH3

HO CH3
Epinephrine
Pronethalol
4.6. Combinatorial synthesis
Automated synthesis of large number of compounds with different
structures in as short time span as possible.
4.7. Computer-aided design
Design drugs using computer software
 4.8. Serendipity and the prepared mind
O Cl
 Mustard-like drugs for leukemia after N
HN Cl
mustard gas explosion
O N
H
Uracil mustard

ONO2
 Nitroglycerin for angina pectoris after ONO2
discovering dilatation effect of TNT ONO2

Nitroglycerin

 Disulfiram for chronic alcoholism after CH3

S

discovering that antioxidant prevent H C

3 N S
S N CH3

oxidation of acetaldehyde H 3C
S

Disulfiram
 4.9. Computerized search

• Computerized searching of structural databanks

– Database mining
– Possible only if pharmacophore of the drug is known
– Requires large structural database
 4.10. Design by Using NMR

• Design instead of discover a lead compound by using NMR spectroscopy

– Require >200 mg of 15N labeled target protein with molecular weight of
<40,000
 5

Finding a
Choosing a Choosing a Identifying a Isolation and Structure
Lead
Disease Drug Target Bioassay Purification Determination
Compound
 5. Isolation and Purification
-The lead compound (or active principle) :
+ is present in a mixture of other compounds
+ obtained from a natural source
+ source or from a combinatorial synthesis

Need to be isolated and purified.

Method depends on:
+ structure
+ stability
+ quantity of the compound.

• New techniques are needed for

unstable compounds
– Freeze drying for penicillin
• Diverse chromatographic separation
techniques are frequently used
 6

Finding a
Choosing a Choosing a Identifying a Isolation and Structure
Lead
Disease Drug Target Bioassay Purification Determination
Compound
 6. Structure Determination

• X-ray crystallography

• NMR spectroscopy
• Mass spectroscopy
• Total synthesis
7. Herbal Medicine
 Advantages
– Some plant extracts have a wide variety of different active principles
which act together to produce a beneficial effect:
• Aloe preparations are still used today to treat burns, irritable bowel
syndrome, rheumatoid arthritis, and asthma, etc.
• Aloe preparation contain analgesic, anti-inflammatory, antimicrobial,
and many other agent which all contribute to the overall effect, and
trying to isolate each active principle would detract from this.
 PART 2
GETTING THE DRUG TO THE MARKET
Preclinical and clinical trials

Patenting and regulatory affairs

Chemical and process development

1. Preclinical and clinical trials
1.1. Toxicity testing

1.2. Drug metabolism studies

1.3. Pharmacology, formulation, and stability tests
1.4. Clinical trials

1.5. Ethical Issues

 1.1.Toxicity Testing

• Animals are needed

– It is impossible to predict whether a potential drug will be safe by
in vitro tests alone
– Usually measured by its LD50
• Lethal dose required to kill 50% of a group of animals

• Therapeutic ratio or therapeutic index.

The ratio of LD50 to ED50 (the dose required to produce the desired
effect in 50% of test animals)
Comparison of therapeutic and lethal dose curves of a sedative drug
- Different types of toxicity test
Acute toxicity test
Sub-acute toxicity test
Chronic toxicity test
 Acute Toxicity Study

• Designed to determine the short-term effects of a drug when

administered in a single dose or in multiple doses within 24 hours
or less
• Exaggerated doses by two different routes of administration
• Observed for at least one week after the initial dosing mainly
central nervous system (CNS), and cardiovascular system (CVS)
toxicity
• Potential overdose situation
 Sub-acute Toxicity Study

• Three dose levels, doses are therapeutic, medium, expected to

cause some toxicity but are not lethal
• Takes one to six months
• Two species (rodent and a non-rodent, e.g. rat or mouse and
dog)
• Check for the signs of toxicity
• Regularly examine blood and urine samples
• Histopathologic examination
 Chronic Toxicity Study

Long-term toxicology tests are carried out over a period of years at lower
dose levels to test the drug for chronic toxic effects, carcinogenicity,
special toxicology, mutagenicity, and reproduction abnormalities.

• One year duration for intended to be taken orally on a chronic

basis, either regularly or as needed
• Study protocol is same as sub-acute study except duration
 More Toxicity Studies

• Carcinogenicity studies
– Check for cancer causing effect
– Rat or mice for two years
– Postmortem examination
• Mutagenicity studies
– Gene mutation, chromosomal damage, or primary DNA
damage
• Reproduction studies
– Fertility and general reproductive performance
– Teratology drugs administered during pregnancy
– Prenatal/postnatal study during the last trimester through
delivery and on into the early period of lactation
 1.2. Drug metabolism studies

• Drugs are xenobiotics

– Xenobiotics are substances which are foreign to the particular
biological system
– Metabolism means degrade or modify xenobiotics to facilitate
excretion
– Metabolism needs a whole range of metabolic enzymes
– Primary site of metabolism is the liver
• Testing for drug metabolites
– It is important to ensure that no toxic metabolites are formed
• Metabolites should be inactive and quickly excreted
– Drug metabolism studies can sometimes be useful in drug design
• Inactive in vitro but active in vivo indicates that metabolism
converted inactive parent drug to active metabolite (prodrug)
1.3. Pharmacology, formulation, and stability tests
1.3.1.Pharmacology tests

- To see whether the drug has activity at targets other than the intended one.
- To gain a better insight into the drug's mechanism of action.
- Determine a dose-response relationship
- Define the drug's duration of action.
1.3.2. Formulation tests

- Involve developing a preparation of the drug which is both stable and

acceptable to the patient.
- Preformulation involves the characterization of a drug's physical,
chemical, and mechanical properties in order to choose what other
ingredients should be used in the preparation.

- Consider such factors as particle size, polymorphism, pH, and solubility, as

all of these can influence bioavailability and hence the activity of a drug.

- Consideration has to be given to what is called the drug load—the ratio of

the active drug to the total contents of the dose.
1.3.3. Stability tests

- To test whether temperature, humidity, ultraviolet light, or visible light

have any effect, and the preparation is analysed to see if any
degradation products have been formed.

- To check whether there are any unwanted interactions between the

preparation and the container.
 1.4. Clinical Trials

• Clinical trials involve testing the drug on volunteers and patients.

– Phase I study
– Phase II study
– Phase III study
– Phase IV study
 a. Phase I Clinical Study

• 20-80 healthy volunteers

• Designed to provide a preliminary evaluation of drug safety and
dosage, pharmacokinetics (absorption, distribution, metabolism
and elimination) of the active ingredient(s), and indication of
possible drug-drug and drug-food interactions
• Also includes pharmacokinetic studies in volunteers with
impaired renal or liver functions
• Takes about one year
 b. Phase II Clinical Study

• Phase IIa utilize a limited number (100) of subjects

• Phase IIb greater numbers (300) and well-controlled comparative
designs
• Looking for efficacy and side effects
• Therapeutic studies are performed to demonstrate the
pharmacodynamics of the new drug, and to access its short-term
safety in patients suffering from the intended disease or condition
• Also explore possible effective dose ranges and regimens and, if
feasible, clarify dose-response relationship to guide the design of
larger therapeutic trials (phase III)
• Takes about two years
 c. Phase III Clinical Study

• The initial phase III (phase IIIa) studies are intended to provide
sufficient proof of efficacy and adequate safety of the new drug in a
large number of patients (1,000-3,000) prior to its registration with the
FDA
• Compared with a placebo
– A preparation which has no effect at all
• Randomized and double-blind
– Neither the patient nor the investigator knows which treatment is
being applied
• Later-staged Phase III (phase IIIb) studies are conducted after the
NDA is submitted to the FDA, but prior to new drug's approval
• Frequently in phase IIIb trials the new drug is compared with others
already established as effective, or augment earlier trials
• Takes about three years but the drug shows a clear beneficial effect
early on, the phase III trials may be terminated earlier than planned
 d. Phase IV Clinical Study

• Performed after the FDA grants marketing approval

• Continue documenting the safety of the drug (post marketing
surveillance)
• Although in general, the clinical trial phases are performed in
chronological order, not all phase I studies are necessarily completed
before the start of phase II and not all phase II studies are completed
before the start of phase III
• Examples of market withdrawal
– Tienilic acid (diuretic)
• Damage liver cells in one out of every 10,000 patients
– Phenylbutazone (antiinflammatory agent)
• Caused fatal side-effect in 22 patients out of every million
treated

Cl O O
Cl O
OH N

N
S
O CH3
O

Tienilic acid Phenylbutazone

 1.5. Ethical Issues

• Ethical problems concerning patient selection during clinical trials

– Patients who can not make decision whether to participate clinical
trials or not but can be benefited from the trials
• Unconscious patient
• Mentally ill patient
• Children (most licensed drugs have been licensed for adults)
2. Patenting and regulatory affairs
2.1.Patents

• Patent grants the exclusive right to the use of intellectual property for
a limited term
• Requirements for patents
– Absolute novelty
– Utility
– Unobviousness
• Patent should cover specific products, the medicinal use of the
products, and the synthesis of the products
2.2. Regulatory affairs

- Good laboratory, manufacturing and clinical practice

3. Chemical and process development
3.1. Chemical development

Once a compound goes forward for preclinical tests, it is necessary to

start the development of a large-scale synthesis as soon as possible.

A synthetic route has to be devised which is straightforward, safe,

cheap, efficient, and high yielding, has the minimum number of
synthetic steps, and will provide a consistently high-quality product
which meets predetermined specifications of purity.
 3.2. Process development

To ensure that the number of reactions in the synthetic route is as

small as possible and that all the individual stages in the process are
integrated with each other such that the full synthesis runs smoothly
and efficiently on a production scale.

3.3.Choice of drug candidate

The issues surrounding chemical and process development can affect

the choice of which drug candidate is taken forward into drug
development.