This document provides an overview of preclinical drug discovery and development processes. It discusses rational drug design, screening approaches, molecular modification of lead compounds, pharmacokinetic and toxicology studies in animal models, and regulatory requirements for data on a drug's primary pharmacology, secondary effects, and interactions prior to clinical trials. The goal of preclinical research is to obtain sufficient safety and efficacy data on new chemical entities to justify testing in humans.

1. PRECLINICAL DRUG DISCOVERY
& DEVELOPMENT

2.  Preclinical development encompasses ‘all of the activities that must
take place before a new chemical entity (drug) can be administered
to humans.’
 It spans the gap between drug discovery and clinical testing.

3. RATIONAL DRUG DESIGN
 Basic research approach to drug discovery.
 Thorough knowledge of biochemical and physiological mechanisms
(responsible for the normal functioning of a particular organ system)
will allow an understanding of any pathophysiology of the same
system.
 Permits drug design that affects the altered target (enzyme, receptor
or cell) and correct the deficiency (pathological state).
 Result: Targeted-screening approaches
• Receptor-oriented drug research; reversible and irreversible enzyme
inhibitors.
• Inhibitors of voltage- and receptor-operated systems, transporter
systems.
• Eg.: Nucleoside analogs (5F, Cytosine arabinoside, 6-MCP) were
synthesized based on the concept that they inhibit nucleic acid
synthesis, causing disruption of cell replication and cell death.

4. GENERAL SCREENING
 Aim: to find any therapeutically useful property.
 Random screening of a large no. of diverse compounds through > 1
bioassay.
 Invitro (enzymes or binding assays); OR
 Invivo animal model.
TARGETED SCREENING (disease-oriented approach)
 Compounds are tested in bioassays selected to reveal specific
therapeutic activity.
 Eg.: National Cancer Institute – large scale cell-based assays for
potential antitumor agents.
 60 human tumor cell lines from 8 human cancer types (lung, colon,
breast, melanoma, kidney, ovary, brain and leukemia).
 ‘Lead agent’ (agent with disease-specific activity) is subjected to
further tests.
 Computerized programmes also aid in this process.

5. MOLECULAR MODIFICATION
 A lead structure/agent rarely yields a compound with all properties
needed for full clinical development.
 Compound is modified / optimized to:
• ↑ potency and BA
• ↓ metabolism, toxicity and side effects.
 Lead structure optimization is done by
• Enlightened opportunism
• Unenlightened opportunism
 Enlightened opportunism :
• Combine the important structural features of 2 or more classes of
compounds into 1 molecule to achieve a superior therapeutic agent.
• Eg.: Cisplatin, Carboplatin, Oxiplatin, etc…

6.  Unenlightened opportunism / ‘Me-too approach’:
• Done at a later stage of development.
• Attempt/s to make a close chemical variation in a therapeutic area
where multiple agents already exist.
• Eg.: Development of tricyclic antidepressants.

7. CLINICAL OBSERVATIONS
 Physicians observe an ‘apparent side effect’ and may recognize it as
a novel therapeutic effect.
 Egs.:
• Antidepressant activity of Iproniazid (an anti-TB drug).
• Antirheumatic effects of penicillamine
• Anxiolytic property of the neuroleptic Buspirone
DRUG DEVELOPMENT
 A ‘lead molecule’ is made to undergo many tests in vivo (to determine
it’s potency) in the appropriate animal models.
 Results: Oral BA , therapeutic ratio, preliminary data (indicates
whether primary activity resides in the parent molecule or
metabolite), toxicity profile.
 These tests are always designed with a focus on the primary
indication intended.
 Necessary for obtaining the data needed to fulfill the IND regulatory
requirements.

8. PHARMACOLOGICAL DATA
 Regulatory agencies require the p'cological props. of the
compounds presented in 3 sections:
Primary p’cology
Secondary p’cology
Drug interactions
Primary Pharmacology
 Info. on all pharmacological actions relevant to the proposed
therapeutic use.
Guidelines:
• Establish the mechanism of the principal p’cological action (where
possible).
• Validity of animal models should be established. [Models must be
accepted (through literature / earlier similar studies)].
• Results must be in quantitative terms (dose and time-related; to
be correlated with p’cokinetic and p’codynamic data).

9. Secondary Pharmacology
 Are effects additional to primary p’cological action.
 More investigations are required if doses producing 2° effects
approach those producing 1° effects.
 In-vitro and in vivo data are required.
Drug Interactions
 Interaction of the drug substance with other compounds, when
relevant to the proposed therapeutic usage should be investigated.
 Interactions with other drugs, food, etc…

10. TOXICOLOGICAL STUDIES
 Usually carried out in vivo (in mice, rats, dogs or monkeys).
 In-vitro models are now gaining popularity…..
• ↑ed availability of human tissues
• Rapid and functional multiplicity of mammalian drug-metabolizing
enzymes.
 Acute and subacute toxicology studies are initially performed.
Acute, single dose toxicity studies:
 A new drug candidate must be tested in atleast 2 animal species
(usually rats and dogs).
 Route of admn. should be the same as that intended for human use.
 Purpose: to study the adverse effects of the drug and extrapolated
estimate of LD50. Lasts from 1 – 2 weeks
 Male and female animals: 10 – 30 rats and 2 – 4 dogs per gender
and dose;.
Repeated dose toxicity studies: 2 species (one non-rodent) tested and
followed-up for 1, 3 or 6 months.

11. P’COKINETIC & ADME Studies
 Absorption, t1/2 and metabolism are detected.
 These tests are to exclude those compounds which are poorly
absorbed, rapidly metabolized or eliminated.
 In vivo (usually in mice, rats and dogs).
 In vitro studies are also becoming popular.
 Results….
• Drug candidate’s metabolic profile.
• Species differences in the metabolism of the drug candidate.
• Can get an idea about the enzyme(s) responsible for metabolic
clearance of the drug candidate in humans.

13. WHY ARE FORMULATION STUDIES IMPORTANT?
 For drugs requiring special routes or methods of administration (egs.:
transdermal, inhalational or topical therapies, timed-release
formulations).
 Eg.: a) Paclitaxel - mitotic inhibitor
- is poorly soluble in standard aqueous I.V. solutions.
- Clinical trials commenced only when oleaginous I.V.
formulation CREMEPHOR EL (polyoxyethylated castor oil) was
formulated.
- Clinically, this caused potential life-threatening anaphylactoid
hypersensitivity reactions.
b) 9-aminocamptothecin(9-AC) – topomerase inhibitor
- Clinical development commenced in 1989. BUT….
- Clinical trials only started in 1993.
- Time required to develop a compatible vehicle was the cause

14. IN VIVO STUDIES
 Why are mice and rats preferred for targeted studies?
• The sequence of the mouse genome (discovered in 2002) and the
‘almost complete’ genetic code sequence of the rat make them the
best possible candidates.
• Short generation times and modest maintenance costs.
 In vivo cancer studies:
• The selected animal models must suitably demonstrate the drug’s
antitumor efficacy.
• It must be possible to evaluate systemic toxicities in intact organs.
• Animal models must be genetically stable over time.
 Animal models: Spontaneous models
Engineered models
Transplanted tumor models

15. Spontaneous models
 Sometimes, animals develop diseases similar to humans either
naturally or induced by invasive interventions (treatment w/ drugs,
chemical toxins or radiation).
 Used very successfully in CV research.
 Egs.: Spontaneously Hypertensive Rat (SHR) for CV studies
Engineered models
 Use of genetically-engineered animal models;
 Genetic alterations are performed in the animal models;
 Permits organ and site-specific targeting, better growth rates and
patterns, can obtain better immuno-competent animals;
 Disadvantages:
– High cost of animals;
– Requires commercial license.
– Tumors often develop late in the animal’s life span or even in
the next generation.

16. Transplanted Tumor Models
 Most widely used these days.
 Involves various systems and techniques to propagate tumor tissues
in different hosts for controlled studies in vivo.
 Rodents are the preferred species.
Allograft transplant models
 Also known as Syngeneic models;
 Tumor tissues are derived from animals with the same genetic
background of the given animal model’s genetic strain.
 The ‘transplant’ is not rejected by the recipient (due to shared genetic
ancestry).
 Researchers then monitor the cancer tissue(s) for growth changes
(shrinkage, metastasis and survival rates)
 Therapeutic interventions (new drugs potency) can be performed.
 Disadv.: The transplanted mouse tissue may not fully represent the
clinical situations observed in human tumors.

17. Xenograft transplant models
 Involves actual human cancer cells or solid tumors which are
transplanted into the rodent.
 The recipient rodents have impaired immune systems (induced). The
‘transplant’ is not rejected.
The transplant (tumor) can either be…
• Orthotopic: the tumor is placed in the site it would be expected to
arise in humans (human liver cancerous cells are transplanted
into the liver of the rodents)
• Subcutaneous: placed just below the rodents’ skin
 Adv.:
• These studies of the cancerous tissues employ real human cancer
cells; more representative of the properties and mutations in human
cancer cells.
Disadv.: Due to changes in the rodents’ immune system, it may not
mimic the actual clinical situation.

18. MELD10
Mouse Equivalent LD10 (in mg/m2
) is scaled to MELD10 dose for dogs
by the following formula:
MELD10 in dogs = (Km dog / Km mouse) x MELD10 in mice
Km = surface-to-weight ratio for each species
Km values: 3 (mice);
6 (rats);
20 (dogs);
humans: 25 (children);
37(adults)

19. Humane Endpoint vs Experimental Endpoint
 Experimental Endpoint
• Planned endpoint when animal will be euthanized
and tissues harvested for in vitro analysis
 Humane Endpoint
• Unplanned endpoint (earlier than Experimental
endpoint) if something goes wrong.
• The animal must be humanely euthanized if, in
distress, which cannot be treated

20. Three R’s
 Reduction
 Refinement
 Replacement
Reduction
 Animal numbers must be reduced to the absolute minimum
to achieve necessary result(s).
 Greater focus is placed on study objectives, achieving better
experimental design, and minimizing the need for repeat
studies.
 Animal testing can be reduced by…
• prescreening;
• using in vitro tests where possible;
• promoting greater sharing and dissemination of test data
worldwide;
• reusing animals for multiple tests (eg.: for ocular and dermal
tests).

21. Refinement
 Refine or modify the testing to make it more humane,
without reducing scientific validity.
 Examples:
• Laparoscopy instead of laparotomy
• Blood collection from vein instead of cardiac puncture
Replacement
 Replacing animals with in vitro models
• cell and tissue cultures
• computerized models
 Replacing a higher more sentient animal with a lower less
sentient animal
• Instead of a monkey, using the less sentient rat/mice
is a preferred alternative.

22. To Calculate drug dose
Body weight (kg) x Dose (mg/kg)
Concentration (mg/ml)

23. • To calculate an
individual animal dose,
multiply its weight (kg)
by the drug dosage and
divide by the
concentration.
• Eg: 25 g x 100 mg/kg
10mg/ml
• Don’t forget to convert
the weight to like
units!!!

24. HAZARDS
SPECIES-SPECIFIC CONCERNS
Most rodents, rabbits: Minor concerns
Cats: Toxoplasmosis
Non-human Primates: Herpes-B
Shigella
Tuberculosis
Other zoonotic diseases

25. THE END