Drug development involves basic research to identify drug targets and applied research to develop treatments. Preclinical trials test drug safety and efficacy in animals prior to human trials. They involve pharmacokinetic, pharmacodynamic and toxicology studies in two animal species. This provides data on effective and toxic doses, screens the drug's activity, and identifies formulation. Preclinical trials help determine if a drug warrants further development or should be terminated. They aim to predict potential human adverse effects and provide guidance for initial human clinical trials. However, extrapolating animal data to humans has limitations due to interspecies differences.

2. Drug design – Trial and error (earlier); Structure-based drug design (new).

3. There are two types of Research:
Basic and Applied
Basic Research: discovering new facts about existing
concepts (how things work, how they are made, or what
causes a biological event to occur). Basic research can
explore / explain / describe a topic.
Eg.: Andrew Fire and Craig Mello discovered that genes
can be turned off or on by small RNA molecules in the
body. This study was conducted on worms. It led to the
Nobel Prize in 2006.
(
http://www.oxbridgebiotech.com/review/science-basics/nobel-prize-20
)

4. Applied Research: Taking the information discovered in
basic research and investigating how to use it to treat and
prevent sicknesses.
Eg.: A researcher can use the information about turning
genes off and on to find a drug that is used to turn off
genes that cause diseases and disorders in humans.

5. Several steps involved Pre-Clinical
Trials
File for approval as an Investigational
New Drug (IND)5
4
3
2
1
Establish Effective and Toxic Doses
Screen the Drug in the Assay
Develop a Bioassay
Identify a Drug Target

6. What is a Preclinical Test/ Trial?
• Preclinical trial - a laboratory test of a
new drug or a new medical device,
usually done on animal subjects, to see if
the ‘hoped-for treatment’ really works and
if it is safe to test on humans.

7. INTRODUCTION
 One of the initial processes of drug development.
 Research stage prior to commencement of clinical trials in humans.
 Risk-based exercise that extrapolates non-human safety and efficacy
to potential human outcomes.
 Main goals: * to determine a product's ultimate safety profile.
* to identify a formulation that achieves the highest drug
exposure with the lowest dosage (yet tolerated by the
animal species involved), for the duration of toxicity studies.
 P’cological; p’codynamics; p’cokinetics (ADME) and toxicological studies.
 In vitro and in vivo tests are performed.
 Toxicity studies focus on which organs are targeted by the drug; any long-term
carcinogenic effects, or toxic effects on mammalian reproduction.
 Pre-clinical studies generate evidence and confidence whether a drug is worthy of
further development or should be terminated from the development.

8. Introduction (contd.)
 Scope of Preclinical studies:
• Preliminary evaluation of the p’cology, p’cokinetics, p’codynamics, dose–
response profiles, safety and toxicological potential of a drug.
• Analyze physicochemical characteristics of the test compound.
• Determines the optimal formulation and dose for phase I clinical trials.
• Provides the rationale for the proposed therapeutic indication.
• Preclinical p’cological and animal toxicity results from studies →
Approval or disapproval to initiate human clinical trials.
 Major tests performed on a drug candidate during preclinical trials :
• PK and PD studies
• Bioequivalence and bioavailability
• Toxicity studies [Acute and chronic toxicity; Mutagenicity or
carcinogenicity; Immunotoxicity and other tests].

9. Issues that frequently arise during planning pre-clinical trials
 Preliminary formulation of test; frequency and routes of drug
administration.
 Materials required; selection of animal species and number; duration
of toxicity studies.
 Proposed human clinical dose in relation to animal studies, and
validation of a modified test system.
 Information from animal safety tests
• Initial guide for comparing clinical benefits and risks for human trials.
• To predict and characterize potential adverse effects in humans.
• Provide general methods of toxicological studies (appropriately
evaluate drug safety and applications for drug approval);
• To meet the requirements of regulatory agencies.
• To serve as a basis for establishing Quality Controls and product
specifications.

10. P’cokinetic studies
• To establish parameters for drug actions; how ADME affects the drug
and vice-versa.
• Estimates the most appropriate method and optimum effective
dosage of drug administration .
• Helps to identify any toxic effect.
P’codynamic studies
• How a drug exerts its pharmacological effects.
• How a drug interacts with cells or organs; drug effects and adverse
reactions, and characteristics of dose–response curves.
Usefulness
• Selecting dose levels, dose regimens, design of toxicology studies as
well as in evaluating safety and extrapolating toxicological data to
humans.

12. ANIMAL SPECIES
 Ethical & cost reasons → ↓ed animal testing in research-based
p’ceutical industries (recently).
 Animal-based testing is still conducted (similarity in anatomy &
physiology).
 Animal species: Murine (rats, mice); Canine; Primate and Porcine.
 Species are selected based on which gives the best correlation to
human trials.
 Parameters: Differences in gut, enzyme activity, circulatory system,
etc. make certain models more appropriate based on the dosage
form, site of activity, or noxious metabolites.
• Underdeveloped carnivore intestine (vs omnivores)
↓
↑ed gastric emptying rates (canines may not be good models for
studying solid oral dosage forms).
• Rodents can’t act as models for antibiotics (resulting alteration to their
intestinal flora causes significant adverse effects).

13. Preclinical safety assessment
 2 different animal species (rodents and non-rodents);
 Appropriate dosage form that delivers the drug in a way that
maximizes the drug availability for thorough evaluation of drug-
related toxicity or adverse effects.
Toxicological evaluation
 Identify the optimized drug formulation to deliver the drug to the
animal species and also to minimize any non-drug related
issues.
 Must have Placebo / control.
 Dose- and time-dependent toxicities are also studied.
 Unpredictable idiosyncratic adverse effects are considerably
more difficult to identify in preclinical drug evaluation.

14.  Similar or variable drug metabolism between species (based on a
drugs’ functional groups) → affects efficacy and toxicology.
 Larger species (dogs, pigs, sheep) are usually used for most tests
(similar-sized model as that of a human).
 Similarity in specific organs or organ system physiology
• swine (dermatological & coronary stent studies)
• goats (mammary implant studies)
• dogs (gastric studies).
 Pre-clinical trials enable in establishing the ‘No Observable Effect
Levels’ (NOEL) / ‘No Observable After Effect Levels’ (NOAELs) on
drugs.
 NOEL / NOAEL
• Indicates the optimal initial phase I clinical trial dosage levels on a
mass API per mass patient basis.
• Achieving the highest drug exposure levels (systemic / local) with the
lowest dosage administered.
[API – Active Pharmaceutical Ingredient]

15.  Minimize any non-drug-related events and outcomes.
• Physicochemical drug properties → impose limitations
↓
Toxic level exposures not achieved.
• Eg.: any solution or suspension has a limit volume that can be
administered safely to a given animal species for a given route of
administration.
• Administration of volumes or amounts > those limits → non-drug-
related observations (effects not due to the active ingredients).
 The pharmaceutical scientist must strive to achieve a balance
between maximum tolerated drug dose in a given volume of vehicle
and maximum tolerated volume of the vehicle.

16. LIMITS TO EXTRAPOLATING ANIMAL DATA
 Species differences (in anatomy, physiological functions, drug tolerance and
enzyme induction).
 P’cokinetic differences between test animals and humans.
 Idiosyncratic adverse events in humans ( mechanisms not fully understood; not
demonstrable in animals by ordinary toxicological and p’cological investigation) –
Steven-Johnson’s syndrome, Phocomelia
 Underlying pathological condition/s — drugs may exacerbate underlying diseases
in humans, that do not exist in animals.
• Beta blockers, antibiotics (myasthenia gravis)
• Antimalarials, Beta blockers, Ibuprofen, Naproxen (psoriasis)
 Drug (and it’s metabolites) + underlying disease relationships cannot adequately
be investigated or predicted from studies conducted in healthy animals.

17. Acute toxicity testing
 By a route of administration that allows for adequate systemic
exposure.
 Usually only one animal species must be tested.
 Anticipated drug use determines frequency & duration of
administration.
 Duration of exposure normally does not exceed 4 weeks.
 Evaluation of reversibility should be included.
 Controls
• are vehicle or excipients;
• positive controls should be used where possible.
• The similar actual concentrations should be used in animal tests as
are proposed for use in humans.
 Caution: to minimize painful and traumatic exposure and to terminate
experiments (if severe adverse reactions).

18. Skin
 Little value in skin irritancy testing (enormous variability in the skin
response of different animal species to toxic chemicals).
 If animal dermal tolerance testing is justified,
1] Single-dose dermal tolerance test
• in rabbits (on shaved intact skin and shaved abraded skin).
• examine exposed skin for erythema, edema, desquamation, scab
formation and other lesions.
• note changes at 24, 48 and 72 hours.
• follow up (upto 8 days) may be necessary.
2] Repeated-dose dermal tolerance test
• also done in rabbits (intact and abraded shaved skin);
• follow-up for periods of upto 4 weeks.

20. KEY WORDS
Basic Research
Applied Research
Target
Bioassay
Investigational New Drug (IND)
NOEL / NOAEL
API
Limit Volume
Positive Control
Single dose and repeated dose dermal
tolerance test

21. Eyes
 Any chemical with irritant or corrosive properties when
applied to the skin can irritate the cornea and conjunctiva
 Draize test in rabbits: most widely used test for prediction
of ophthalmological irritancy; it is injurious to the
experimental animals.
• Local ocular toxicity and tolerance testing – ophthalmic
products (topical) or products applied close to the eye [face
or hair (medicated shampoos)],
• single administration (in rabbit).
• also examine for anesthetizing properties.
• evaluation of the eyes, lids, conjunctivae, nictitating
membrane, cornea, and iris in one eye is necessary; other
eye (control).
 Repeat ocular dosing:
• daily administration in the rabbit for not more than 4 weeks.
• based on the results of the acute, single-dose study.

22. Mucosal Surfaces
 Irritancy testing is necessary when substances are intended
for application to mucosal surfaces such as the vagina,
where local factors such as pH have to be considered.
Lethal Dose 50 (LD50) Test
 Aimed at determining the dose of a toxic substance (drug)
that kills 50% of the animals that receive it.
 Is a standard part of the early assessment of a new
medicine.
 Killing animals in this way has proved objectionable to
many, necessitating a critical review of the justification for
the test.

23. Value of the LD50 Test
 Twofold:
1] To determine the Therapeutic margin (margin between
effective and toxic doses).
2] Comparing lethal effects with blood levels of the active
principle.
Pointers
 Always consider in conjunction with other relevant
information.
 Conducting the LD50 test on large animals should be
discontinued.
 Conduct the test on a limited number of small animals, w/
detailed recording of symptoms and pathology.
 LD50 test should not be conducted with p’cologically inert
subs. [max. of 5g/kg (oral admn.); 2g/kg (parenteral) is
sufficient, if death or acute symptoms are not produced].
 Don’t conduct in newborn animals.

24. LONG-TERM (CHRONIC) TOXICITY TESTING
 Value of long-term testing has been seriously questioned.
 A more complete understanding of the relevant p’cology and
of the physiological changes caused by acute exposure to a
new drug might provide sufficient information to anticipate
adverse long-term effects.
 With repeated-dose testing ….
• most probable structural lesions should be identifiable.
• knowledge can also be gained of functional disturbances.
 BUT, Long-term animal studies…
• influence of drugs and variables (aging, disease, and diet)
on toxicity remains uncertain.

25. Mechanisms of Drug Injury from Long-Term Exposure
 Accumulation of the parent drug or its metabolites in the tissues (with
consequent toxic injury).
 Repeated / low-grade continuous injury to DNA.
 Disturbance of the adaptive properties of cell receptors.
 Damage to repair responses, rendering the animal sensitive to
additional toxic substances (drug / it’s metabolite).
Dose Considerations
 Minimum of 3 treatment groups (divided according to dose).
• 1 control group, 1 test group.
• An additional group (if it’s necessary to examine a toxic effect in
relation to a particular dose).
 OD dosing for 7 days is adequate to expose the experimental animal
to a drug.
 More frequent administration may be necessary for drugs with a very
short t1/2 or brief duration of action.

26. Route of Administration
 Should ideally be the same as that proposed for clinical use.
 Can be problematic if a high dose is required that cannot be
tolerated when given by a particular route.
 If alternative route is also studied, comparative p’cokinetic
data will be required.

27. PHARMACOKINETICS AND TOXICOKINETICS
General Principles
 P’cokinetic results from single-dose kinetic studies help in
the choice of formulation and in prediction of the rate and
duration of exposure during a dosing interval.
 This may assist in the selection of appropriate dose levels for
use in later studies.
 Select treatment regimen and species (whenever possible)
with regard to p’codynamic and p’cokinetic principles.
Toxicokinetic data
 Focuses on kinetics of a new therapeutic agent under the
conditions of toxicity.
 Toxicokinetics is the generation of pharmacokinetic data to
assess systemic exposure in the conduct of nonclinical
toxicity studies.
 The data may be used in interpretation of toxicology findings
and their relevance to clinical studies.
 Can be single and repeated-dose toxicity studies;
reproductive, genotoxicity, and carcinogenicity studies.

28. Immunotoxicological studies
 Indicators of immunosuppression:
• Myelosuppression (leucopenia, lymphopenia, or other blood
dyscrasias).
• Alterations in immune system organ weights and histology
(thymus, spleen, lymph nodes, or bone marrow).
• Decreased globulin levels.
• Increased incidence of infections and tumors.
 Organ weights and histological examination are be
conducted on the spleen, thymus, lymph nodes, and bone
marrow.
 The lymphoid tissue that drains or contacts the site of drug
administration (exposed to highest drug conc.) should be
specifically examined.
 Changes in T-cells or B-cells.

29. DEVELOPMENTAL & REPRODUCTIVE TOXICITY
TESTING
 Combination of studies that allows exposure of
animals at all stages of development to toxic doses of
the drug (from conception to sexual maturity and
beyond).
 Major manifestations of developmental toxicity:
(1) death of the developing organ
(2) structural abnormalities
(3) altered growth
(4) functional deficiency
 Rats – preferred rodent species (large amount of
background knowledge already available).
 Rabbits – used as a second mammalian species;
required in embryotoxicity studies only.

30.  The results depend on…
• extent, duration, and time of exposure and on the
chemical entity concerned.
 Results: impaired ability to conceive, abortion,
dysmorphogenesis, premature birth, low birth weight,
perinatal mortality and morbidity, cancer, and
dysfunctional growth and development after birth
 The scope of reproductive & developmental toxicity tests:
• determining the effects of test drug on reproductive
competence of adult animals;
• determine developmental toxicity (any adverse effect prior
to attainment of adult life); embryotoxicity, fetal toxicity,
and embryo–fetal toxicity.
• enable extrapolation of the results to humans.
• detect immediate and latent effects of exposure (continue
observations thru one complete life-cycle - from conception
in one generation through conception in the following
generation).

31. THANK YOU