PRECLINICAL DRUG DISCOVERY & DEVELOPMENT

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.

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.

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.

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…

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.

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.

PHARMACOLOGICAL DATA Regulatory agencies require the p'cological props. of the

compounds presented in 3 sections: Primary p’cology Secondary p’cology Drug interactionsPrimary 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).

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…

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.

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.

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 for this delay.

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

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.

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.

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.

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 speciesKm values: 3 (mice); 6 (rats); 20 (dogs); humans: 25 (children); 37(adults)

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

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).

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.

To Calculate drug dose

Body weight (kg) x Dose (mg/kg)Concentration (mg/ml)

• 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/kg10mg/ml

• Don’t forget to convert the weight to like units!!!

HAZARDS

SPECIES-SPECIFIC CONCERNS

Most rodents, rabbits: Minor concerns

Cats: Toxoplasmosis

Non-human Primates: Herpes-BShigellaTuberculosisOther zoonotic diseases

THE END