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

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-2006-from-petunias-to-potential-cure-all-in-the-discovery-of-rna-interference/)

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.

Several steps involved Pre-Clinical Trials

File for approval as an Investigational New Drug (IND)5

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Establish Effective and Toxic Doses

Screen the Drug in the Assay

Develop a Bioassay

Identify a Drug Target

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.

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.

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].

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.

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.

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).

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.

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]

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.

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.

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).

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.

KEY WORDSBasic ResearchApplied ResearchTargetBioassayInvestigational New Drug (IND)NOEL / NOAELAPILimit VolumePositive ControlSingle dose and repeated dose dermal tolerance test

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.

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.

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.

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.

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.

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.

PHARMACOKINETICS AND TOXICOKINETICSGeneral 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.

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.

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.

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).

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