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By: Maged Wasfy

Lecturer of Pharmacology Faculty of Pharmacy and Drug Manufacturing

Pharos University in Alexandria

Regulatory Aspects of Safety in Process of drug Discovery & Development

Forensic Chemistry (PHR 476) Lecture of week 12


Drug discovery and development is a long and complex process.

Only one of every 5,000-10,000 molecules screened in the

laboratory ever makes it to market as a new drug.

FDA & Research-based pharmaceutical companies take

extraordinary measures to ensure the safety and efficacy of all

approved prescription medicines.

To ensure patient safety, FDA and the pharmaceutical company

follow careful scientific procedures in four distinct stages:

1. Preclinical safety assessment

2. Pre-approval safety assessment in humans

3. Safety assessment during FDA regulatory review

4. Post-marketing safety surveillance.


1. Preclinical Safety Assessment

The relative safety of newly synthesized compounds is initially

evaluated in both in vitro and in vivo tests.

If a compound appears to have important biological &

pharmacological activity →→ →→ special tests are conducted to

evaluate its safety in the major organ systems (for example, the liver;

the central nervous, cardiovascular, and respiratory systems).

Other organ systems are evaluated when potential problems appear.

The goal of preclinical animal studies are:

A. To ensure that a drug is safe enough to be tested in humans.

B. Characterize any relationship between increased doses of a drug

and toxic effects in the animals.


2. Pre-Approval Safety Assessment in Humans (clinical trials)

A drug sponsor can begin clinical trials in humans once FDA is

satisfied that the preclinical animal data do not show an

unacceptable safety risk to humans.

New Drug Application (NDA) seeking FDA regulatory review to

market a new product.

Each clinical trial evaluates:

A. Safety & adverse effects regardless of the stated objective of

the trial.

B. Effect of new drug on the quality-of-life

C. Pharmacoeconomic of the new drug.

All harmful reactions are reported to FDA and, when appropriate, the

information is incorporated in a drug’s package labeling.


2. Pre-Approval Safety Assessment in Humans (clinical trials)

The average NDA for a novel prescription medicine is based on

approximately 70 clinical trials involving over 4,000 patients.

Clinical trials are conducted in three stages:

A.Phase I:

Drugs are evaluated for safety in healthy volunteers using single

dose &multiple dose

B.Phase II:

To evaluate the safety and efficacy of a drug using patients instead of

healthy volunteers.

C.Phase III:

These large trials evaluate safety and efficacy in groups of patients

(with the disease to be treated) from different populations (elderly,

patients with multiple diseases, those who take other drugs, and

patients whose organs are impaired).


3. Safety Assessment During FDA Regulatory Review

A sponsor submits an NDA to FDA for approval to manufacture,

distribute, and market a drug based on the safety and efficacy data

obtained during the clinical trials.

FDA usually completes its review of a “standard” drug in 10 to 12

months. 120 days prior to a drug’s anticipated approval, a sponsor

must provide the agency with a summary of all safety information in

the NDA, along with any additional safety information obtained

during the review period.

The addition of 600 new reviewers made possible by the user fees

paid by pharmaceutical companies has enabled FDA to approve new

drugs while maintaining high safety standards.


4. Post-Marketing Safety Surveillance

Monitoring and evaluating a drug’s safety become more complex

after it is approved and marketed as drug will be taken by many

more patients and physicians are free to use it in different doses,

different dosing regimens and different patient populations.

Adverse reactions that occur in fewer than 1 in 3,000 to 5,000

patients are unlikely to be detected in pre-approval investigational

clinical trials, and may be unknown at the time a drug is approved.

These rare adverse reactions are more likely to be detected when

large numbers of patients use a drug after it has been approved.

If the drug shows serious adverse effects, FDA has its authority to

RECALL the drug from the market.


Preclinical Safety Assessment (Toxicity Testing )

Toxicity testing is the structural unit of two branches of

toxicology:

A.Descriptive toxicology:

It is concerned with toxicity testing in experimental animals and

used to evaluate the risks posed to humans by exposure to specific

chemicals.

B.Regulatory toxicology:

It is concerned with deciding, on the basis of the data provided by

mechanistic and descriptive toxicologists, whether a drug or

another chemical poses a sufficiently low risk to be marketed for a

stated purpose.

Preclinical Safety Assessment (Toxicity Testing )

Classification of Toxicity Studies

1. Acute Toxicity studies (Limit Tests, LD50, LC50, ED50, MTD,

Irritation)

2. Subchronic Toxicity studies

3. Chronic Toxicity studies

4. Special toxicity studies:

A. Tests for Developmental toxicity ( teratogenicity).

B. Tests for Reproductive Toxicity

C. Tests for Mutagenicity

D. Tests for Carcinogenicity

E. Tests for Neurotoxicity & Behavioral toxicity.

F. Tests for Immunotoxicity

1- Acute Toxicity Studies

Definition:

It is studying of the adverse effects occurring within a short

time of administration of a single dose or multiple doses

given within 24 hours.

Objectives of Acute toxicity studies are:

1.Identify the symptoms & possible mode of action of relative

toxicity of the compound.

2.To determine the existence of species differences.

3.Give information for further toxicity testing (i.e define more

precisely the doses to be used in subchronic toxicity tests)


Parameters obtained from Acute toxicity testing:

1. LD50 (Median lethal dose)

The calculated dose of a chemical that causes death in 50% of

the tested animals after acute, single exposure.

For inhaled chemicals, median lethal dose is expressed as LC50

(median lethal concentration) which is median concentration to

which animals are exposed for a fixed time that will kill 50% of the

animals.

2. NOAEL ( Non Observed Adverse Effect Level).

The highest dose in a toxicity study at which no toxic or adverse

effects are observed.

3. MTD (Maximum tolerated dose)

The highest dose of a chemical that when administered to a

group of test animals does not increase the death rate during a

long-term study.

1- Acute Toxicity Studies Experimental Design of Acute toxicity testing: 1. Animal species: At least 2 species are required

Rats and mice are usually used, but sometimes dogs and rabbits are also included.

2. Animal number & gender: At least (8-12) of each species, (4-6) of each sex.

3. Route of administration: The chemical should be administered by the same route with which

human would be administered. (e,g: Oral gavage, Ocular, Dermal, Intraperitoneal, lnhalation, intravenous, Intramuscular, Intranasal, Subcutaneous)

Sometimes, we use more than two route of administration to assess potential hazardous effects of chemical handling by human.

e.g: if the route of human exposure would be oral, acute dermal, eye, and inhalation studies may be indicated to assess the hazard to personnel's handling the compound.

1- Acute Toxicity Studies Experimental Design of Acute toxicity testing: 4. Dosing : single dose or multiple doses

5. Animal Monitoring: The animals are observed for 14 days after chemical administration.

Observations include:

A. Recording of any serious symptomatic adverse effects.

B. Recording the number of animals dying during observation period, to

calculate LD50.

C. Any animals that haven't already died are then sacrificed and

dissected. Postmortem examination

• In general, all animals dying during the observation period and all surviving

animals should be autopsied by a qualified pathologist. The autopsy should include gross and histopathological examination of all organs.


Examples of Acute toxicity testing

A. Inhalation test

It is used for assessment of the toxic hazard of agents whose

principal route of exposure is via inhalation and to Study of the

comparative toxicity of agents administered by different routes.

LC50 (median lethal concentration) is the parameter of choice of

this test. (where C = concentration of inhaled chemicals for a

fixed time T

Comparative LC50 data are often obtained for 4-hrs exposures

and a 14-day post-exposure observation period.

The most species used for inhalation studies include rats, dogs,

and monkeys.


Examples of Acute toxicity testing B. Draize test It is an acute toxicity tests that initially used for testing of topical

preparations.

It includes:

1. Ocular test

In this test, conscious rabbits are immobilized while chemicals are put

directly into their eyes. They are often left for as long as a week.

Injuries to the eyes after the exposure are determined by an irritancy

value (score). After the test, the rabbits are usually killed and internal

effects on the rabbits are examined.

2. Dermal irritation test

Skin corrosion and dermal irritation tests are typically conducted by

placing a chemical or chemical mixture on the skins of animals, usually

rabbits.


Limitation of Acute toxicity testing:

1. Parameters obtained from acute toxicity testing as LD50 is

subjected to interspecies difference (differences between toxic

effects of chemicals on human and other mammals)

e.g: LD50 of caffeine = 192 mg/kg in human & 620 mg/kg in mouse

The causes of interspecies difference include:

a. Differences in pharmacokinetics of the chemicals in different

species.

b. Existence of different physiology, organ or tissue types.

2. Can not predict the long-term organ toxicity.

Thus, several modifications have been done on Acute toxicity testing


Modified Acute toxicity testing:

1. In vitro cytotoxicity test

It is done using human cell culture taken from specific human tissue

that was expected to be affected, for example the skin, or the liver.

Advantages:

a. Obtained results are more reliable and more predictable than LD50

data as they avoid species differences.

Parameters used in In vitro cytotoxicty assays include:

1.Cell viability.

2.Cell proliferation.

3.Cellular protein content.

4.Cellular metabolism.


Modified Acute toxicity testing:

2. Repeated high-dose studies (Subacute studies)

The design of these studies based on repeated daily administration

of a series of doses to groups of animals for 5-21 days.

Advantages:

1. Obtained results are able to predict long-term toxicity, rather than

LD50 .

2. They define more precisely the doses to be used in subchronic

tests.

2- Subchronic Toxicity Studies

Definition:

It is studying of the adverse effects occurring as a result of daily or

frequent exposure of the compound over a period up to about 90

days (10% of animal life time).

Objectives of Subchronic toxicity studies are:

1. Identify the major toxic effects & target organ toxicity of the test

compound.

2.To determine the potential development of delayed toxicity.

3.To determine the potential reversibility of developed toxic effects.

4.Give information for further toxicity testing (i.e define more

precisely the doses to be used in chronic toxicity tests).


Experimental Design of subchronic toxicity testing:

1. Animal species:

At least 2 species are required, one rodent and one nonrodent.

Rats and dog are usually used (because of their availability &

complete physiological background of these species).

These tests should be done during the period of the most rapid

growth of the animal. (e.g: just after weaning of rats)

2. Animal number & gender:

At least 10 animals of each sex of each species.

3. Route of administration:

The chemical should be administered by the same route with

which human would be administered.


Experimental Design of subchronic toxicity testing:

4. Dosing : (4 dose levels)

Selection of doses for subchronic studies obtained from the results

of acute and repeated high-dose studies.

Doses include

1. The highest dose that provide a distinct toxic effect

2. The lowest dose that should not produce any detectable toxic

reaction.

3. At least two intermediate doses should be included.

2- Subchronic Toxicity Studies Experimental Design of subchronic toxicity testing: 5. Animal Monitoring : The animals are observed at 3-week intervals during exposure and

last up to 3 months following termination of exposure.

Parameters to be tested include:

A. Animal weights

B. Daily Food and water consumption

C. Biochemical organ function tests

D. Metabolic studies

E. Hematologic studies

F. Postmortem examination

These parameters are statistically compared to those obtained from control groups of animal.

Control groups include Negative control and vehicle received control.


Biochemical organ function tests

Using dog instead of a rodent species offer larger samples of

blood for biochemical organ function tests.

Organ function studies should be done prior to initiation of the

test, 3 and 10 days after the start of dosing, at 30-day intervals

throughout the test, and terminally.

Metabolic studies

The toxic nature of the metabolites and the rate of metabolic

transformation of the test compound are of essential parameters t

be evaluated in this toxicity studies.

Urine and faeces can be collected and examined for the presence

of metabolites.


Hematologic studies

Hematological studies should be started prior to initiation of the

test, at 30-day intervals, and on all animals terminally.

Bone marrow should be examined terminally.

Postmortem examination

All animals should be subjected to autopsy that include gross and

histopathological examination of all organs.

3- Chronic Toxicity Studies

Definition:

It is studying of the adverse effects occurring as a result of

exposing the animals to the chemical for the greater part of their

life span (i.e. exposure occurs from early life).

Objectives of Chronic toxicity studies are:

1.To determine precisely the major toxic effects of the test

compound and its mode of action.

2.To determine the potential carcinogenic action of the test

compound.


Experimental Design of chronic toxicity testing:

1. Animal species:

A wide variety of animal species should be used in this type of work.

At least 2 animal species are required, these include:

A.Rodents are the animals of choice, since large numbers can be

used to aid in the statistical interpretation of the results.

B.Dogs and monkey (Larger animals) should also be used for such

species have the advantage that larger samples of blood can be

obtained on a routine basis.

• N.B: for studying of the carcinogenic potential of a compound, the

rat, mouse, or hamster is usually chosen because of its:

Shorter lifetime.

Ability of using large numbers (to increase the sensitivity of the

test).



2. Animal number & gender:

Large number should be used.

3. Route of administration:

The chemical should be administered by the same route with

which human would be administered.

N.B: For food additives, pesticides, and other chemicals likely to

come into contact with food or water, it is recommended to

incorporate the test chemical into the diet or drinking water.



4. Dosing :

Selection of doses for chronic studies obtained from the results

of subchronic toxicity studies.

At least 2 dose levels required.

5. Animal Monitoring :

The same as subchronic toxicity studies.

Assignment Assignment

For students with ID: from 108291 – End of students IDs including transfer students (106……. & 107……..).

Discuss the following topic:

1.Example of the recently FDA recalled drugs

Presentation: 1 – 2 page word document, font type: arial, font size 14, line spacing 1.5

Thank you