Experimental Techniques For Evaluation Of New Drugs.ppt

EXPERIMENTAL TECHNIQUES FOR
EVALUATION OF A NEW DRUGS
Dr. Karabi Adak
MBBS, MD

• A new drug is defined as a new substance of chemical,
biological or biotechnological origin for which
adequate data is not available for the regulatory
authority to judge its efficacy and safety for the
proposed claim.

Purpose of New Drug Evaluation
1) The substance’s potential for toxicity with short-
term (acute effects) or long- term use (chronic
effects).
2) The substance’s potential for specific organ toxicity.
3) The mode, site, and degree of toxicity.
4) Gender, reproductive, or teratogenic toxicities.

Stages of drug development-
• Stage I : Hit and lead compound development
phase(identification of lead compound amongst
the million compounds and selection for further
study)
• Stage II : Preclinical studies (done in form of in vitro
and in vivo animal experiments)
• Stage III : Clinical studies (Phases 0,I, II, III, IV)

Toxicity Studies In Animal
 Identify any toxic substance prior to clinical use.
 Qualitative (nature of effect of drug seen in the
target
organs) and quantitative(plasma and tissue
drug
levels) assessment of drug use.
 To know the cumulative toxicity of the drug under
study.
 To allow a careful selection of doses for further
study.

Types of Toxicity tests
 Acute toxicity study (single dose toxicity study)
 Subacute/subchronic study
(repeated dose from 2 weeks -6
month)
 Chronic toxicity study
(repeated dose study for 6 months-
12

Acute toxicity test
Acute toxicity refers to those adverse effects
occurring following oral administration of a single
dose of a substance, or multiple doses within 24 hr.
Effects are observed within a short time of
exposure to the chemical.
Two rodents species(mice and rats); in each group
at least 5 animals of either sex.

In conditions where rodents are known to be poor
predictors of human toxicity (e.g., antifolates), or where the
cytotoxic drug acts by a novel mechanism of action, MTD
should be established in non-rodent species.
Route of administration-
 Same as intended for humans.
 At least one more route should be used in one of the
species to ensure systemic absorption of the drug.

Doses-
At least three graded dose. A limit of 2g/kg (or 10
times the normal dose that is intended in humans,
whichever is higher) is recommended for oral
dosing
Treatment- given in a single bolus or several doses
or by continuous infusion within 24 hr.

Observations-
Animals should be observed for 14 days after the
drug administration, and minimum lethal dose
(MLD) and maximum tolerated dose (MTD) and
LD50 should be established. Other features to be
observed are-
1. Signs of intoxication
2. Effect on body weight
3. Gross pathological changes

Subacute Studies
• Animal toxicity studies of a minimum of 2 weeks of
daily drug administration at three or more dosage
levels to two animal species are required to support
the initial administration of a single dose in human
clinical testing.
Aim-
 Aim is to identify target organ toxicity and
establishment of MTD for subsequent study.
 Repeated dose study is most appropriate for
compounds which have an apparently long half
life,incomplete elimination or unanticipated organ

Animals-
 At least two mammalian species (1 rodent
+1nonrodent)
 Younger and still growing animals are preferred
at the initiation of a subchronic study.
 If a species is known to metabolize the drug in
the same way as humans, it should be preferred
for toxicity studies.

Selection of dose-
 Highest dose should produce observable toxicity.
 Intermediate dose should cause some symptoms, but
not gross toxicity or death, and should be placed
logarithmically between the other two doses.
 Lowest dose should not cause observable toxicity.

Treatment and duration-
 Given in 14, 28, 90 or 180 days(duration depends on
therapeutic indication)
 Group I; Control group- normal or vehicle group.
 Group II; Treatment group- drug treatment group.
Route of administration-
Same as intended for humans.

Observations-
 Behavioral, physiological, biochemical and
microscopic observations.
 Site of injection should be subjected to gross and
microscopic examination if given parenterally.
 ECG and fundus examination in non rodent species.

Chronic toxicity studies
 Adverse effects are observed following repeated
exposure to a chemical during a substantial fraction
of an organism's lifespan (usually more than 50%)
or some time up to the full span of the animal life.
 For humans, chronic exposure typically means
several decades; for experimental animals, it is
typically more than 3 months and up to 2 years.
 Chronic exposure to chemicals over a period of 2
years using rats or mice may be used to assess the
carcinogenic potential of chemicals.

 Animal used is 2 species- one rodent and a non-
rodent. In rodent chronic study is of 6 months to 2
year duration, in non-rodent it is usually for 1 yr.
 Observations include body weight, food/water intake
, hematology, study of the viscera etc.
 In-life observations including ophthalmology and
Electrocardiogram should be done in nonrodent
species.

Results of chronic toxicity test can be used for-
1) To characterize the toxicity of a food ingredient
following prolonged and repeated exposure.
2) To determine toxicological dose-response
relationships needed to establish the maximum dose
that produces no adverse effects (i.e., NOEL or
NOAEL). The following guidance is written primarily
for rats or mice,

Carcinogenicity Studies
Carcinogenicity studies should be performed for all
drugs that are expected to be clinically used for
more than 6 months and for drugs used in the
treatment of chronic conditions.
Carcinogenicity studies should be done in a rodent
species (preferably rat).
The selected strain of animals should not have a
very high or very low incidence of spontaneous
tumors.

The highest dose should not reduce the life span of
animals by more than 10% of normal.
The lowest dose should be comparable to the intended
human therapeutic dose or a multiple of it.
The drug should be administered 7 days a week.
Generally, the period of dosing should be 24 months
for rats and 18 months for mice.

Observations should include macroscopic changes
observed at autopsy and detailed histopathology of
organs and tissues.
Additional tests for carcinogenicity (short-term
bioassays, neonatal mouse assay or tests employing
transgenic animals) may also be done.

Genotoxicity or Mutagenicity Studies
• Genotoxicity tests are in vitro and in vivo tests
conducted to detect compounds which induce
genetic damage directly or indirectly with respect
to damage to DNA.
Both in-vitro and in-vivo studies should be done.
In-vitro studies should include Ames’ Salmonella
assay and chromosomal aberrations (CA) in
cultured cells.
In-vivo studies should include micronucleus assay
(MNA) or CA in rodent bone marrow.

The following standard test are conducted-
(i) A test for gene mutation in bacteria.
(ii) An in vitro test with cytogenetic evaluation of
chromosomal damage with mammalian cells or an in
vitro mouse lymphoma tk assay.
(iii) An in vivo test for chromosomal damage using
rodent hematopoietic cells.

Knockout mouse
• A knockout mouse is a genetically engineered
mouse in which researchers have inactivated, or
"knocked out," an existing gene by replacing it or
disrupting it with an artificial piece of DNA.
• The loss of gene activity often causes changes in a
mouse's phenotype, which includes appearance,
behavior and other observable physical and
biochemical characteristics.

• They are widely used in knockout experiments,
especially those investigating genetic questions that
relate to human physiology.
• Humans share many genes with mice.
• Examples of research in which knockout mice have
been useful include studying and modeling different
kinds of cancer, obesity, heart disease, diabetes,
arthritis, substance abuse, anxiety, aging and
Parkinson's disease.

Transgenic Mouse
• Transgenics -the transfer of a gene into an
organism has great power because of its
specificity.
• The transferred gene can be derived from a variety
of sources, including the species itself.
• It allows new approach to life science research that
general cell culture techniques cannot deliver.

• The manufacture of large quantities of complex,
bioactive proteins like hormones or growth factors, for
the study of genetic regulation, animal development
or disease pathology.
• Examples currently in development include the
production of antithrombin III which is currently in
phase three clinical trials and alpha-1-antitrypsin.

BIOASSAY
It can determine the effect of any natural source of
unknown substances without affecting the
complete system.
Bioassay determines the quantitative relationship
between the concentration and magnitude of
response
The popularity of bioassay among the other
methods for assessment of any drug is due to
improved reliability, specificity, accuracy, sensitivity.

Application of bioassay
1) Estimate potency of natural drug
2) Standardization of drugs of natural origin
3) Screening of new compound for biological
activity
4) Estimation of biologically active substances like
histamine, acetylcholine, 5-hydroxytriptamine,
adrenaline, bradykinin, substance-P, PG.

Evaluation of analgesic method-
1) Tail clip method
2) Hot plate method
3) Tail flick method
4) Tail immersion test
5) Writhing test
Evaluation of anti-inflammatory method-
1) Rat paw oedema
2) Granuloma pouch
3) Experimental pleurisy
4) Adjuvant induced arthritis

Evaluation of drug acting on CNS-
1) Elevated plus maze, Hole Board apparatus, Light
dark expression induced anxiety in mice.
2) Forced swim test, tail suspension test induced
depression.
3) MES(maximal electric shock) and
Pentylenetetrazole induced convulsion in mice.
Evaluation of anti-Parkinsonian agents-
1) MPTP model
2) Oxotremorine model
3) LON-954 model

Evaluation of anticarcinogenic agents-
1) Ehrlich Ascites carcinoma(EAC)
2) Azoxymethane induced colon cancer
3) Solid tumour induced in Balb-C mice by 3-
methylcholanthrene(3-MC)

Evaluation of GIT functions-
Anti-ulcer agents assay-
1) Pylorus ligated rat
2) Restraint ulcer in rat
3) Drug induced gastric mucosal damage in rat
4) Histamine induced gastric ulceration in guinea
pig
Assay of Anti-secretory agents-
1) Continuous recording of gastric acid secretion in

Assay of drug affecting intestinal motility
1) Charcoal meal test in rat/mice
2) Castor oil induced diarrhoea in mice/rat
3) Intraluminal fluid accumulation in rat
Assay of drug affecting IBD
1) TNBS(Tri Nitro benzene sulphonic acid) induced
colitis in rat
2) Acetic acid induced colitis
3) Carrageenan induced colitis

Evaluation of anti-diabetic agents
1) Alloxan induced
2) Streptozotocin induced
Experiment on CVS
1) Doxorubicin induced cardiotoxicity
2) Isoproterenol induced myocardial infarction
3) Deoxycorticosterone acetate salt induced HTN.

Alternatives To Animal Experiment For
Evaluation Of New Drugs
 The scientific and lagislative authorities and
animal right activists throughout the world have
been demanding the abolition of the animal
experiments in the laboratory & development of
some alternatives.
 Ressell and Burch in 1959 developed the concept
of 3 R’s alternatives that can minimize to a great
extent the use of animals---
1) Refinement
2) Replacement

Acute toxicity tests-
Normal Human Keratinocyte (NHK) and other
standardized cell lines are being increasingly use
in Acute toxicity tests.
In addition there are several computer software
packages for predicting acute toxicity from the
chemical structure of the test compound.

Repeated dose toxicity test-
The repeated dose toxicity study in animal is now
a days replaced by a combination of
computerized biokinetic modeling and organ
specific in vitro assays(eg. Brain, liver, kidneys
etc).
Carcinogenicity test-
Syrian Hamster Embryo(SHE) and Balb/c 3T3 assay
can be used as non-genotoxic method for testing
carcinogenicity.

Experimental Techniques For Evaluation Of New Drugs.ppt

Experimental Techniques For Evaluation Of New Drugs.ppt

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