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Animal Experimental Study

Rovina Ruslami

Outline
introduction (definition, history)
why doing AES
how to do AES
objects
animal model
sample size

ethical consideration
some examples
where to publish
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INTRODUCTION
definition
history

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what is AES?...
the use of non-human animals in experiments
although some research about animals involve only pure observation/
natural behavior
pure research: genetics, developmental biology, behavioral studies
applied research (experiment): biomedical research,
xenotransplantation, drug development, cosmetic testing
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terminology
animal experimental study (AES)
animal experimentation
animal research
animal model
animal testing
in vivo testing
vivisection

drug development: preclinical trial

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history of AES
since 2nd 4th centuries BC
2nd-century: vivisection by Galen
12th-century: animal experimentation (surgery)
18th-century: discovery of drugs insulin, AB, vaccine
Laika (Sovyet dog); Dolly the sheep
20th-century: toxicology
1960s:

Thalidomide safety testing on animal

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as usual
Debatepro- & anti-animal testing
increasing of AE on animal controversy

ethical issue
harm (for animal) vs. potential benefit (for human)
animal protection law

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WHY DOING AES?

for the science
for the benefit of humanity

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the need of
understanding of physiology, pathology, pathophysiology,
genetics of diseases
exploring and developing of new intervention (drugs, device,
etc.) of health problems
drugs:
ecacy (incl. pharmacokinetics, dose)
pharmacodynamics (mechanism of action)

testing (safety) before applying to human

drugs
safety
toxicology
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in general
1. Diseases process in human and animals have similarities
2. Cell systems contain or manipulate only a part of the
organ system.
3. Computer models lack the complexities of living entity

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...in drug development...

test of a new drug or a new medical device, done on
animal subjects, to see if the hoped-for treatment really

works and if it is safe to test on humans.

Its about ecacy (including pharmacodynamics: m.o.a)
and safety ( clinical trial)
before it goes into clinical trial (higher stage)

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AES in drug development

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HOW TO DO AES?
objects
animal model
sample size

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what animal?...
invertebrates vertebrates
non-human primates
50-100 million vertebrates/yr.

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invertebrates
fruit flies, worm
J short life cycle, ease with large numbers
L dierent immune system, simple organs
to identify active compound

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vertebrates
mice, rat, zebra fish, amphibians, cats, dogs, non-human primates
best model of inherited human disease
J genetic engineering model for a range of human disease
physiology, pharmacology, toxicology, cancer research, neurological
research, education J, endocrinology, reproduction, genetics,
HIV-AIDS drugs, vaccine, etc.
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source & fate

Source:

bred for a purpose lab, specialist suppliers (e.g. genetically

modified animal)
wild animal, bunching L

normal/healthy ones OR mutant

experiment could be:
usually one procedure
minutes, days, months or even years

fate: die because of

the experiment
euthanized after experiments
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Animal model
(of disease/condition)
to solve specific or practical problems
early stage of drug development
transgenic animal (inserted/modified/removed)
how and why disease develop?
test of new treatments

induced animal model

by virus, agent
inoculation of cancer cells
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Animal model: induction

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ETHICAL
CONSIDERATION
e 3-Rs

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Care and Use of animals

Replacement

use non-animal methods over animal methods

(if possible)
use lower class of animal

Reduction

use fewer animal for comparable levels of

information
use healthy animal, genetic homogeneity
obtain more information from the same no. of
animal

Refinement

alleviate /minimize potential pain, suffering, or

distress
enhance animal welfare for their used
caring, treatment, non-invasive
(Russell & Burch, 1959)

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how to do 3-Rs?...
Replacement

Reduction

Refinement

cell culture

experimental
techniques

less invasive
techniques

computer models

data analysis

better medical
care

human volunteers

sharing
information

better living
conditions

epidemiological
studies
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pain & suering

does animal feel pain suering???
testing causes distress & pain!
use of analgesia or anesthesia (local/general anesthesia)
what to use? what dose?
what if

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5-freedoms (Animal Welfare)

The council believes that the welfare of an animal ... should
be considered with reference to Five Freedoms.
v

Freedom from hunger and thirst

Freedom from discomfort

Freedom from pain, injury and disease

Freedom to express normal behaviour

Freedom from fear and distress

(Animal Welfare Council UK, 1993)

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post-mortem examination OR euthanized how?

injection/inhalation

physical (decapitation/cervical dislocation)

maceration grinding into small pieces
irradiation of the brain
captive bolts concussion of brain

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only after
animal is
unconscious
(anesthetized)

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EXPERIMENTAL
DESIGN
Basic principles
Sample size calculation

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...basic principles...
remember, this an
experimental study
1.
2.
3.
4.
5.
6.

RQ
comparison/control
replication
randomization
stratification
factorial experiment

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...basic principles...
1. RQ: does salted drinking
water aect BP in mice?

remember, this an
experimental study
1.
2.
3.
4.
5.
6.

Experiment:

RQ
comparison/control
replication
randomization
stratification
factorial experiment

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1. provide a mouse with water

containing NaCl 1%
2. for 14 days
3. measure BP
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2. Comparison/control:
good experiments are comparative
compare BP in mice fed with salt water to BP in mice fed plain water
compare BP in strain A mice fed salt water to BP in strain B mice fed
salt water

ideally they are compared to concurrent controls (rather than to

historical controls)

3. Replication
will increase precision decrease the sample size J
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4. Randomization:
to avoid bias and to control the chance
RAL J

5. Stratification
replication measure BP in the morning and some in the
afternoon (limited by personnel)
dierent of BP between morning & afternoon measurement?
ensure that within each period: equal number of objects in
each treatment group
latertake account of the di. between periods in stat. analysis
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example

20 M and 20 F mice randomized to be treated & untreated

can only work with 4 mice/day

Q: how to assign individuals to treatment group & to days?

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example

20 M and 20 F mice randomized to be treated & untreated

can only work with 4 mice/day

Q: how to assign individuals to treatment group & to days?

RANDOMIZED

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example

20 M and 20 F mice randomized to be treated & untreated

can only work with 4 mice/day

Q: how to assign individuals to treatment group & to days?

STRATIFIED DESIGN
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6. Factorial experiments:
to assess eect of > 1 treatment/intervention
exp.: eect of both salt water & high-fat diet on the BP (2
intervention
ideally: look at all 4 treatments in one experiments (&
interactions among them)
plain water normal diet
x
salt water
high-fat diet
J we can learn more
more ecient
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in terms of sample size

Remember again:
too few animals a total waste! (no power to show the eect)
too many a partial waste

AND..3-Rs reduction
variability
reduce number of treatment group, if possible
more homogenous object (i.e. in breeding, control other
variables- BW, age, sex, etc. if possible)
use stratification
multiple measurement more precise the mean of measurement
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sample size: formula?

Federers formula

(t-1) * (n-1) > 15

t: no. of treatment group (t.g)

n: n/treatment group
exp. t=3 n/group > 8.5 = 9
N total = 27
Federer WT. Experimental design, theory and application, 1967

Meads equation

E=NBT

E: error D.o.F (10 20)

N: total samples needed
B: blocking/stratification
T: no. of treatment group

Festing MFW, et al. Reducing the animal in lab BMR, ATLA, 1998
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sample size: formula?

Meads equation

E=NBT
Exp.
T = 3, B = 0 (10-20) =N-3
N = 13 23 5 7/group
N total: 15 21

E: error D.o.F (10 20)

N: total samples needed
B: blocking/stratification
T: no. of treatment group

Federers formula
T = 3 (t 1)* (n 1) > 15
2 * (n 1) > 15
n 1 > 15/2 (= 7.5 = 8)
n > 9 N total: 27

T (f.d) = 2x2x2 = 8
(10 20) = N (8)
N = 18 28 3/group
N total: 24
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Meads equation
N total: 15 - 21

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TWO TYPES OF AES

Pharmacodynamics study
Toxicity study

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Pharmacodynamics study(1)
Route of administration:
Per Oral - similar to human use

Dose:
Based on Dose-Response Relationship
One or more doses that provide a desired eect
Dose conversion from human to animal may be used
Calculation of ED50
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Pharmacodynamics study(2)
Control group:
Negative control
Solvent / vehicle group

Positive control
Standard drug group
To validate that a method works
To obtain Relative Potency of drug candidate or herbal medicines

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Toxicological studies(1)
General toxicity test
Short-terms
Acute Toxicity Test (calculation of LD50)

Long-terms:
Sub Acute Test ( up to 1 month)
Sub Chronic Test ( up to 3 months)
Chronic Test ( up to 6 months)
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Toxicological studies(2)
Local toxicity test:
Dermatological Preparation

Special toxicity test:

Mutagenicity Test
Carcinogenicity Test
Reproductive& Development
Toxicity Test (teratogenicity)

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Animal model: infectious

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Animal model: infectious

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disease

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disease

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Animal model: infectious

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Animal model: metabolic

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disease

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disease

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like other studies/research..

methods are very crucial
as well as ethical aspects
validity of the data
statistical analysis
important path before next
step: clinical trial

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WHERE TO PUBLISH?

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Even the guidance

is there

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take home message

AES is a path of finding the answer for health problems
avoid doing AES only for a narrow purpose
just do it for the right reasons
animal are a living creature
do it correctly, appropriately the 3-Rs

thank you
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