Animal Experimental Study

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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

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drug development: preclinical trial

history of AES

! since 2nd 4thcenturies 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

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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

! understandingof physiology, pathology, pathophysiology,genetics of diseases

! exploring and developingof new intervention (drugs, device,etc.) of health problems!

drugs:! efficacy (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 reallyworks and if it is safeto test on humans.

! Its about efficacy(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?...

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invertebrates vertebrates

"non-human primates

50-100 million vertebrates/yr.


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invertebrates

! fruit flies, worm

!#short life cycle, ease with large numbers

!

$different 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! #genetic engineering"model for a range of human disease

! physiology, pharmacology, toxicology, cancer research, neurologicalresearch, education#, 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$

! 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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ETHICALCONSIDERATION

e 3-Rs

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Care and Use of animals

use non-animal methods over animal methods(if possible)

use lower class of animalReplacement

use fewer animal for comparable levels ofinformation

use healthy animal, genetic homogeneity

obtain more information from the same no. ofanimal

Reduction

alleviate /minimize potential pain, suffering, ordistress

enhance animal welfare for their used

caring, treatment, non-invasive

Refinement

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(Russell & Burch, 1959)

how to do 3-Rs?...

Replacement

cell culture

computer models

human volunteers

epidemiologicalstudies

Reduction

experimentaltechniques

data analysis

sharinginformation

Refinement

less invasivetechniques

better medicalcare

better livingconditions

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pain & suffering

! does animal feel pain !suffering???

! 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.

%

Freedom from hunger and thirst

% Freedom from discomfort

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Freedom frompain, injury and disease

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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)

EXPERIMENTALDESIGN

Basic principles

Sample size calculation

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...basic principles...

remember, this anexperimental study

1. RQ

2. comparison/control

3.

replication4. randomization

5. stratification

6.

factorial experiment

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...basic principles...

remember, this anexperimental study

1. RQ

2.

comparison/control3. replication

4. randomization

5. stratification

6.

factorial experiment

1. RQ: does salted drinkingwater affect BP in mice?

Experiment:

1. provide a mouse with watercontaining 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 fedsalt water

!

ideally they are compared to concurrent controls (rather than tohistorical controls)

3. Replication

! will increase precision"decrease the sample size #

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4. Randomization:! to avoid bias and to control the chance

! RAL#

5. Stratification!

replication"measure BP in the morning and some in theafternoon (limited by personnel)

! different of BP between morning & afternoon measurement?"ensure that within each period: equal number of objects ineach treatment group

! latertake account of the diff. 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




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RANDOMIZED


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example




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STRATIFIED DESIGN#

#

#

6. Factorial experiments:

! to assess effect of > 1 treatment/intervention

! exp.: effect of both salt water & high-fat diet on the BP (2intervention

!

ideally: look at all 4 treatments in one experiments (&interactions among them)

plain water normal diet

salt water high-fat diet

! #we can learn more

more efficient

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x


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in terms of sample size

! Remember again:

!

too few animals"a total waste! (no power to show the effect)

! 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 othervariables- 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

!

Meads equation

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

E = N B T

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

n: n/treatment group

exp. t=3"n/group > 8.5 = 9

N total = 27

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

Federer WT. Experimental design, theory and app lication, 1967


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sample size: formula?

Meads equation

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E = N B T

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

N: total samples needed

B: blocking/stratification

T: no. of treatment groupExp.

T = 3, B = 0"(10-20) =N-3"

N = 13 23"5 7/group N total: 15 21

T (f.d) = 2x2x2 = 8

(10 20) = N (8)

N = 18 28"3/group

N total: 24

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

Meads equation

N total: 15 - 21

TWO TYPES OF AES

Pharmacodynamics study

Toxicity study

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Pharmacodynamics study(1)

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!

Route of administration:! Per Oral - similar to human use

! Dose:! Based on Dose-Response Relationship

! One or more doses that provide a desired effect

! Dose conversion from human to animal may be used

! Calculation of ED50

Pharmacodynamics study(2)

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! 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:

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Mutagenicity Test! Carcinogenicity Test! Reproductive& Development

Toxicity Test (teratogenicity)

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Animal model:infectious disease

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Animal model: metabolic 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 nextstep: clinical trial

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WHERE TO PUBLISH?

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Even the guidanceis 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

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thank you

Documents

Animal Experimental Study