Bio-Ethics In Animal and Human

Studies
A/Prof Colin Dunstan
colin.dunstan@sydney.edu.au
 In Vivo Research
• In vivo research (literally “in life”) is research in
living animals
• in vitro research (literally “in glass” is research
done in glass (or plastic ...). Does include cell
culture
• A major difference for In Vivo Studies is the
ethical issue of conducting research in animals
capable of sensing pain, stress, fear, isolation
and environmental deprivation.
 Advantages and Disadvantages
Compared to organ culture, cell culture
and test tube
Advantages
• Direct relevance to whole animal physiology •
• Model complex interactions of several
systems ie assess pharmacokinetics of
dosed factor
• Model disease processes
• Can identify novel actions
• Effects likely to be predictive of human
physiology or pathology
Disadvantages
High variability due to low numbers, genetic
and environmental heterogeneity
• Complexity makes interpretation difficult ie
direct versus indirect effects
• Compensatory mechanisms may modify or
nullify effects
• Confounding responses are frequent
• Experimental animals may not reflect
human response
 How are the ethical issues
addressed in science

• Animal experimentation is accepted only when

strict guidelines can be met.
• The use of animals is regulated by legislation and
by Animal Welfare Committees.
• All animal studies must be conducted strictly
according to an approved protocol.
• Animal care and usage is monitored and
outcomes are reported. (In particular adverse
outcomes for the animal must be reported and
protocols reviewed to prevent repetition)
 Ethics – Should you do an Animal
Study

• Can the study be done another way?

• Is the study likely to produce novel and
scientifically valuable results, or have some other
significant value?
• Does the importance of the results justify the
level of invasiveness of procedures or potential
pain and suffering in the animal?
• Do investigators have the skill, knowledge and
facilities to conduct the study ethically
 Code of Practice
• It is the responsibility of the investigator to
ensure that all facets of animal care and use meet
the requirements of the Australian Code of
Practice for the Care and Use of Animals for
Scientific Purposes.
• This includes a responsibility to protect and
promote the welfare of animals used.
The Code of Practice embodies the
principles of:

– Reduction of animal use

– Replacement of animal use

– Refinement of animal use

 Ethics – Planning a study - 1
• Obtain ethical approval for Institutional Animal Care
and Use Committee

– This includes all procedures planned

– Amendments for any change in protocols need
approval
– More invasive procedures will receive higher
scrutiny and require more rigorous justification

• Use appropriate and minimal numbers

– Do not use excessive numbers

– Do not use insufficient animals to obtain
scientifically meaningful results
 Ethics – Planning a study - 2

• As much as possible prevent pain and suffering

pre-emptively

– Plan to use appropriate anesthesia, and analgesia

– Kill animals humanely
– Plan euthanasia for animals showing signs of distress.
– Use endpoints that precede induction of distress
– Provide environmental enrichment for the animals
 Ethics – Planning a study - 3

• Monitor animals
– frequently for signs of pain, distress, or other morbidity
– Continually while anesthetized for temperature,
respiration and recovery
– For changes in appearance
– For changes in behaviour – eating, drinking, grooming
– For weight loss
– Respond to adverse indications with euthanasia, or with
analgesia, suspension of experiment etcetera as
covered by protocol.
IRMA animal ethics application
 Transgenic Overexpression
Introduce multiple copies of a gene into the genome of a mouse under a
promotor to induce high levels of local protein expression.
• Constitutive promotor – protein expressed in all tissues ie actin
promotor
– Advantage: High exposure to the protein as expressed in all tissues
– Good for discovery studies where gene function unknown
– Disadvantage: Significant probability of lethal effects. Can occur
during development of the embryo. Phenotype can be complex.
• Tissue specific promotor eg apoE promotor to give liver expression
– Advantage: effects in a particular cell type or tissue can be isolated
– Disadvantage: Developmental effects can obscure normal
physiological role
• Conditional promotor – promotor regulated by nonphysiological
chemical administered by investigator eg tetracycline
– Advantage: Avoids the effects of compensatory pathways that
develop over time
– Disadvantage: Expression levels often low
Inducing transgenic overexpression of
a gene

Attach coding region of gene to

a promoter
Promoter can be universal,
tissue specific, or inducible

Inject multiple copies into a

fertilised egg for multiple
site incorporation in DNA

Select high expressing

mouse for breeding
Gene Knockout - Universal
 Generating a Knockout Mouse
Embryonic Stem Cell

Blastocyst

Surrogate

Heterozygote Heterozygote

Homozygote (knockout)
 Vitamin D Receptor Knockout

Aimling et al, Endocrinology 1999 140:4982-7

 Mice develop rickets
Normal VDR-/- Normal VDR-/-

Rickets in VDR-/- can be cured by high Ca/lactose diet but

not by vitamin D
Aimling et al Endocrinology. 1999 140(11):4982-7.
Gene Knockout - Selective
 Breed Loxp mice with Cre recombinase expressing
mice to produce tissue specific gene knockout

Make a mouse with Loxp sites

around gene to be excised
Overexpress Cre
with a tissue
specific promoter
+/- inducible

Homozygous for Lox P insertion

Mate to make a mouse with gene

deletion only in target tissue
 Develop genetically modified mice to determine
gene function
Whole Body Radiographs Show Osteoporosis and Vertebral Deformity in OPG
Knockout Mice

+/+

-/-
6 months
 OPG Deficiency in Mice Produces High
Bone Turnover Osteoporosis and
Changes in Cortical Architecture

Wildtype

OPG-/-
 In Vivo Tools - Genetic Manipulation
• Gene Knockout
– Disrupt gene in germ line to produce no expression or a
totally inactive product
Advantages Disadvantages
• Guaranteed gene deficiency • Redundancy or compensation
• Phenotype reveals function may mean no phenotype
• Phenotype may reveal • Deletion may be lethal
unanticipated function • Phenotype could be complex
• Provides animal model for • Challenge may be needed to
challenge studies unmask phenotype

– Selective gene disruption of a gene in a particular cell type

of tissue
Advantages
• Avoid lethality
• Address role in specific tissue
• Knockout can be timed to
occur in adult tissues
Disadvantages
• knockout may be incomplete
• Methodology is complex
• Knockout may not be as
completely tissue restricted
 In vivo Methods in Drug Development

Nucleotide sequence from human or mouse genome for protein of unknown function
Discovery
•Tissue distribution of expression
•Effects of overexpression in a transgenic mouse
•Effects in vivo of recombinant protein
•Effect of gene deletion
Translation
•Action of drug (recombinant protein, antibody, small molecule of antisense
construct) in animal models of human disease
•Determine pharmacokinetics of handling of drug (2-3 species)
•Determine safety margin between efficacy and toxicity in animal models (usually 2
species)
 Use of Challenges

Includes dietary, metabolic or hormonal

perturbations, induction of disease,
• Demonstrate function
– Unmask phenotype
– Exaggerate effects
• Demonstrate link to disease processes
• Illustrate therapeutic potential of a gene as a
target.
Osteoprotegerin blocks cancer
induced bone destruction

No cancer cells Cancer cells Cancer cells and

OPG
 Ultimate In vivo Methods in Drug
Development Involve Human Studies

• Test for safety in human subjects “Phase 1

studies” 20-100 volunteers
• Test for safety and biological activity in human
subjects “Phase 2 studies” 100-500 patients
• Test for safety and efficacy in a large human
population “Phase 3 studies” 500 to 5000
patients
Trial Documentation required by TGA at
time of trial commencement

• Patient informed consent and any other information documents supplied

• Documentation of financial agreements between the sponsor and investigators
• Insurance statement – to ensure compensation for trial-related injury or
misadventure.
• Signed and Dated approval from the Human Research Ethics Committees
concerned of protocol, investigator’s
brochure, consent forms, Case Report Forms (CRFs).
• Copy of Clinical Trial Notification (CTN) form or Clinical Trial Exemption (CTX)
approval where applicable.
• Curriculum Vitae of investigator(s) and sub-investigators.
• Certificates of Analyses and compliance with Good Manufacturing Practice (GMP)
for investigational products (GMP certification not required for first experimental
studies in human volunteers).
• Decoding procedures for blinded trials.
• Master Randomisation list.

The Australian Clinical Trials Handbook (March 2006)

 Human Research Ethics
Committee
a) The trial is designed to answer a specific question or questions;
b) There is a scientifically reasonable hypothesis to be investigated which suggests any
experimental treatment is at least as good as the current treatment available
(including no treatment);
c) Where the research is intended to be therapeutic, there is an acceptable risk/benefit
balance based on the known scientific information available;
d) The methodology is adequate to demonstrate selection rationale; recruitment
method(s); appropriate informed consent; a clear description of the proposed
intervention(s) and observation(s); and a sample size adequate to demonstrate
clinically and statistically significant effects.
e) There is adequate expertise available to advise on the safety of the medicinal
products or other interventions available;
f) The protocol conforms to the National Statement, the ICH Note for Guidance on GCP
(CPMP/ICH/135/95), and any other Commonwealth, State or Territory law.
g) Any financial arrangement on the part of investigator(s) and sponsor(s) is disclosed;
h) Aspects of budget for the trial that impact on ethical acceptability have been
addressed, including capitation fees, payments to researchers, institutions or
organisations involved; current and consequential institutional or organisational
costs and costs incurred by participants;
i) Adequate compensation is available to subjects in the event of any trial-related injury.
j) Procedures are in place for regular reporting of trial progress; serious or unexpected
adverse event reporting; reporting of significant new information regarding the trial
products; reporting of premature trial discontinuation, and in the case of medical
devices, that a system is in place to track the recipient for the lifetime of the device.
The Australian Clinical Trials Handbook (March 2006)
 Protocol
• General and Background Information
• Trial Objectives and Purpose
• Trial Design
• Selection and Withdrawal of Subjects
• Treatment of Subjects
• Assessment of Efficacy
• Assessment of Safety
• Statistics
• Direct Access to Source Data/Documents(audits)
• Quality Control and Quality Assurance
• Ethics
• Data Handling and Record Keeping
• Financing and Insurance
• Publication Policy

The Australian Clinical Trials Handbook (March 2006)

 Principles of Informed Consent

The informed consent process relies on three

principles:
• adequate information is provided (generally, what
a "reasonable person" would want to know to
make a decision)
• participants comprehend the information
• consent is given voluntarily.

National Cancer Institute http://www.cancer.gov/clinicaltrials/conducting/informed-consent-guide

 Informed consent is a process
• An initial meeting during which a member (or
members) of the research team provides you with
the informed consent document and explains its
content to you.
• Time to digest the information.
• Assessment of your understanding. The research
team should take some steps to ensure that you
comprehend the information
• Opportunities to ask questions.
• Continuing updates on new information.

National Cancer Institute http://www.cancer.gov/clinicaltrials/conducting/informed-consent-guide

 Informed consent should contain
• TITLE
• PURPOSE
• DESCRIPTION OF PROCEDURES
• DURATION
• RISKS
• BENEFITS to you or to others which may reasonably be
expected. A trial may or may not involve direct medical
benefits to you, but it might lead to new knowledge that
can help others in the future.
• ALTERNATIVES TO PARTICIPATION
• CONFIDENTIALITY
• COSTS/ ADDITIONAL EXPENSES (level of insurance)
• PARTICIPANT'S RIGHTS
• CONTACT INFORMATION
• SUPPLEMENTAL INFORMATION
• THE SIGNATURE
National Cancer Institute http://www.cancer.gov/clinicaltrials/conducting/informed-consent-guide
CHILD PARTICIPANT ASSENT FORM
(approximate ages 7-12)
[TITLE OF STUDY]
[SAMPLE WORDING OF A DRUG STUDY ASSENT]
Dr. __________ is doing a research study to find out how a new medicine works in kids who
have (name of condition in common term). You are being asked if you want to be in this
research study because you have (name of condition).
If you decide that you want to be in this research study, this is what will happen to you:
1. Dr. ______ will give you some medicine to take for the next 5 days. It might be the new
medicine or it might be the medicine that you would get if you weren’t in this research study. You
won’t know which one you get and Dr. ______ won’t know what you get either.
2. You will have to have a needle stick today (like a shot) to take some blood out of your arm.
Dr. _______ has to take some more blood out of your arm in 5 days to find out if the medicine
you are taking is making you better. And even if you don’t want to do the research study, you
are still going to get a needle stick today because you’re sick.
3. If you still are not getting better after 5 days, Dr. ____________ will (tell them what to expect
such as being given another medicine or other treatment).
Sometimes kids don’t feel good after they take the medicine. They might feel these things:
•an upset stomach, very sleepy, a headache, get a rash on their skin
If you feel any of these things, be sure to tell your mom or dad.
You don’t have to be in this research study if you don’t want to. Nobody will be mad at you if you
say no. Even if you say yes now and change your mind after you start doing this study, you can
stop and no one will be mad.
Be sure to ask Dr. ______ to tell you more about anything that you don’t understand.
§Yes, I will be in this research study.
§No, I don’t want to do this.
_____________________________________________ National Cancer Institute
Write your name on this line http://www.cancer.gov/clinicaltrials/conducting
____________________ Date /informed-consent-guide/page5
 OPG Deficiency in Mice Produces High
Bone Turnover Osteoporosis and
Changes in Cortical Architecture

Wildtype

OPG-/-
 Pharmacokinetics: Animals are used to
determine how drugs are absorbed and
cleared to help determine dosing

• Animals are given a drug (typically orally, intravenously or

subcutaneously)

• Time dependent changes in drug concentration are

determined by frequent timed blood sampling

• Kinetics of absorption are calculated from the

concentration curves
Serum Concentration Profile of AMG 162
Mean + SE
0.01 (n=6)
ng/mL 0.03 (n=6)
0.1 (n=6)
0.3 (n=6)
4 1.0 (n=6)
10 3.0 (n=6)

3
10

2
10

1
10

0
10

10 -1
0 1 2 3 4 5 6 9
Study Month
 Toxicology Studies
(one of the more contentious forms of
animal experimentation)
• Generally two species used: rats and one larger species (rabbit or
dog are common)
• Animals receive escalating doses of the drug
• Dosing continues until the maximum planned dose reached or
toxicity is detected (illness or death in a proportion of animals)
• After euthanasia, animals are thoroughly investigated to identify
tissue pathologies
• Results direct dosing in humans, and monitoring for adverse
effects during clinical trials.
 Conclusions
• In vivo (in animals) methods can be very powerful experimental
tools
• Research with animals must be ethically justified
• Can study be done another way?
• Is the study likely to produce novel and scientifically
interesting results, or have some other significant value?
• Does the importance of the results justify the level of
invasiveness of procedures or potential pain and suffering in
the animal?
• Do investigators have the skill, knowledge and facilities to
conduct the study ethically
• Research must be approved by an animal welfare committee
• Minimal numbers must be used
• Pain and suffering must be anticipated and minimised
• Animals must be monitored closely

• Be aware of the limitations, there are many pitfalls

 Topic-specific Learning Objectives
• Students should understand the ethical issues in using
animals
• They should understand the questions researchers should
ask themselves before undertaking animal research
• The principles of the three R’s; Replacement, Reduction
and Refinement, in animal experimentation should be
understood.
• The students should be generally familiar with the
approaches for genetically modifying mice for research .
• The students should understand the concept of the use of
animals to model human diseases and the advantages
and disadvantages of these animal models.