CHAPTER 6.

Introducing clinical
trials

1
Introduction: Experimental design
• An experiment can be defined as planned research conducted to obtain new
facts, or to confirm or refute the results of previous experiments
• An experiment helps a researcher to get an answer to some question or to
make an inference about some phenomenon
• In a narrow sense, an experiment is conducted in a controlled environment in
order to study the effects of one or more categorical or continuous variables
on observation
• The planning of an experiment begins with an introduction in which a
problem is generally stated, and a review of the relevant literature including
previous results and a statement of the importance of solution of the problem
• An experimental design declares how to obtain data. Data can come from
observations of natural processes, or from controlled experiments

2
4.1. Clinical trial (under controlled conditions)
• A controlled trial is a planned experiment carried out on subjects maintained
in their normal (i.e. usual) environment

• A clinical trial is a systematic study in the species, or in particular categories of

the species, for which a procedure is intended (the target species) in order to
establish the procedure's prophylactic or therapeutic effects, safety and
efficacy

• Clinical trials date back to the 18th century, when they provided clues to the
cause of disease
• The provision of citrus fruits to English sailors prevented scurvy, indicating that
the cause (subsequently shown to be vitamin C deficiency) was nutritional
3
4.1. Clinical trial…
• In veterinary medicine, effects may include improvements in
production as well as amelioration of clinical disease
• The procedure may be a surgical technique, modification of
management (e.g., diet), or prophylactic or therapeutic
administration of a drug
• A clinical trial would also include studies of pattern of absorption,
metabolism, distribution of drugs within the body and excretion of
active substances
• The term clinical trial is often used synonymously for controlled trial.
However, some authors restrict its use to trials of therapeutic
products and/or trials carried out in a clinical setting

4
4.1. Clinical trial…
•Controlled trials: the best way for evaluating animal-health interventions
(better control of potential confounders than observational studies)
•CL is prospective study and is the best study design to address the questions about
treatment or prevention

•Clinical trial uses generally the Randomized Controlled Trial (RCT) format
•Controlled trials are valuable for assessing:-

• Factors affecting animal health and productivity, e.g.

• Management practices
• Nutrition
• Environmental changes
• Pharmacological products (therapeutic and preventive)
5
4.2. Phases of Clinical Trial
•Clinical pharmaceutical research can be divided into 4 phases
• Phase I trial (formulation trials)
• Are pharmacological and toxicity trials /studies carried out
in healthy animals (or lab animals) primarily to evaluate
safety of the drug, e.g.,

• To determine safe dosage ranges

• To identify adverse reactions etc.

6
4.2. Phases of Clinical Trial…
•Phase II trials are the first evaluation of the drug in a small number of animals
from the target population (e.g. sick animals)

• Initial trials of therapeutic effect and safety (Used to document the activity of
the drug)

• Usually conducted on the target species on a small scale (n=100 subjects) in a

controlled environment (e.g., on research establishments),
• Often with the object of selecting the potentially most attractive drugs from
those that are available

• Involve before/after comparisons

7
 4.2. Phases of Clinical Trial…
•Phase III trials are large-scale experimental studies to determine the efficacy of a drug in a normal
clinical population, to monitor side effects and to compare the drug with other available treatment

• Phase III trials are much larger (thousands of patients with the condition)

•These studies should be based on randomized controlled trials

•Carried out before the registration of products for human use

•Studies carried out for the purpose of registration of animal products (Phase III trials)

8
4.2. Phases of Clinical Trial…
•Phase III trials
• Need to be carried out according to good clinical practice (GCP) standards

• GCP is a standard for the design, conduct, monitoring, recording, auditing,

analysis and reporting of clinical studies (right of patients, confidentiality,
credible and accurate result

• Are randomized trials comparing the medication with established treatments to

show if it is safe and effective

• The results of several randomized trials are often combined, sometimes using a
systematic review and sometimes a meta-analysis.

9
4.2. Phases of Clinical Trial…
• The basics on how to comply with GCP
1. Write a good protocol -Weigh risks vs. benefits
2. Obtain IRB/IEC approvals
3. Protect the subjects –
Obtain Informed Consent,
Ensure safety, rights & confidentiality
4. Use qualified study team
5. Handle investigational products appropriately
6. Implement quality systems
7. Record and analyze information appropriately
8. Follow the protocol and trial SOP’s!!!
10
4.2. Phases of Clinical Trial…
•Phase IV trials
• Are post-registration trials (after a license has been issued) designed to evaluate the
most effective way of using a product

• Carried out as randomized controlled trials

• Post-authorization surveillance of a drug after it has been licensed, to monitor

adverse drug reactions and provide more information on longer-term safety and
side effects, and also on how well it works with other conditions.

11
4.3. Randomized Controlled Trial (RCT)
• Also called "Randomized Clinical Trial"
• The mainstay of experimental medical studies, normally used in
testing new drugs and treatments
1. A sample of patients with the condition, and who meet other
selection criteria, are randomly allocated to receive either the
experimental treatment, or the control treatment (commonly the
standard treatment for the condition). 
2. Occasionally, a placebo or sham treatment will be used in the
control group, but where there already is a standard treatment, it is
unlikely to be ethical to use a placebo.
3. The experimental and control groups are then followed for a set
time, and relevant measurements are taken to indicate the results (or
'outcomes') in each group. 12
4.3. Randomized Controlled Trial (RCT)…
• Not possible to use RCTs to test effects of exposures that are expected to be
harmful, for ethical reasons
• Advantage
• Controls for all main forms of bias
• Good for both etiologic and evaluative research
• Disadvantage
• Often uses selected populations: issues of generalizability?
• Very expensive
• Ethical concerns in etiological applications
• Not appropriate to answer certain types of questions
• it may be unethical, for example, to assign persons to certain treatment or
comparison groups
13
14
4.3. Randomized Controlled Trial (RCT)…
• The "random" of RCT refers to:-
• Random allocation to either experimental or control group;
• It does not refer to random selection or sampling of the patients to include in
the trial
• Do not confuse these two concepts:
• Random selection of a sample ensures that the sample is representative of the
broader population; it is typically used in a survey (an observational study)
•  Random allocation ensures the experimental and control groups are
equivalent, but does not ensure they are representative of all patients with the
condition.
• Random allocation is used mainly to avoid confounding
• The major type of experimental study
15
 4.3. Randomized Controlled Trial (RCT)
…
• To make sure that any differences in the final outcome
measurements were due to your experimental treatment and
not to a confounding factor, you want the two groups to be
comparable on all other factors (you want to control all other
factors)
• If you know about a confounder before beginning the
experiment, you could match the two groups on it (e.g.,
ensure equal numbers of males and females in each group)
• Randomization protects against all potential confounding
factors, both known and unknown
• A basic rule in running a trial is to include everyone in the
analysis, including those who drop out 16
17
4 .4.Clinical field trial (Under natural field conditions)

• It is conducted under operational conditions;

• It is frequently prophylactic, and therefore relies on natural

challenge to the treatment that is being assessed
• e.g., assessment of the efficacy of a bacterial pneumonia
vaccine would rely on vaccinated animals being naturally
exposed to infection with the relevant bacterium during
the period of the trial
• Field trial is invariably conducted on clinically healthy
individuals to determine prophylactic effect (primary
prevention) 18
4.5. Importance of design in the clinical trial

• An important feature in the design of a controlled trial is the

development of a detailed study protocol which covers all
elements of the study design and execution

• Important elements to be considered include:

• Stating the objectives
• Defining the study population
• Allocation of subjects
• Specifying the intervention
• Masking (blinding)
19
4.5. Importance of design in the clinical
trial…
• Follow-up and compliance

• Specifying and measuring the outcome

• Analysis of trial results

• Ethical considerations

20
4.6. Importance of control group

• Historical control trials are ones in which the outcome after an

intervention is compared with the level of the outcome before the
trial (before/after comparison)

• For example, a vaccine for neonatal diarrhea might be introduced

into a dairy herd and the incidence of diarrhea in the year after
vaccination compared with the incidence in the year before

• Generally unacceptable, no blinding

21
4.6. Importance of control group…
• For a historical control trial to have any validity, four criteria must be
met
• The outcome being measured must be predictable (e.g. constant
incidence of neonatal diarrhea from year to year)

• There must be complete and accurate databases on the disease of

interest

• There must be constant and specific diagnostic criteria for the

outcome

• There must be no changes in the environment or management of 22

4.7. Assignment of animals to the treatment group
• Need for random assignment
• Randomization is the process by which experimental units (the basic objects
upon which the study or experiment is carried out) are allocated to
treatments; that is, by a random process and not by any subjective and hence
possibly biased approach
• Randomization helps avoid bias
• Formal randomization is the preferred method of allocation
• Does not mean 'haphazard' allocation
• The use of this technique should produce a representative sample, typical of
the population under investigation
• It is assumed that through randomization confounding variables will be evenly
distributed across different groups so that they are comparable, with only the
independent variable distinguishing them
23
4.8. Methods of randomization
• 1. Simple randomization
• Is the most basic type of randomization

• When there are only two treatments, tossing a coin is an

elementary method. However, it is usually more rigorous to
randomize in advance using random numbers

• Allocating units identified by odd numbers to one group, and

evenly numbered units to the other

• Randomization should be applied after eligible units have been

identified 24
4.8. Methods of randomization…

•2. Stratified randomization

•Some factors (e.g., age, parity or severity of disease) may be known to
affect the outcome of a trial and may bias results if they are unevenly
distributed between the treatment and control groups
•This can be taken into account during initial randomization by stratifying
(i.e., matching) both groups according to these confounding factors
•The experimental units are then allocated to R x and C groups
within the strata, using simple or block randomization
•helps ensure that a potential confounder (age) is equally distributed
across study groups

25
4.8. Methods of randomization…
2. Stratified randomization…

• One specific form of stratified randomization is random allocation of

animals within herds

• This ensures that all herd factors that might influence the outcome are
balanced across study groups

• Stratification leads to related samples and therefore decreases the

number of units that are required to detect a specified difference
between treatment and control groups
26
4.8. Methods of randomization…

• 3.Blocked randomization

• Requires the random allocation of subjects within blocks

of subjects as they enter the trial

• Enhance statistical efficiency (ensuring equal numbers of

subjects in each study group)

27
 4.9. Blinding
• A key component in the effort to prevent bias in controlled trials is the use of
masking (or blinding)
• single-blind study:
• the participant is unaware of the identity of the intervention going to the
study subjects
• help ensure equal follow-up and management of subjects in the various
intervention levels
• Double blind study:
• both the participant and the study team (i.e. people administering the
interventions and assessing the outcomes) are unaware of intervention assignment
• helps ensure equal assessment of the subjects in different intervention levels
• Triple-blind study:
• the investigators analyzing the data are also unaware as to which group received which
treatment
• designed to ensure that the analysis is conducted in an unbiased manner
28
4.10. A placebo
• Is a product that is indistinguishable from the product being evaluated and which
is administered to animals in the groups designated to receive the comparison
treatment

• In many drug trials, the placebo is simply the vehicle used for the drug, but
without any active ingredient
• A placebo is an inactive treatment or procedure

• When people know they are taking medication or receiving a treatment, they can
gain a psychological and neurobiological boost which improves their condition.
This is known as the placebo effect

• The 'placebo effect' (usually a positive or beneficial response) is attributable to the

patient's expectation that the treatment will have an effect
29
4.11. Increasing the precision of the estimates
• Replication
• In the experimental design it is necessary to define treatments (populations),
size of samples, experimental units, sample units (observations), replications
and experimental error

• The definition of a population (usually some treatment) should be such that

the results of the experiment will be applicable and repeatable

• From the defined populations, random and representative samples must be

drawn.

30
 4.12. Concept of blocks…
• Complete randomized block design
• The term randomized block emanated from agronomic research wherein several
variables or treatments are applied to different blocks of land to study the effect
of replication on experimental effort, such as, yield of different types of
sugarcane by using variable amounts of water to irrigate the fields.

• is a design in which the subjects are matched according to a variable that the
experimenter wishes to control

• The subjects are put into groups (blocks) of the same size as the number of
treatments

• The members of each block are then randomly assigned to different treatment
groups
31
4.13. Quasi-experimental designs
• In these, there is an intervention, but it is often not completely
planned by the person doing the research
• Is observational study, but there was also an intervention, although it
was not the experimenter who decided when and how the change
would occur and to whom it would be applied, so this is a "quasi-
experiment."
• Typically, random allocation is not involved.

32
4.14. Cross-over trials
• A crossover study, also referred to as a crossover trial/Change-over
trial/ is a longitudinal study in which subjects receive a sequence of
different treatments  (or exposures)
• Patients (experimental units) cross over from one treatment to
another during the trial course
• In contrast to a parallel design where patients are randomized to a
treatment and remain on that treatment throughout the trial duration
• Use the participant as their own control
• Each participant gets more than one treatment
• While crossover studies can be observational studies, many important
crossover studies are controlled experiments
33
4.14. Cross-over trials…
•A crossover trial has a repeated measures design in which each patient
is assigned to a sequence of treatments, including at least two
treatments (of which one may be a standard treatment or a placebo)
• Popular in medicine, agriculture, manufacturing, education, and
many other disciplines
• Not preferred routinely because of the problems inherent with this
design
• Subjects are randomly allocated to study arms where each arm
consists of a sequence of two or more treatments given consecutively

34
 4.14. Cross-over trials…
• The simplest model is the AB/BA study. Subjects allocated to the AB
study arm receive treatment A first, followed by treatment B, and vice
versa in the BA arm

• Crossover trials allow the response of a subject to treatment A to be

contrasted with the same subject's response to treatment B.

• Removing patient variation in this way makes crossover trials potentially

more efficient than similar sized, parallel group trials in which each
subject is exposed to only one treatment

• In theory treatment effects can be estimated with greater precision

given the same number of subjects 35
4.14. Cross-over trials…
• Not preferred routinely because of the problems inherent with this design
 In medical clinical trials the disease should be chronic and stable, and
the treatments should not result in total cures but only alleviate the
disease condition

 If treatment A cures the patient during the first period, treatment B will
not have the opportunity to demonstrate its effectiveness when the
patient crosses over to treatment B in the second period

 This type of design works only for those conditions that are chronic, e.g.,
asthma where there is no cure, and the treatments attempt to improve
quality of life
36
4.14. Cross-over trials…
• Nearly all crossover designs have "balance", which means that all
subjects should receive the same number of treatments and that all
subjects participate for the same number of periods
• In most crossover trials, in fact, each subject receives all treatments,
usually in a random order
•The issue of "carry-over" between treatments, confounds the estimates
of the treatment effects
•"carry-over" effects can be avoided with a sufficiently long "wash-out"
period between treatments
•However, planning for sufficiently long wash-out periods requires expert
knowledge of the dynamics of the treatment, which is often unknown.
37
4.14. Cross-over trials…
• Advantages of cross-over designs
 each participant acts as their own control
 removes variability between participants,
 fewer subjects needed.
• Disadvantages of cross-over designs
 Main disadvantage: Carryover effects may be aliased (confounded) with direct
treatment effects, i.e., these effects cannot be estimated separately
 A carryover effect: the effect of the treatment from the previous time
period on the response at the current time period
 Significant carryover effects can bias the interpretation of data analysis, so
it should be proceeded cautiously whenever considering the
implementation of a crossover design

38
4.14. Cross-over trials…
 How to deal with this carryover effect?
 The incorporation of lengthy washout periods in the experimental design
can diminish the impact of carryover effects
 A washout period: the time between treatment periods

 How long of a washout period should there be?

 Based on an investigator’s expertise
 For example, in a trial involving pharmaceutical products a washout
period equivalent to 5 (or more) times the length of the half-life of the
drug concentration in the blood
 Recommendation: avoid the problems caused by differential carryover
effects at all costs by employing lengthy washout periods and/or
designs where treatment and carryover are not aliased with each other

39
4.14. Cross-over trials…
• Cross-over trials are suitable for:
• chronic diseases (asthma, arthritis)
• symptomatic treatment
• quick, quantitative outcome (attack frequency, lung function, pain
scores)
• early stage in treatment development.
• Cross-over trials are not suitable for:
• acute conditions (myocardial infarction, pneumonia),
• treatment to cure (clot-busters, antibiotics),
• slow or qualitative outcomes (time to recurrence, death),
• later stages in development (side effects of long term treatment).
40
4.14. Cross-over trials…
• A sequence: the order of treatment administration in a crossover experiment
• Sequences should be determined a priori, and the experimental units are
randomized to sequences
• A period: the time of a treatment administration
• 2×2 crossover design: 2-sequence, 2-period, 2-treatment, crossover design, with
sequences AB and BA
• Most popular crossover design
• Experimental units, randomized to sequence AB, receive treatments A and B
in the first and second period, respectively; experimental units, randomized
to sequence BA, receive treatments B and A in the first and second period,
respectively
• E.g., to examine the effects of replacing butter with margarine on the
lipoprotein profile of subjects with hypercholesterolaemia
41
4.14. Cross-over trials…
• Patients were randomized to a six week butter diet followed by a six week
margarine diet, or the reverse sequence

• Treatment periods were separated by five weeks' washout in which patients

returned to their usual diet

• The impact on lipoprotein profiles was measured from blood specimens taken in
the last week of each experimental period

• The assumptions are that six weeks is long enough for an

experimental diet to affect lipoprotein profile and that five weeks
is long enough for the effects to dissipate
42
4.14. Cross-over trials…
• Cross-over trials are only suitable if:
1) the effect of treatment is reversible;

2) the period of treatment is not too long;

3) the condition is relatively stable;

4) it may reasonably be believed that carry-over is not a

problem

43
• Table1 : 2×2 crossover design (AB|BA)
Period 1 Period 2

Sequence AB A B

Sequence BA B A

Table 2: 3-period, 2-treatment crossover designs (ABB|BAA)

Period 1 Period 2 Period 3

Sequence ABB A B B

Sequence BAA B A A

44
Table 3: 3-period, 2-treatment crossover designs (AAB|ABA|BAA)

Period 1 Period 2 Period 3

Sequence AAB A A B

Sequence ABA A B A

B A A
Sequence BAA

Table 4: 3-period, 3-treatment crossover designs (ABC|BCA|CAB)

Period 1 Period 2 Period 3

Sequence ABC A B C

Sequence BCA B C A

Sequence CAB C A B

45
Table 5: 3-period, 3-treatment crossover designs (ABC|BCA|CAB|ACB|BAC|CBA)

Period 1 Period 2 Period 3

Sequence ABC A B C
Sequence BCA B C A
Sequence CAB C A B
Sequence ACB A C B
Sequence BAC B A C
Sequence CBA C B A

46
Results from clinical trials
• Typically the outcomes from a clinical trial are summarized using some measure of
incidence
• Incidence is the number of new events in the population or group over a particular
time period
• The event may be death, recovery, occurrence of a disease, relapse, a particular sign
or clinical finding, or any other specific measurement of interest
• The key element here is new events
• Therefore, the animal must be free of that event at the beginning of the study, so that
the event developed after the intervention groups were formed and intervention
initiated

47
Results from clinical trials…
•This means that there will always be at least two assessments for the event of interest, one at the beginning of
the study and one later in the study to confirm or deny the event occurred while the patient was undergoing
intervention
•Two common types of incidence are cumulative incidence and incidence density
•Cumulative incidence is the number of individuals that have the event during the study period divided by the
number of individuals that could develop the event at the start of the study (sometimes called population at risk)
•For example, in a randomized clinical trial on the efficacy of Escherichia coli antiserum in neonatal foals, 30 of 138
treated foals became ill with some health problem. This corresponds to a cumulative incidence of 22%
•Incidence density has the same numerator as cumulative incidence but is divided by the sum of the length of
time of observation for all individuals before the event occurred. This denominator is often measured in animal-
years at risk or animal-months at risk

•This measurement is used infrequently in veterinary medicine. However, it can be extremely useful in a setting in
which the time the animals were included in the study varied, as would happen if animals were entered into the
study across a 2-year time period and the study ended 3 years after the first animal was included

48
Results from clinical trials…
• Relative risk (RR) is the incidence in the treated group, divided by the incidence
in the control or standard treatment group
• RR greater than one indicates that the event occurred more often in the treated
group than the control
• RR of less than one means that it occurred less frequently in the treated group
than the control or that the treatment is protective for the event
• A RR of one means that there is no difference in the incidence between the two
groups and that there is no association or relationship between the event and
the intervention groups
• However, p-value and CI is needed

49
4.15. PILOT STUDY
• A pilot study, pilot project, pilot test, or pilot experiment
• Definitions: A pilot study is
• a mini-version of a full-scale study or a trial run /done in preparation of the
complete study. The latter is also called a ‘feasibility’ study. It can also be a
specific pre-testing of research instruments, including questionnaires or interview
schedules
• a ‘small study to test research protocols, data collection instruments, sample
recruitment strategies, and other research techniques in preparation for a larger
study
• is a small scale preliminary study conducted in order to evaluate feasibility, time,
cost, adverse events, and improve upon the study design prior to performance of
a full-scale research project
• A preliminary investigation intended to collect data to prepare for a larger, more
definitive study
50
4.15. PILOT STUDY…
• Generally small in size, scope, duration and budget

• A pilot study allows you to know what things go wrong so you can fix
them before you start the large study
• The pilot study will thus follow after the researcher has a clear vision of
the research topic and questions, the techniques and methods, which
will be applied, and what the research schedule will look like
• It is “reassessment without tears trying out all research techniques and
methods, which the researcher have in mind to see how well they will
work in practice. If necessary it can then still be adapted and modified
accordingly
51
4.15. PILOT STUDY…
• A pilot study is one of the important stages in a research project and
is conducted to identify potential problem areas and deficiencies in
the research instruments and protocol prior to implementation during
the full study
• Help members of the research team become familiar with the
procedures in the protocol, and can help them decide between two
competing study methods, such as using interviews rather than a self-
administered questionnaire

52
4.15. PILOT STUDY…
The importance of a pilot study

• To determine the feasibility of the study protocol and identify weaknesses in a

study
• To test whether the study instrument(s), is asking the intended questions,
whether the format is comprehensible and whether the selected validated tool is
appropriate for the target population
• To test the appropriateness of data collection using the selected interview
technique (face-to-face or telephone) or self-completed questionnaire (postal or
administered at the center)
• To test the data collection process – the time taken to complete questionnaire, and
the subjects’ willingness to participate in the study
• To test data entry, coding of the items, and appropriateness of statistical tests
• To obtain preliminary data for the primary outcome measure, in order to calculate
a required sample size (especially in randomized control trials).

53
4.15. PILOT STUDY
• Sample Selection
• By definition, working with small samples. Use as rigorous a strategy as possible
but recognize the risk of less representative samples
• Try to make sure that your pilot subjects cover the entire range of subjects in
your full study
• Do not slap on the label of pilot study when your sample size is too small
• Feasibility
• Beware of the tendency to propose or do too much
• Sample size
• Depends on the objective of the study
• Some pilots don’t require formal sample size calculations
• Enough observations to provide useful information
54
4.15. PILOT STUDY
• Design of the pilot is different from that actually employed in the trial
(leading to different effect sizes)
• Pilot is run by the interested, skilled, and dedicated researcher while
the follow-up confirmatory experiment is run by less interested,
skilled, and dedicated personnel (leading to reduced effect sizes in the
follow-up study)
• Analysis
• Be clear about how the data will be interpreted and utilized
• Analyses mainly descriptive although p values or confidence intervals
appropriate in some situations
• Treat results as preliminary and interpret with caution

55
4.15. PILOT STUDY
• Budget
• Usually for specific expertise (data management, statistics, consultant), supplies,
part of study assistant effort
• Usually not for investigator salaries, full time study coordinators, equipment, travel
• Pilots help understand resource requirements in full study
• Challenges
• Short time frame
• IRB approval: A pilot takes as long as a large study
• Competing demands on your time: Teaching load changes, more clinic or rounding
time
• Supply chain problems : Animals, reagents, databases hard to get
• Data inconclusive: Uninformative numbers
• Equipment breakdown: Flow cytometer, multiplex assay system not working?

56
Key Elements for Studies of Clinical Trials of Treatment or Prevention

1. Was there a concurrent control group?

2. Did the control group receive the current best intervention
rather than placebo (if appropriate)?
3. Were the subjects randomly assigned to the groups?
4. Were the groups similar at the beginning of the trial?
5. Was there blinding (single, double, or triple) to intervention
assignment?
6. Was there relatively complete follow-up of all subjects (>80%)
that entered the trial?
7. Were the results in the different groups relatively large and
clinically important?
8. Was a formal (and appropriate) statistical analysis performed?57