Success of the Research Work
Upon completion of the study, the research work should have made a worthwhile contribution to your field, providing (at least one of the following)2,3
In addition, the work should yield publication(s).
Algorithm for Developing a Protocol
In addition, all experimental protocols involving animals should be approved by the Institution’s animal ethics committee before a study begins. For a study involving human subjects, investigation shall not be initiated unless that study has been reviewed and approved, and remains subject to continuing review, by an institutional review board (IRB). Prior approval by an IRB may not be needed for an emergency use of a test substance provided that such emergency use is reported to IRB within five working days.9 For details, see Chapters 13 and 14. Some of the following elements have been discussed in the first four chapters.
Draft Research Protocol
You may need several drafts before the protocol is finalised by you and your guide/mentor.
Final Research Protocol
There are variations in the formats. Guidelines for writing protocols and conducting research are largely determined by the research guide/mentor, department and/or university. Most research protocols, however, contain the following elements:
Elements of a Research Protocol
Research Topic and Question
The most important component of a study protocol is the research topic and is best formulated as a research question(s). The research topic is the keystone of the entire scientific enterprise. It begins the project, drives the entire study and is crucial in moving the project forward.2,3,6 It dictates the remaining elements of the study and, thus, should not be too narrow (i.e. should not yield trivial results) or too broad (i.e. not researchable) or unfocused (i.e. should not drift). Because of these potential pitfalls, it is essential that a good or novel scientific idea should be based on a sound concept.
Selection of a research topic is based on assimilation of several ideas or, at best, developed in a step-by-step process. The problem is formulated based on (i) critical or intelligent observations made from an ongoing study in the investigator’s laboratory; (ii) discussion with your guide/supervisor and other teachers; (iii) conversation with your colleagues; (iv) identification of gaps in the literature on a topic or idea of study in which a researcher is interested and (v) adding additional important variables.2,5,8 Once you identify the niche area that is not overworked, you may want to develop a preliminary idea into a researchable topic. A topic must have a focus and, when formulated into a question, sets the framework. A research attitude is to continually question what is believed to be true or factual.6 A clear research question should have the following characteristics. First, it should concisely and clearly describe the study. Second, it should define realistic and achievable objectives that can be investigated using appropriate testing methods. Finally, the objectives should be translated or reframed into a workable or testable hypothesis (prediction).6,7,10,11
Choosing a Thesis Topic1–3,5,7–10
This is one of most crucial decisions that you will make in your research career as a postgraduate or a doctoral candidate.
Literature Search
The importance of a thorough ‘literature review’ in research is discussed in Chapter 20. The literature review is a theoretical orientation of the topic at hand to the current state of science in that area. It helps the researcher to identify a suitable topic for research, creates an awareness of studies already carried out, initiates arguments in developing a logical and reasonable hypothesis, adds variables to the study, identifies types of methods available to answer the research question and describes the tools available to collect and measure data generated in the study.2,5,11 Some of the best ideas come from reading research papers outside your area of interest.8
Significance
This is the rationale for the proposed research. It is written after an extensive review of the relevant literature and highlights the current state of knowledge relevant to the proposal. It is important to make a case for why the study is important at this time, what gaps in the knowledgebase it is intended to fill and how the results will be applied to further research in this field or related areas. Justify the need for conducting the study. Is this a study to confirm, disprove or extend previous findings, or is it intended to provide new findings? Will some kind of benefit accrue with the application of new knowledge derived from the proposed study? Will this study bring improvements or refinements in existing methodology? The researcher should provide arguments as to why the intended study is a step forward, that the final results will be meaningful, and will add to the published literature in the field.3 For example, ‘the results of the current study are intended to provide information on the relative bioavailability of losartan under fasted and fed conditions in support of an ongoing clinical study’.
Objectives (Aims, Goals or Purpose)
The research question or topic is divided into specific objectives. Be sure that all objectives are related. They should fit together to form a unified strategy. The objectives require us to be precise about methods and to define key terms. They are developed logically from a description of the research problem based on gaps in the knowledge. The objectives provide a new explanation of the related theory, and errors or shortcomings in methodologies and intervention, variables and data collection and analysis.
Objectives are sometimes organised into hierarchies: primary, secondary, tertiary or exploratory. This is common practice in clinical studies (see Chapter 14). Examples in pharmacology research include (i) determining the cause and effect relation between activation of extracellular signal-regulated kinase (ERK) 42/44 and the effect of protein kinase C (PKC) on PE-induced contractions in non-pregnant and pregnant ewe uterine arteries12 and (ii) determining if α-1 adrenoceptor-induced vasoreactivity is reduced by increasing endothelial nitric oxide (NO) bioavailability in spontaneously hypertensive rats (SHR). (Refer Chapter 3 for more discussion on ‘Objectives’.)
1. Title of the study: A randomised, double-blind, placebo-controlled ascending single-dose study of compound X in healthy male volunteers.
2. Objectives
(a) Primary: To assess safety and tolerability in healthy male subjects after a single dose of compound X.
(b) Secondary: To evaluate the pharmacokinetic profile in healthy male subjects after a single dose of compound X.
(c) Exploratory: To assess plasma pharmacodynamics such as plasma renin activity and plasma immunoreactive renin in healthy male subjects after a single dose of compound X; or to evaluate the incidence of peripheral oedema reported as an adverse event in healthy male subjects after a single dose of compound X.
Hypothesis
Development of each objective into a valid hypothesis will make all of the steps in the research process much easier and manageable while maintaining a focus on the study. For Durbin,7 a hypothesis is a formal statement of the research question. A hypothesis forces us to think carefully about what comparisons will be needed to answer the research question, and it establishes the format for applying statistical tests to interpret the results.13 Studies that utilise statistics to compare groups of data should have hypotheses. A good hypothesis helps in selection of an experimental design and drives the study to completion. Proposals are largely funded by the granting agency based on the intellectual strength of the hypothesis and the preliminary study data. All biological research, including discovery science, is hypothesis-driven. However, not all studies need be conducted with a hypothesis.14 For example, a complete genome sequence can be determined independent of a hypothesis. However, finding relationships among genes, between gene expression and subsequent protein levels, and between specific cell signalling pathways and gene/protein expression generally require a hypothesis.14 Likewise, studies in pharmaceutical sciences (including Pharmacognosy and Phytochemistry, Pharmaceutical and Medicinal Chemistry and Pharmaceutics) are commonly done without a hypothesis.
The hypothesis is tested using different designs, as there is no single correct design.15 The researcher should explain how the results to be obtained will be used to test the hypothesis. The hypothesis is the premise that the study is designed to prove or disprove (i.e. hypothesis testing).10,12 There are two kinds of hypotheses. The first is the null hypothesis. In this case, the hypothesis is that there is no difference between the groups being compared (e.g. a group treated with the test antihypertensive drug versus a group of control animals treated with an alternative drug or placebo), with respect to the measured variable (blood pressure [BP]). For example, both losartan and enalapril lower BP to a similar extent and thus there exists no difference between these two drugs. The second is the alternative hypothesis. In this case, the hypothesis is that a difference does exist between the groups being compared (e.g. a possible effect of treatment), with respect to the measured variable (e.g. BP). Losartan lowers BP by 20 mm Hg in the experimental group relative to control. This gives a magnitude of difference (e.g. 20 mm Hg) and direction (i.e. lowers BP).
The alternative hypothesis has two types. The two-tailed or non-directional alternative hypothesis gives the magnitude of the difference without indicating a direction, leaving open the possibility of whether it is positive or negative relative to the control. The claim is not of a superiority or inferiority to the control, but only that they are different. For example, there is a difference between the two treatments, such as losartan and amlodipine in lowering the BP in hypertensive individuals. Further, the treatment effect may be of a difference of 20 mm Hg. The one-tailed or unidirectional alternative hypothesis gives a direction in addition to the magnitude. It states that one treatment is better than the other, in effect, claiming superiority of a drug, since inferiority is ruled out. For example, amlodipine is better (i.e. direction) than losartan in controlling BP. A more detailed description on types of hypothesis2,4,13 is given in Section D (see Chapter 17).
1. Research Topic: Polyphenols improve endothelial dysfunction in SHR: involvement of NO dynamics and NADPH oxidase activity.
(a) Objectives
- Specific: To investigate endothelial function in isolated thoracic aortas obtained from SHR pretreated with polyphenols for 6 weeks by assessing the physiological response, NO and superoxide production, nitrotyrosine formation and the protein expression of endothelial NO synthase and NADPH oxidase.
- General: To verify that endothelial dysfunction in SHR is a result of heightened state of oxidative stress caused by increased production of reactive oxygen species and uncoupling of NO synthase.
(b) Hypothesis
- Endothelial dysfunction is associated with a heightened state of oxidative stress in spontaneously hypertensive rats.
- Polyphenols prevent the vascular oxidative stress, thus contributing to the beneficial effect on the vascular system.
(c) Questions
- Does treatment with polyphenols normalise endothelium-dependent relaxation in SHR?
- Does attenuation of vascular reactive oxygen species production contribute to the drug’s reduction in peroxynitrite formation?
- Do polyphenols prevent vascular oxidative stress by inhibiting NADPH oxidase activity?
- Can the study demonstrate that production of reactive oxygen species from the NADPH oxidase leads to uncoupling of NO synthase?
- Are there more than one mechanisms by which polyphenols protect endothelium, such as promoting eNOS activity and expression, inhibiting iNOS activity, increasing bioavailability of NO and attenuating oxidative or nitrative stress?
Experimental Methodology (Research Design and Methods)
The purpose is to describe how the study will be carried out. It includes elements such as study outline, experimental design, statistical analysis, materials, methods, instrumentation, measuring techniques, experimental procedures, data recording and collection, difficulties and limitations, and ethical and safety issues.
(Note: Similar steps are described for a clinical study, see Chapter 14.)
1. Objectives
2. Hypotheses
3. Choose variables: Dependent variables (e.g. response, character, outcome of treatment or surgery, such as measuring BP, heart rate, pain, inflammation) and independent variables (e.g. experimental treatment or pharmacological intervention, such as treatment of ileum with a receptor blocker or NO synthesis inhibitor, pretreatment of aortic rings with or exposure to phorbol ester, animals fed with cholesterol-rich diet for 4 weeks or coronary artery ligation). An independent variable has levels, whereas a dependent variable has values. The latter must be operationally defined, and methods for measuring it must be defined.16
4. Experimental design
(a) Experimental unit1,17: Before choosing an animal for the study, consider any ethical issues involved. Use the ‘3Rs’ principle,18,19 whether the animal model can be replaced by cell systems or in vitro studies (discussed in Chapter 11). The experimental unit includes the animal (species- and/or strain-specific characteristics for a specific study, sex, age, weight), animal model (disease model, e.g. spontaneously hypertensive rats, double transgenic rats), surgical interventions (e.g. descending coronary artery ligation to produce myocardial infarction), a group, a piece of tissue suspended in an organ bath, a flask, dish or well of cells that can be assigned at random to a treatment.18,17
(b) Experimental group size: Sample size per group. The number of animals for each group is critical (see Chapter 17 for determination of sample size and power).
(c) Control(s): Define the type of controls needed for the study based on variables encountered. A number of controls are identified: vehicle, negative or positive (e.g. used in genetic toxicology studies to demonstrate that a response can be detected and to validate the assay), sham (e.g. to mimic the surgical procedures), dietary (e.g. where a drug is administered to animals in the diet for fixed duration (e.g. carcinogenicity study) and comparative (e.g. an experimental drug is compared with a reference drug).
(d) Randomisation of animals to control bias, confounding variables and unknown sources of variation (see Chapters 17 and 18). Experiments should be conducted blind with respect to the treatment.
(e) Study design: On the basis of the experimental subject, nature of the investigation and variables chosen, different types of designs are available, including completely randomised, randomised block, Latin square, crossover, repeated measures, split-pot, incomplete block and sequential and factorial designs.4,17–21 (Also see Chapters 17 and 18.)
(f) Evaluating the logistics of the actual performance of the experiments.18
(g) Identifying the most suitable statistical analyses.
5. Experimental procedures (methods and materials): Preparation of drug solution, solubility and stability issues, selection of appropriate doses (based on pilot or dose-range-finding studies), treatment schedule, route of administration (oral or parenteral), setting up an in vitro/in vivo/in situ preparation, type and frequency of observations after drug treatment, sampling (blood, urine and other media collection) and processing of sample and analysis.
6. Instrumentation and measurements techniques.
7. Data recording and collection.
8. Data analysis and statistics (as in 4(f) above).
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