BMS 3008: Integrated Biomedical Sciences Lecture 3: Experimental Design and Clinical Trials

Part 1: Hypothesis-Driven Laboratory Project

• This section discusses a hypothesis-driven laboratory project that uses experimental procedures for data interpretation.

• It investigates the mechanisms of neural blast cell differentiation regulation by a growth factor, acting as a Local Mediator.

• As an organism develops, more stringent restraints are placed on cells to prevent uncontrolled growth.

• Growth factors are peptides released by signaling cells to affect target cells.

• Several growth factors control the differentiation of neural cells.

Regulating Neural Cell Differentiation

• Changes in gene expression are key for differentiation.

• Two approaches to identify genes involved:

  • Candidate approach: Informed analysis of genes thought to change with growth factor treatment, using quantitative PCR (limited output).

  • Global analysis: RNA-sequencing to interrogate all gene expression changes and identify gene cohorts involved in differentiation.

Principles of RNA-Sequencing

• RNA sequencing involves:

  • Isolating RNA from samples.

  • Fragmenting RNA into short segments.

  • Converting RNA fragments into cDNA.

Outcomes of RNA Sequencing

• By aligning sequencing reads to the human genome, all transcripts expressed in cells can be identified; this is called a Transcriptome.

• Transcriptomes can be compared between different cell populations (e.g., treated with and without growth factor) to identify global transcriptional changes during differentiation.

• Heatmaps are used to display changes to gene expression, with individual replicate samples as columns, genes on the right, and expression level indicated by color.

PhD Project Example: Identifying Key Regulators

• A PhD student, James, is identifying genes regulating neural cell differentiation.

• He treats undifferentiated neural cell populations with and without growth factors and performs RNA-seq analysis.

• He identifies numerous genes showing differential expression in response to Growth Factor 2 treatment.

• One of these genes is Synaptophysin.

• Question: What is the direction of change?

Validating Synaptophysin mRNA Levels

• The effect of growth factor treatment on synaptophysin mRNA levels has not been previously reported.

• Key question: Does it lead to an increase in synaptophysin protein level?

• Validating synaptophysin mRNA levels are elevated upon cell differentiation using Quantitative RT PCR (reverse transcriptase PCR).

• Steps:

  • Isolate RNA

  • Generate cDNA by reverse transcription

  • Determine level of cDNA for sequences of interest by quantitative-PCR

Techniques to Assess Synaptophysin Protein Level Changes

• Techniques include:

  • Western blotting: detect protein hybridised to a membrane by antibodies.

  • Immunofluorescence: detect proteins directly in intact cells by antibodies, providing cellular distribution information.

  • ELISA: quantifies proteins bound by antibodies on plastic.

  • Flow cytometry: quantifies proteins in cells using fluorescently-labelled antibodies.

Synaptophysin Western Analysis

• Synaptophysin Western analysis indicates synaptophysin levels increase in response to growth factor.

• Additional controls are required for this assumption to be correct, such as α-Tubulin.

Indirect Immunofluorescence

• Indirect Immunofluorescence used to detect native synaptophysin in cells.

• Process:

  • 1° Antibody Binding

  • 2° Antibody Binding

  • Fluorophore activation

  • Light emission detected by fluorescent microscope

• Advantages:

  • Allows multi-colour imaging.

  • Proteins are in physiological state

• Disadvantages:

  • Protein of interest may not have suitable Ab.

  • Expensive and time-consuming.

Direct Immunofluorescence

• Direct immunofluorescence to detect fluorescently-labelled Synaptophysin.

• Process:

  • Transfection of cells with plasmid

  • GFP-Synaptophysin fusion protein generated in cells

  • GFP fluorophore activated

• Advantages:

  • Quick

  • Multi-colour imaging

  • Cheap

• Disadvantages:

  • Signal variation between samples

  • Relies upon cells being transfectable

  • Fluorophore may effect normal protein activity.

Confirming Red Fluorescence is Synaptophysin

• How to confirm that the red fluorescence is synaptophysin?

  • Perform experiment in cells lacking expression of synaptophysin.

  • Block primary antibody binding with specific peptide/epitope

Quantifying Synaptophysin Level Increase with ELISA

• After western analysis and immunofluorescence, PhD student James demonstrates that synaptophysin levels increase during cellular differentiation

• Enzyme-linked immunosorbent assay (ELISA) is a useful technique to quantify the level of a specific protein in a complex protein mixture (cell lysate).

Sandwich ELISA

• Sandwich ELISA steps:

  • Cell lysate added to vessel containing capture antibody

  • Target protein (analyte) specifically binds to capture antibody

  • Vessel is washed and primary antibody is added, which binds target protein

  • Secondary antibody is added and selectively binds to primary antibody

  • Secondary antibody-conjugated enzyme is activated to generate a colour change

• The intensity of colour change is proportional to the quantity of target protein in the mixture.

Utilizing ELISA to Quantify Synaptophysin Levels

• James utilizes ELISA to accurately quantify synaptophysin levels

• ELISA plate set-up:

  • Standards: These wells contain a range of known concentrations of synaptophysin

  • Samples to be assayed

Preparing for ELISA

• Collect cell lysate

• Measure [protein]

• For ELISA, the student requires $50 \mu g$ total protein per well in a total volume of $50 \mu l$

• After determining the concentration of his lysates, he needs to calculate the volume of each lysate that contains $50 \mu g$ protein.

ELISA Calculations

• Example 1:

  • Concentration of untreated cell lysate = $2.3 mg/ml$

  • Student requires $50 \mu g$ for his experiment

  • What is the volume of lysate required to provide $50 \mu g$?

  • $2.3 mg = 2300 \mu g$

  • Calculate dilution factor: $\frac{2300 \mu g}{50 \mu g} = 46x$

  • Calculate volume for $50 \mu g: \frac{1 ml}{46} = 0.0217 ml$ or $21.7 \mu l$

  • Then add $28.3 \mu l H_2O$ to generate $50 \mu l$ solution

• Example 2:

  • Concentration of treated cell lysate = $3.7 mg/ml$

  • Calculate the volume required for $50 \mu g$ total protein

  • $3.7 mg = 3700 \mu g$

  • Calculate dilution factor: $\frac{3700 \mu g}{50 \mu g} = 74x$

  • Calculate volume for $50 \mu g: \frac{1 ml}{74} = 0.0135 ml$ or $13.5 \mu l$

  • Then add $36.5 \mu l H_2O$ to generate $50 \mu l$ solution

ELISA Plate Set-Up Considerations

• Each sample, including standards, are plated in triplicate.

• Standards: These wells contain a range of known concentrations of synaptophysin.

Reading ELISA Plate and Standard Curves

• The ELISA plate is read using a spectrophotometer for each unknown and standard samples

• Optical density is directly proportional to protein concentration

• Plot curve of standard samples (O.D on y-axis; [Protein] on x-axis)

• Estimate synaptophysin conc. in the 2 lysates.

Calculating Synaptophysin Concentration

• Firstly calculate mean O.D of the two unknown samples

• Plot O.D against protein concentration and calculate protein concentration of unknowns

• Important to draw a line of best fit

Determining Synaptophysin Amount per mg of Total Protein

• James would like to determine amount of synaptophysin per mg of total protein in his samples

• Amount of total protein per well in untreated sample was $50 \mu g$

• In $50 \mu g$ total protein, we have 3 ng synaptophysin

• Therefore, amount of synaptophysin in 1 mg of total protein is:

  • $\frac{1 mg}{50 \mu g} or \frac{1000 \mu g}{50 \mu g} = 20$

  • $3 ng \times 20 = 60 ng/mg$ in untreated cells

• Amount of total protein per well in untreated sample was $50 \mu g$

• In $50 \mu g$ total protein, we have 71 ng synaptophysin …and in his treated samples

• Therefore, amount of synaptophysin in 1 mg of total protein is:

  • $\frac{1 mg}{50 \mu g} or \frac{1000 \mu g}{50 \mu g} = 20$

  • $71 ng \times 20 = 1420 ng/mg$ in treated cells

Conclusion

• James has discovered that during neural cell differentiation, synaptophysin gene expression increases that results in a $23.7$ fold increase in cellular levels of the protein

• Calculating fold change in synaptophysin level:

  • $\frac{Growth factor treated cells}{Control cells} = \frac{1420}{60} = 23.7$ fold increase in expression

Studying Synaptophysin Activity with siRNA

• Using siRNA to assess Synaptophysin activity in neural stem cell differentiation

• siRNAs are short double-stranded RNA molecules (~21 base-pairs)

• Specific hybridisation of siRNA to complementary mRNA causes degradation of mRNA

• This reduces protein levels encoded by that degraded mRNA

Confirmation of Synaptophysin Depletion

• Synaptophysin levels are depleted in cells transfected with a synaptophysin-targeting siRNA.

Does Synaptophysin Depletion Block Neural Stem Cell Differentiation?

• What experimental readout do you think you could use?

• What experimental control would you need?

Part 2: Introduction to Clinical Trials

Aims of Clinical Trials

• To improve current treatments available to patients.

• To prevent disease.

• To better screening and diagnoses techniques for diseases.

Importance of New Therapies

• New therapies are vital for many diseases.

• Lectures 1 and 2 covered aspects of laboratory research akin to target discovery and validation experiments.

• These represent the early stages of drug discovery and development.

• After 5-6 years of comprehensive drug development and pre-clinical testing, one may be in a position to treat patients with a novel compound.

Ultimate Aim of a Clinical Trial

• To determine if a new drug is:

  • Safe and effective for a defined disease: only treatments in animals have been conducted at this stage so adverse effects in humans is a major trial determinant

  • More effective / safer than existing treatment: a current therapy may have numerous side-effects and not be particularly effective- is the new drug better?

  • Effective for additional diseases: the drug target may be important in other diseases- is the new treatment effective in these cases?

Information Gained from Animal Models

• For many diseases, informative mouse models exist which reflect the processes involved in disease initiation and progression

• These not only give researchers information on whether their new compound is effective against tumours in a physiological context (better than clonogenic assays on plastic!!)

• They also allow predictions to be made for dosing and scheduling in man and what toxicities may be encountered

Clinical Trial Design Considerations

• Once a drug has shown great promise in mammalian cell culture and animal model systems, a clinical trial may be conducted.

• However, the process of clinical trial design is complex, and numerous criteria must be considered:

  • What research questions are being addressed in the trial?

  • Why should the trial be conducted?

  • Is the trial adequately designed to answer the questions it addresses?

Clinical Trial Design: Key Questions

• What research questions are being addressed in the trial?

  • How likely is the trial to lead to change in clinical practise?

  • How many future patients are to be impacted by this trial?

  • Will blood/tissue samples from patients be available for scientific research?

• Why should the trial be conducted?

  • Has this drug been utilised in other disease types? Why is it appropriate in this one?

  • Is there strong evidence that the drug works in animals?

  • How strong is the underlying biology- is the drug targeting a relevant biological mechanism, how effective is the drug at interfering with this mechanism?

• Will the trial answer the questions it sets out to address?

  • This is dependent on patient selection, types of controls used, choosing an appropriate measurement/end-point (e.g. overall survival)

Example of Concept Development

• A clinician treats patients with Disease X. In talking to a subset of her patients who had especially favourable outcomes she noticed that many of them were big coffee drinkers.

• This raised the research question: “Does caffeine intake improve the survival of patients with Disease X?”

• The experimental design that is used to test this hypothesis is articulated in a clinical trial protocol

Control Arms in Clinical Trials

• In many clinical trials, the control arm receives no intervention or a placebo.

• However, due to the nature of cancer clinical trials, in which it is unethical not to provide treatment, the control arm represents the current standard of care while experimental arm receives the new agent

Controlling Bias in Clinical Trials

• The key to any clinical trial is to prevent bias as this will impact massively on the outcome of the study

• Two methods in clinical trial design are utilised to prevent this issue:

  • Randomisation

  • Blinding

Randomization in Clinical Trials

• Assigns patients to treatment arms by chance

• If it is a trial with an experimental and control arm only, patients will be assigned to a single group by tossing a coin!

Blinding in Clinical Trials

• Ensures that neither patients, doctors and researchers know to which group each patient has been assigned and is important to prevent false interpretation of data or patients receiving different care.

• Trials are said to be single- (patient only), double- (patient and doctor) or triple- (patient, doctor and researcher) blind

Phase 1 Clinical Trials

• To evaluate safety, i.e. determine safe dose range and identify side effects

• Generally involve small groups of healthy volunteers

• BUT: Phase 1 trials for anti-cancer drugs generally involve patients who have failed conventional treatments (anti-cancer agents are generally too toxic to give to healthy volunteers)

• Identifies the most appropriate route for administering drug

• Gradual increasing doses are generally given to successive individuals (dose escalation)

• For cancer trials, Phase I trials tend to be across multiple cancer types

Maximal Tolerated Dose (MTD)

• 3+3 model is used to identify Maximal Tolerated Dose

• Maximal tolerated dose (MTD) is defined as the highest dose of drug that does not elicit significant toxicity

• Dose-limiting toxicity (DLT) is where the patient experiences any number of unacceptable adverse events (reactions) to the drug

Monitoring During Phase 1 Trials

• During the course of the trial, patient are monitored routinely to assess:

  • Drug levels in blood: information on drug kinetics in the body

  • Predicted effects of drug on patient: biomarker analysis (e.g. blood pressure is measured for an anti-hypertensive drug, prostate specific antigen measured for a prostate cancer drug).

  • Toxicity - i.e. unwanted side effects

PK and PD Measurements

• Pharmacokinetic studies:

  • What the body does to the drug.

  • e.g. what concentrations are achieved in blood? How long are they maintained?

• Pharmacodynamic studies:

  • What the drug does to the body.

  • e.g. measure change in enzyme activity to show the drug acts on planned target.

• Toxicity.

Phase 2 Clinical Trials

• To see if the new drug or treatment is effective using defined biomarkers/treatment response in patients (e.g. tumour size in cancer; alleviation of symptoms)

• To further evaluate its safety

• These generally involve larger groups of patients (40-100)

• For cancer trials, they normally occur in one specific cancer type (unlike Phase I)

Phase 3 Clinical Trials

• To further determine effectiveness and side-effects of the new drug or treatment

• Phase III trial has strict and well-defined eligibility criteria (i.e. inclusion and exclusion criteria); patients enrolled in the trial must meet these criteria

• Generally by comparison with standard treatments: Superiority Trial

• Also to collect information that will allow it to be used safely

• Generally involve large groups of people (more than 200) for statistical validity etc

Phase 4 Trials/Studies

• Continued testing after the drug or treatment has been approved or marketed.

• e.g. to collect information about their effect in specific populations and side effects from long-term use.

Clinical Trials in Practice: Examples

• Example 1: Testing efficacy and side-effects of three anti-depressants

Example 1: Efficacy of 2nd Generation Antidepressants

• One of the main purposes for conducting a clinical trial is to improve current clinical practice and better the therapies currently available

• Although many anti-depressants are on the market and have shown clinical benefit, there is a need to provide new, more beneficial and safer drugs to the patient

• Tricyclic anti-depressants (TCAs) are first generation drugs

• Selective serotonin re-uptake inhibitors (SSRIs) and Serotonin -noradrenalin reuptake inhibitors (SNRIs) are the new class of agents

Efficacy of Antidepressants (TCAs, SSRIs, SNRIs)

• SSRIs, SNRIs and TCAs exhibit equivalent anti-depressant efficacy

Side-Effect Profiles of Antidepressants

• TCAs and SSRIS/SNRIs display different side-effect profiles

Clinical Trials in Practice: Example 2

• Example 2: Assessing the effectiveness of a new treatment patch for Alzheimer's disease

Alzheimer's Disease Treatment Patch Study

• A 6-month, double-blind, placebo-controlled study of the first skin patch for Alzheimer disease

• Randomized 1,195 patients

• Brain function measured by assessment of: ADAS-cog Alzheimer's Disease Assessment Scale-cognitive subscale

Cognitive Improvement with Treatment Patch

• Improvement to patient cognitive ability in response to treatment

• Changes in cognitive abilities over 24 weeks compared to abilities at start of the study:

  • p < 0.05 versus placebo

Side-Effects of Alzheimer's Treatment Patch

• Comparable side-effects observed across treatment methods

Enzalutamide for Prostate Cancer Treatment

• Enzalutamide is a newly developed treatment for prostate cancer

• Enzalutamide targets multiple steps in the androgen-receptor-signaling pathway, the major driver of prostate-cancer growth

• Evaluate whether enzalutamide prolongs survival in men with castration-resistant prostate cancer after chemotherapy

Data Interpretation: End Point Analysis

• PSA is Prostate Specific Antigen and is a biomarker for prostate cancer:

• More PSA = Greater disease burden

Data Interpretation: Side-Effect Profiles

• Table summarizes adverse events according to grade for Enzalutamide and Placebo groups

Ethical Considerations for Clinical Trials

• 7 Ethical Requirements

  • Collaborative partnership

  • Social Value

  • Scientific Validity

  • Fair subject selection

  • Favourable risk-benefit ratio

  • Independent review

  • Informed consent

Collaborative Partnership

• To be ethical, clinical research must involve the community in which it occurs.

• This requires:

  • community participation in planning, conducting and overseeing research, and integrating research results into the health system.

  • avoidance of supplanting existing health care services and the sharing of rewards with the community.

Social Value

• To be ethical clinical research must lead to improvements in health or advancement in generalizable knowledge.

• Must consider how the research will improve health of:

  • Participants in the research

  • Community in which research is conducted

  • Patients worldwide

Scientific Validity

• Research must be conducted in a methodologically rigorous manner that is practically feasible.

• To be ethical the research must produce reliable and valid data that can be interpreted.

• Invalid research includes underpowered studies, studies with biased endpoints, instruments, or statistical tests, and studies that cannot enrol sufficient subjects.

Fair Subject Selection

• The scientific objectives of the study—not vulnerability or privilege—should guide inclusion criteria and targeted populations.

• Lowering risk and enhancing generalizability can then be considered.

• Groups cannot be excluded without scientific reasons.

Favorable Risk-Benefit Ratio

• Clinical research must be conducted in a manner consistent with the standards of clinical practice.

• Evaluate the likelihood and magnitude of harm

• Identify mechanisms to minimize risks:

  • Additional diagnostic tests

  • Hospitalizations

• If potential benefits to the individual outweigh risks to the individual then proceed.

Ethical Considerations: Independent Review

• Because investigators have multiple legitimate interests, they have potential conflicts of interest. Independent review of the research minimizes these conflicts.

• Independent review also assures society it will not benefit from abuse of subjects.

Informed Consent

• Researchers have to prepare an information leaflet about their trials for patients. The research ethics committee checks that this is clear and accurate and written in easy to understand English.

• What information is included?

  • the research questions the trial is trying to answer

  • who can take part in the trial and who cannot

  • how those taking part in the trial will be treated and what they will need to do

  • what treatment or other intervention is being used

  • what type of research it is

  • what the possible risks are to participants

  • what the possible benefits are to participants

  • who is carrying out the trial and who is funding it

Informed Consent Elements

• Informed consent consists of 4 elements

  • Competence of the subject

  • Disclosure of information to the subject

  • Understanding or comprehension by the subject

  • Voluntariness of the decision

Research Ethics Committees

• Every clinical trial is covered by regulations that protect the health, safety and dignity of the people taking part.

• All medical research involving people in the UK, whether in the NHS or the private sector, has to be approved by an independent research ethics committee.

• The committees are often based at local hospitals and are formed of local people, such as health professionals, patients, lawyers and members of the public. They have to include members who are not health professionals.

• All clinical trials of medicines need to be authorised by the Medicines and Healthcare products Regulatory Agency (MHRA)

Summary of Clinical Trials

• Clinical trials are conducted for several reasons, but mainly to improve current clinical practise

• Numerous types of clinical trials are conducted- be aware of the differences e.g. placebo-controlled, single-blinded, double-blinded

• Although we haven’t investigated statistics utilised in these studies (due to the sheer complexity of them!) be aware that they are key when interpreting results and statistical significance is required to draw conclusions from the data