Lecture 10: Cardiovascular Saftey Testing

What are the Key Features of the Drug Discovery Phase in Drug Discovery and Development?

• Takes around 5-7 years

• Find a target of interest relevant to the mechanism underlying the disease pathology

• Thousands of compounds are screened to find hits

• Hit compound is optimised and improved by a chemist to create a lead compound

• Lead optimisation aims to avoid safety issues and improve the compound

Why is Lead Optimisation a Critical Phase in Drug Discovery and Development?

• It is cheap to fail → not costly if a project is stopped due to safety concerns associated with the compound

• It produces the greatest chemical choice → thousands of compounds generated (lots of choice)

What are Key Features of the Drug Development Phase of Drug Discovery and Development?

• Takes around 6-8 years

• If successful, a drug will move into pre-clinical testing, entering

  • Phase 1 Clinical trials: healthy volunteers; assess drug safety and metabolism properties

  • Phase 2 Clinical trials: small cohort of patients with disease of interest

  • Phase 3: Clinical trials: large cohort of patients with disease of interest

What is the Attrition Rate in Drug Discovery and Development?

• Attrition rate during development is high: only ~1 in 12 compounds entering development reach the market

• Cardiovascular side effects are a common cause of attrition within this process

What are Key Features of the Cardiovascular System?

• It is essential for life

• It matches supply to demand

• Cardiac output can increase and supply blood where it is needed, e.g. when running (heavy exercise) CO increases and is directed towards the skeletal muslce

Why is cardiovascular safety important when developing new drugs?

• Many patients have damaged cardiovascular systems or cardiovascular disease

• This is especially common in elderly populations (due to long lifetime, bad diet, smoking)

• WHO: CVD accounts for 33% of deaths

• Common conditions:

  • Ischaemic heart disease or coronary artery disease e.g heart attack

  • Cerebrovascular disease e.g. stroke

  • Hypertension & peripheral vascular disease

• Therefore, new drugs must have minimal cardiovascular side effects

What is an Investigational New Drug (IND) Application?

• An application filed by a company before giving a candidate drug (CD) to humans for the first time

• This is submitted to the Regulatory Agency in the country where the trial is conducted (e.g. EMA (EU), FDA(US))

• Includes a section summarising safety studies

• Must include a minimum safety data package

• This includes effect of CD on cardiovascular:

  • Function (e.g. HR, BP)

  • Structure of the cardiovascular system

• This protects first human participants (usually healthy volunteers) taking the CD

  • Exception: Oncology drugs (tested directly in patients due to high risk)

What monitoring and safety measures are used in Phase 1 clinical trials?

• Typically conducted in young males

• ECG used to monitor the electrical activity of the heart

• EEG used to measure the electrical activity of the brain

• Blood samples are also taken

• Governed by ICH S7A regulatory guidelines describing the cardiovascular pre-clinical functional assessments required

• Aims to protect trial participants from the adverse drug effects of pharmaceuticals

How is cardiovascular function assessed in pre-clinical CD regulatory studies?

• Assessed in dogs (more similar to humans than rats)

• Parameters measured:

  • Blood pressure (BP)

  • Heart rate (HR)

  • Electrocardiogram (ECG)

  • Left ventricular pressure (LVP)

• Dogs used because rat cardiac electrophysiology significantly differs from humans

What is the procedure for cardiovascular function assessment in pre-clinical studies?

• A colony of ~20 dogs developed with an implant (telemetry device) in the abdomen to monitor their CVS

• Conscious dogs monitored using a device which records and transmits cardiovascular parameters (BP, HR, ECG, LVP)

• 4 dogs receive: vehicle (solvent/no drug) /low/medium / high dose of CD

• Clinical route of administration used (oral, most common)

• Data recorded for 24 hours post administration

• Blood samples taken to measure plasma drug concentration

• After a recovery/ washout period, dogs receive a different dose or vehicle

• By the end of the study, each dog had received all treatments over time

• Plasma concentration measurements were used to plot CVS effects vs plasma concentration

What is a dog telemetry study in cardiovascular function assessment?

• Dogs housed in pen, scientists monitor remotely

• Telemetry device implanted in the abdomen

• Measures:

  • Blood pressure (BP)

  • Heart rate (HR)

  • ECG

  • Left ventricular pressure (LVPP)

• Data transmitted to the computer screen

• Example: Moxifloxacin (antibiotic) prolongs QT interval on ECG

• Dose-dependent QTc prolongation

What does a lack of safety margin look like in a dog cardiovascular function safety study?

• Dogs given low / medium / high doses of drug

• X-axis: Blood concentration of drug

• Y-axis: QT interval parameters

• Concentration-dependent increase in QT interval observed

• Problematic: Indicates pro-arrhythmic risk

• No safety margin: Predicted plasma concentration required to treat the disease falls within a range where problematic side effects occur

• If observed, the drug may not progress further

What does a safe outcome look like in a dog cardiovascular function safety study e.g. Left Ventricular Pressure ?

• Measure effect on dP/dt max (index of cardiac contractility)

• Drug Y shows a concentration-dependent decrease in contractility (problematic)

• The estiamed peak efficacious plasma concentration occurs before this effect

• A safety margin present between efficacy and adverse effects

• Therefore, the drug may progress further

• Aim: assess the cardiovascular effects of the compound

How is cardiovascular structure assessed in dog and rat toxicology studies?

• 28-day study in two species required (typically, rats & dogs)

• Aim: Determine the effect of CD on the structure of the heart and blood vessels

• 6 Dogs given daily doses (1x/day for 28 days) :

  • Low

  • Medium

  • High (max tolerated dose)

  • Vehicle

  • (Sometimes recovery group)

• After the study: animals euthanised with an overdsoe of anaesthetic

• Standardised tissue sections taken from all over the body

• H&E staining is used to visualise the structure of the heart & vessels

• Veterinary pathologists assess if and how the drug causes structural damage, by comparing what they know about normal hearts

• Similar procedure used in rats

What was the outcome of the CV structure assessment for ZD1611 using light microscopy?

• Effect of endothelin receptor antagonist (ZD1611) studied on canine coronary artery sections

• Examined using Light microscopy (400×)

• Miller’s stain applied

• Found breaks in the internal elastic lamina (normally uninterrupted) → Indicates structural damage to blood vessels (not stained black)

• Evidence of damage visible at this level→ drug likely to have been discontinued

What was the outcome of the CV structure assessment for ZD1611 using electron microscopy?

• Effect of endothelin receptor antagonist (ZD1611) studied on canine coronary artery sections

• Examined using electron microscopy

• In control animals:

  • Smooth muscle cell visible

  • Endothelial cell visible

• In ZD1611-treated animals:

  • inflammatory cell and erythrocyte infiltration

  • Smooth muscle cell with unusal electron dense granules

• Evidence of damage visible at this level→ drug likely to have been discontinued

How are cardiovascular structural effects analysed in toxicology studies?

• Structural effects are harder to quantify with high resolution compared to functional effects (BP, QT)

• Therefore a semi-quantitative approach is used when assessing pathology

• Veterinary pathologist scores lesion severity

  • Severity of lesion is plotted against dose administered

  • Enables dose–response relationships to be established

How is data from regulatory cardiovascular safety studies used to decide if a drug progresses to humans?

• Decision is based on a risk–benefit balance

• CV risk may be acceptable in severe disease (e.g. cancer) but not mild disease (e.g. rheumatoid arthritis) → dependent on the condition being treated

• Safety margins are a critical factor

  • Small margin (CV effect in dogs occurs at similar concentrations required for therapeutic effect in humans) → may stop development (esp. if disease is not acutely life threatening)

  • Large margin → may allow progression to Phase 1

• Phase 1 can use a low starting dose with dose escalation to ensure no issues are observed

• These decisions depend on whether the effects align to the SMART framework

What is the SMART Framework Used to Assess Before Phase 1 Trials?

• SMART criteria determine whether a candidate drug with CV effects (on function and/or structure) is suitable for testing in healthy volunteers.

• This depends on whether the effects are:

  • S — Non-serious (not acutely life-threatening)

  • M — Monitorable (can be measured in humans)

  • A — Anticipatable (clear dose-response relationship)

  • R — Reversible (effect stops when drug is stopped)

  • T — Treatable (manageable with known interventions)

• If effects are SMART → drug may progress to Phase 1

Give an Example of Where the SMART Framework Would Allow the Progression of a CD To Phase 1:

• Functional effect: Small, transient, dose-dependent increase in blood pressure in dog study

• SMART assessment:

  • S — Non-serious (unlikely a transient increase in BP is acutely life-threatening)

  • M — Monitorable (via BP/sphygmomanometer cuff)

  • A — Anticipatable (dose-response relationship present)

  • R — Reversible (transient)

  • T — Treatable (well characterised anti-hypertensives available)

• Decision: Progress to humans

  • Carefully monitor BP

  • Slowly escalate the drugs dose

Give an Example of Where the SMART Framework Would Stop the Progression of a CD To Phase 1:

• Structural effect: Irreversible cardiac necrosis at high doses

• SMART assessment:

  • S — Serious (life-threatening at high doses)

  • M — Not Monitorable (No test present to detect when heart cells are dying)

  • A — Not Anticipatable (Steep dose response curve may be present → only occurs at high doses)

  • R — Not Reversible

  • T — Not Treatable (No drugs can bring cardiac cells back to life)

• Decision: Unlikely to progress to humans

  • May only progress if the drug is for an acute life-threatening indication

  • Even then, would need evidence of a good saftey margin

What is the purpose and outcome of regulatory cardiovascular safety studies?

• Aim: Minimise risk to first recipients of candidate drug (CD) in the first clinical trials

• Regulatory agencies require CV safety assessment of:

  • Function (usually dogs)

  • Structure (dogs & rats)

• Overall CV safety data are combined, and an integrated risk assessment considers:

  • SMART criteria

  • Target population for CD (patients vs healthy volunteers)

  • Safety margins

• Approach has improved the safety of Phase 1 trial patients in short-duration clinical trials (few serious CV issues)

• However, this does not guarantee safety in later stages of development or post-marketing, when the drug is given to a large number of people or for a long period of time

What are the key features of Grepafloxacin?

• Primary Mechanism: Bacterial topoisomerase inhibitor (enzyme required for duplication, transcription and repair of bacterial DNA)

• Use: Treatment of bacterial infections

• Approved for sale: 1997

• Withdrawn from sale: 1999

• Reason: Cardiac arrhythmia (Torsades de Pointes) → safety issue

Why did grepafloxacin cause Torsades de Pointes?

• Grepafloxacin blocks cardiac hERG K⁺ channels

• hERG channels normally conduct large K+ currents at the end of the cardiac AP → gives rise to short ventricular action potential, with short Q-T interval

• Blockage of the hERG channel leads to an elongation/prolongation of ventricular action potentials and a reduction in the K⁺ current

• Visualised as QT interval prolongation (ECG)

• Linked to an increased risk of Torsades de Pointes (rare, but fatal cardiac arrhythmia)

• Led to drug withdrawal

What are the key features of Norefluramine?

• A metabolite of Fenfluramine

• Often co-administered with Phentermine (Fen‑Phen)

• Primary Mechanism: Modulate serotonin (5-HT) release

• Use/Treatment: Obesity

• Approved for sale: 1973

• Withdrawn from sale: 1997

• Reason: Valvular heart disease → Safety issue

Why was Fenfluramine withdrawn?

• Healthy cardiac valves are essential for optimal Cardiovascular function

• Values are structurally complex → modifications to this can significantly affect their ability to function efficiently

• Valve thickening and stiffening impair function, leading to:

  • Loss of unidirectional blood flow

  • Ventricular overload

  • Congestive heart failure

• Fenfluramine was withdrawn for causing caused Valvulopathy

• This resulted in some patients requiring artificial valve replacement

• This risk was not detected in pre-clinical toxicology studies in 1970s

What is the Mechanism Behind Norfenfluramine’s Cardiotoxicity?

• Adverse effects are likely a consequence of “off-target” activity at 5-HT2B receptors expressed on Valvular Interstitial Cells → failed to be recognised

• Norfenfluramine acts as a 5-HT2B receptor, leading to PKC activation

• This leads to

  • Changes in gene expression

  • Increased proliferation of vascular interstitial cells

  • Increased secretion of the extracellular matrix

• This causes cells to become less floppy and no longer function properly (Spontaneous Valvulopathy → thick and unflexible)

What are Key Features of Rofecoxib?

• Primary Mechanism: Cyclooxygenase 2 inhibitor

• Use: Treatment for pain associated with Arthritis

• Approved for sale: 1999

• Withdrawn from sale: 2004

• Reason: Increased risk of myocardial infarctions and stroke → safety issue

Why was Rofecoxib developed as a selective COX-2 inhibitor?

• Selective for COX-2 over COX-1

• COX‑1 is constitutively active in the stomach → protects gastric mucosa

• COX‑2 activity is induced during tissue inflammation (e.g. arthritis)

• Selective inhibition allows for pain relief (arthiritic flare up) + reduced incidence of gastric damage (side effects in the stomach)

• More selective than non-selective NSAIDs

• Potent and selective COX-2 inhibition, achieved at micromolar concentrations

Why was Rofecoxib withdrawn despite being an effective painkiller?

• Epidemiological studies showed an increased risk of cardiovascular adverse events

• Demonstrate an increased risk of Myocardial infarction (MI) or Stroke

• Relative risk interpretation:

  • 1 = no increased risk

  • >1 = increased risk

• Large number of patient are needed to detect the effect

• Its withdrawal was disappointing for many as the drug was effective for arthritis

What is the Mechanism Behind Rofecoxibs Cardiovascular Safety Issues?

• COX-2 Mediates pre-conditioning and reperfusion in cardiac tissues → leads to the generation of PGI₂ (cardiac protective)

• Hypothesis for safety issue: Rofecoxib removes the cardiac protective effect of PGI₂

What are examples of serious cardiovascular safety issues and how can they be prevented?

• Grepafloxacin → QT prolongation

• Fenfluramine → Valvulopathy

• Rofecoxib → ↑ Myocardial infarction & Stroke

• Post-marketing detection is very bad

• Pre-clinical detection is not ideal

• The goal is to identify safety issues earlier to screen out unsafe drugs

What is the synthesis–screening cycle and how is it used in lead optimisation?

• An iterative process in lead optimisation

• Involves:

  • Synthesis → screening → redesign → repeat

• Screens desirable properties in and undesirable properties out

• Includes in vitro assays (e.g. assessing primary target activity and drug metabolism)

• Adding CV safety screens early and in vitro testing helps identify any potential issues with drugs and remove toxic compounds early on

• Goal: detect safety issues before clinical trials

How are ion channel safety risks (e.g. hERG block) now detected early in drug development?

• Ion channels underlying cardiac action potentials are well characterised

• Drug-induced channel modulation and block are a known safety risk

• Cell lines expressing ion channels are available and are used in automated electrophysiology systems for the rapid high-throughput screening of drugs

• New technology allows testing capacity to be integrated into the synthesis–screening cycle → allows ion channels to be inserted and test whether they are blocked by drugs

• Drugs that block channels can be screened out early

• Example: hERG channel screening reduces the risk of QT-prolonging drugs early on in the drug development and discovery process via synthesis and screening cycle (e.g. Grepafloxacin case avoided)

How are off-target cardiovascular safety risks screened during drug development?

• Certain enzymes, receptors, ion channels, and GPCRs are linked to CV side effects

• These targets are well-known and can be expressed in cell lines

• Drugs are screened for off-target activity against them

• Helps identify and remove compounds with undesirable pharmacological activity

• Integrated into the early synthesis–screening cycle

• Allows for drugs to be removed from development before reaching animal studies or healthy volunteers

What is a “black box” (phenotypic) screen in early cardiovascular drug safety?

• Used because not all CV safety mechanisms are known

• Involves monitoring one or more activities in a cell-based system, and whether the test compound changes the activity

• If an effect is observed, the molecular mechanism is not known from a black box system

  • It pulls together multiple mechanisms and only phenotypic changes in cells, not the specific mechanisms → Mechanism is unknown (“black box”)

• If a drug alters cell function, it indicates potential toxicity

• Used to screen out unsafe compounds early in development

How have “black box” cardiac safety screens improved in early drug discovery?

• Historically, black box options for cardiac systems were limited, using only animal cardiac cells/tissues

• These were not ideal as:

  • Low throughput → not fast enough

  • Ethical concerns → animal use

  • Not suitable for early screening

• Advancement of commercially available human stem cell–derived cardiomyocytes → enabled the use of black box screening

• Allows drugs to be tested against human-cardiac-like cells, generated from fibroblasts

  • Enable human-relevant screening

  • Allows early high-throughput phenotypic screening

• Care required when setting up assays as stem-cell derived cardiomyocytes do not fully replicate native heart biology

How is a phenotypic (black box) screen used to detect negative inotropic effects?

• Uses human stem cell–derived cardiomyocytes

• These cells beat spontaneously

• Compounds can be added to assess whether they affect the rate of beating.

  • Drug added → measure change in beating rate

• Compounds that decrease the rate of contraction are associated with reduced cardiac contractility in humans (negative inotropy)

• Used to deselect unsafe compounds early in drug development and discovery

What is the value and limitation of phenotypic cardiac screening?

• It predicts potential negative inotropic effects in humans → Useful for excluding harmful compounds early

• But this is not 100% predictive

• Shows effect on phenotype, not mechanism (“black box”)

• This combination of molecular and phenotypic screens allows for the rapid exclusion of bad compounds and a focus on good ones

What are New Approach Methodologies (NAMs) in drug safety testing?

• Introduction of initiatives by US and UK governments to reduce animal testing in 2025

  • NC3Rs in the UK have aimed to reduce, replace and remove animal studies for years.

• New Approach Methodologies are innovative, non-animal, human-relevant technologies for chemical and drug safety assessment

• Used in the pharma, agrochemical, and cosmetic industries

• It aims to improve translation for human safety prediction

What are examples of New Approach Methodologies (NAMs)?

• Advanced in vitro cell-based assays, e.g. cell-based systems

• Organ-on-a-chip systems → perspective chip engineered to make a blood vessel

  • Not just one cell type;

  • Formed in vitro

  • Formed from human (derived) cells → no animals involved

  • Mimic organs (e.g. blood vessels)

• Computational modelling

  • Integrates lots of in vitro data

  • Predicts potential biological and toxic effects that may alter the biology of a system

What is the goal and limitation of NAMs in drug development?

• Aim to accelerate the reduction of animal testing

• It combines multiple technologies for prediction

• Must ensure that the systems are sufficient so that drugs are safe enough for testing in Phase 1 trials and healthy volunteers, without animal testing

• Question as to whether they will be accepted as alternatives to animal testing

What is the overall approach and future direction of cardiovascular safety assessment in drug development?

• Regulatory CV studies focus on protecting early clinical trial participants

• Current studies are poor at detecting long-term dosing effects in large population

• As more is known about the molecular basis for adverse effects on the CV system, the use of in vitro assays to detect problems early in the discovery process increases

  • There is a shift towards early in vitro screening in drug discovery

• The increasing prevalence of CVD makes it imperative to find candidate drugs that do not adversly effect the CV system

• New assay systems, e.g. human stem cell–based cardiac assays, may offer new ways to make safer drugs

• Renewed initiatives to reduce animal testing via New Approach Methodologies (NAMs) → may change current approaches to pre-clinical CVS safety assessments