Chapter 5 Safety Pharmacology Studies 

Adverse pharmacodynamic effects may be due to the on-target primary pharmacology effects of the product being tested, which could be related to the given dose, the route and frequency of administration, and systemic exposures, or due to off-target effects termed as secondary pharmacology. The three primary vital organ systems included in the safety pharmacology core battery are physiologically correlated, meaning that effects seen in one system may induce an adaptative response in the others. As such, interpretation of safety pharmacology data typically requires an integrated risk assessment of the totality of effects on organ functions that considers the targeted clinical population, clinical trial plans, and safety margins. The timing of safety pharmacology studies, which are aligned with the intended patient populations, will be discussed in this chapter.

Safety pharmacology has emerged as a discipline to predict risk of adverse pharmacodynamic liability3 through the conduct of core battery studies before performing the first-in-human studies with a new product. These have helped to separate out safer pharmaceuticals with either little or no potential for CV, respiratory, or CNS effects or which have adequate safety margins from those which have a less favorable benefit-risk profile. It should be noted that safety pharmacology assessments may also be required for products in the late stages of development if adverse effects arise in clinical trials or if a marketed product is being repurposed for a new indication, patient population, or administered in a new formulation or route which previously did not require safety pharmacology assessments.

The information presented in this chapter is largely based on US Food and Drug Administration (FDA),1 European Medicines Agency (EMA), and Committee for Medicinal Products for Human Use (CHMP) documents that discuss the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Safety (S) guideline 7A on safety pharmacology studies for human pharmaceuticals. The ICH S7A guidance states that all human pharmaceuticals (small molecules, biotechnology-derived, or biologics) should be reasonably safe to be given to humans during first-in-human clinical trials. As such, the first dose in humans is not expected to have any undesirable pharmacodynamic effects on the physiological function of vital organs at the therapeutic range and above, as evaluated by safety pharmacology studies conducted in accordance with good laboratory practice (GLP) regulations.4 The timing of the safety pharmacology studies is aligned with the guidance from FDA and EMA/CHMP on ICH M3(R2) on nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals.5,6 Currently, cell and gene therapy products or tissue-engineered products are not specially addressed in the Safety Pharmacology guidelines.

This chapter also will discuss the more recently approved and implemented ICH E14/S7B Q&A guidance, which outlines the acceptance of in vitro hERG (human ether-a-go-go related gene) and in vivo QT interval study data (i.e., nonclinical, double negative), both conducted under the Q&A best practice recommendations in support of two clinical scenarios (E14 Q&A 5.1 and 6.1), to apply for a thorough QT (TQT) study waiver.7,8 Case studies are provided at the end of this chapter to demonstrate how safety pharmacology studies may be conducted in practice.

1.General Principles and Importance of Safety Pharmacology Testing

Safety pharmacology guidelines were first developed in 2000 in response to the adverse reports of the effects of the antihistamine terfenadine on the CV system. Terfenadine, which had been introduced into the market by Hoechst-Marion Roussel (now Sanofi-Aventis) in 1985, was marketed as Seldane in the US, Triludan in the UK, and Teldane in Australia as a treatment for allergic rhinitis, among others.3 In the 1990s, however, some people taking terfenadine presented to the hospital with syncope, and in some instances, died. 3,9,10 It was later found that terfenadine is a potent inhibitor of the potassium ion channel (IKr or hERG) resulting in prolongation of the QT interval. 9-11 This had the

potential to develop into a polymorphic ventricular tachycardia termed Torsades de Pointes (TdP), which literally means twisting of the points or peaks (R wave or QRS complex) around the ECG isoelectric line.

The cardiotoxic potential of terfenadine was not detected prior to clinical trials because the drug was completely metabolized by the liver enzyme cytochrome P450 3A4 (CYP3A4), so levels went undetected in the plasma. Moreover, its metabolite terfenadine carboxylate (also called fexofenadine), did not inhibit the potassium ion channel even when levels were at 30 times higher concentrations than the hERG IC50 of terfenadine.11,13 The proarrhythmic potential of the prodrug terfenadine was revealed at very high doses, however, in patients with liver disease affecting the CYP3A4 enzyme or when patients were taking other medications that inhibited CYP3A410,13,14as this resulted in reduced metabolism and thus higher plasma levels of terfenadine and lower concentrations of fexofenadine. Fexofenadine had similar efficacy to terfenadine, but since it only slightly inhibited the hERG channel, it had much lower potential to induce arrhythmia. Fexofenadine was marketed in 1996 under the name Allegra13 for allergic rhinitis and chronic urticaria, among others, and was listed in the WHO Essential list of Medicines,15 while Terfenadine was withdrawn from the market in 1997 due to its potential to cause cardiac arrhythmia.

Hence, the potential adverse effects of small molecules or biopharmaceuticals on all three core organ systems (CV, respiratory,CNS) must be conducted before performing first-in-human studies.1,2 While ICH S7A provides guidance on the conduct of the core battery studies and the functional endpoints to collect, it does not outline the study designs themselves. Safety pharmacology study designs for both in vitro and in vivo studies are discussed in the next section and are provided as examples which may be judiciously applied or modified according to the pharmacology (or class of compound), route and dose, indication and intended patient population of the pharmaceuticals or biopharmaceuticals under evaluation.

2.Core Battery Study Designs and Endpoints

When testing the safety pharmacology of a new product, the potential adverse pharmacodynamic effects need to be tested first on the three core organ systems: the CV system, the respiratory system, and the CNS.

3.Cardiovascular System

Types of studies that may be performed to assess the effects on the CV system include:

• In vitro studies (e.g., cardiac ion channel studies)

• Ex vivo studies (e.g., Langendorff technique)

• In vivo studies (e.g., telemetry in rodent and non-rodent models)

4.In Vitro Cardiovascular Studies

In vitro CV assays mostly involve the study of cardiac ion channels, with the regulatory requirement to conduct a hERG assay7,8,17 (Table 5-1). One such assay is the automated hERG assay to determine the potential of pharmaceuticals to inhibit the hERG potassium channel. Using a single concentration of the test article, this assay is used as a screening tool early in drug development to identify compounds with potential hERG liability during structure-activity relationship research efforts and before leadnomination.  Discovery hERG assays are followed up by using escalating dose concentrations of the test article to get an estimation of its IC50, which is the concentration of the drug that is required to inhibit a biological process by 50%. This is then followed by a rigorous manual patch clamp hERG assay in accordance with GLP regulations.

The recently released S7B Q&As request following up the in vitro hERG assay. Best practices for experimental factors are then conducted on human embryonic cells or Chinese hamster ovary cells overexpressed with human cardiac ion channels (S7BQ&A 2.1)21,22, which includes potassium, calcium, and sodium channels. Highlights of the Q&A best practices for experimental factors set out in ICH guidance include:

• Conducting manual patch clamp experiments at near physiological temperature (35-37°C);

• Measuring the ventricular action potential voltage protocol;

• Monitoring health of the cells overexpressing the cardiac channels;

• Recording seal resistance quality; and

• Reporting the IC50 value and Hill coefficient

Moreover, a validated analytical method should support concentration verification, and one of the three positive controls –dofetilide, ondansetron, and moxifloxacin – should be evaluated as a reference drug. Regarding the latter, repeated evaluations should be done with two or more concentrations of the test article being used for each experiment and including at least four cells at each concentration.

After consideration of protein binding and using free drug concentrations in plasma, the calculated IC50 concentration inhibition should have greater than 50-fold safety margin over that of anticipated clinical exposures. Assessment of additional cardiacion channels is recommended and included in the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, mentioned later in this chapter.

When evaluating oncology products, hERG and cardiac ion channels inhibition assays can be conducted without compliance to GLP regulations because of the likely greater benefit to risk ratio of oncology products versus other pharmaceuticals. For biologics or biotechnology products, the target specificity is usually very high, and so in vitro hERG assays are not required but are recommended. When evaluating these biopharmaceuticals, CV endpoints can be incorporated in vivo in to repeat-dose toxicity studies conducted using non-invasive telemetry (Table 5-2).

5.Ex Vivo Langendorff Technique

The Langendorff technique is an ex vivo assay in which the isolated heart of an animal is perfused with oxygenated, buffered Krebs solution, with and without the drug to be tested. The aim of this is to observe the effects of the drug on ECG parameters, heart rate, left ventricular mechanics, and estimates of coronary blood flow.26,27 Usually, the heart of a guinea pig or rabbit is used, as their hearts possess the IKr potassium ion channel, which is the primary cardiac ion channel responsible for ventricular repolarization (i.e., the QT interval of the ECG) in humans. Studies have demonstrated concordance to human effects of QT prolonging drugs using the ex-vivo assay. Translation of the isolated perfused guinea pig (rodent) heart assay to large animal QTc assay for determination of QTc changes with drugs known to prolong QTc and 13 drugs known to have no effect on the QTc interval was demonstrated.

To perform the technique, the animal’s heart is removed, and the perfusate is introduced via the aorta in a retrograde fashion; this delivers the oxygenated, nutrient-rich perfusate containing either the vehicle or drug through the coronary arteries. Endpoints recorded include ECG parameters recorded from electrodes placed on the heart’s epicardial surface in a Lead II configuration, heart rate, left ventricular contractile parameters (e.g., the maximal rate of rise and maximum rate of fall in ventricular pressure), and mean coronary flow. The effect of the test drug on the function of the myocardium (electrical and contractile activity) and the coronary vessels can be examined by examining the myocardium, smooth musculature, and the endothelium of coronary vessels. Despite having some limitations (e.g., inability to assess metabolites, functional viability of the heart performance isolated for greater than 4 to 6 hours, etc.), the Langendorff technique can provide rapid information as both a CV screening assay and when further interrogation of results from earlier discovery assays (e.g. receptor binding assay on cardiac receptors or hERG results) is needed, as this assay generates an ECG for evaluation of intervals (e.g., RR, PR, QRS, QT and calculated QTc intervals), rhythm and left ventricular pressure curve.

6.In Vivo Cardiovascular Studies

In vivo CV studies involve the evaluation of the CV system in free-moving (non-rodents like canine, non-human primates or minipig) animals via surgically implanted telemetry devices 30,31. Such devices are available with several biopotential and pressure catheter configurations as well as options for impedance leads for measurement of respiratory function 32,33. There are telemetry device options for use in both short and long term studies, as well as reuse of animals on multiple studies (with sufficient washout period), typically based on the battery life of the transmitter. Implanted telemetry, which uses solid tip ECG electrodes that are placed intravascularly, provides high resolution or low signal to noise ratio. This allows for accurate placement of fiduciary marks on the ECG intervals during post-acquisition analysis.

Additional options include non-invasive jacketed external telemetry (JET), which pairs surface electrodes with a small, implanted telemetry device for collection of arterial blood pressure (required for compliance with ICH S7A CV studies) where the electrical components are secured in a jacket pocket.34 It should be noted that the small (rodent) telemetry device lacks the ability to collect continuous body temperature. CV endpoints collected in these studies are listed in Table 5-2. Telemetry studies using internally instrumented animals are convenient to perform, as the animals can move freely, and once surgically implanted and recovered, the animal can be re-used in several CV studies provided the previous test material has already cleared from their system and sufficient washout periods are allowed between studies.

When the implanted telemetry device records both CV and respiratory parameters as set out in the ICH safety guideline 7A, the studies are termed cardio-respiratory studies. Stand-alone CV or cardio-respiratory studies are usually conducted in non-rodents in a 4 x 4 Latin square cross-over design, where each animal is dosed with the vehicle control and three dose levels of the test drug at low, mid, and high doses, in a random fashion following a washout period (typically 3-7 days). The statistical power of this design is superior to parallel or escalating dose designs as each animal serves as its own control, and any sequence effect is mitigated by the inclusion of all dose levels at each session (Table 5-3). This design uses the least number of animals (four in total) which is in keeping with the 3Rs of animal use – replace, refine, reduce – and can be modified for group housing options or to increase statistical power.

Test articles with long half-lives (e.g., oligonucleotides, monoclonal antibodies, and biologics) where a washout period exceeds 7 days can use a parallel study design. This design consists of four groups of four to six animals that can be included in a stand-alone telemetry study (safety pharmacology environment), or alternatively, the CV safety pharmacology assessment can be incorporated in the parallel repeat dose toxicology study (toxicology environment). Males only can be used if there is no difference in exposures; otherwise, use both males and females. Recovery groups are often only in control and high-dose groups, so use four animals with no recovery animals in the low- and mid-dose groups. Considerations include scheduling of surgery for implanted telemetry and costs of implants. In some cases – and based on overall program considerations – escalating study designs can also be appropriate where animals receive a vehicle control, low-, mid-, and high-dose option sequentially (Table 5-4). Safety pharmacology evaluates the acute effects of the test article, hence the recommendation to collect CV parameters on Day 1 following a single dose if integrated in a repeat dose toxicology study. If the kinetics of the test article allow, CV parameters can be collected on Day 2 when all Day 1 activities are done because the excitement generated by Day 1 dosing and all the other activities, such as blood collection for toxicokinetics, clinical pathology, and clinical observations can have undue effects on CV parameters.

CV studies measure arterial blood pressure (systolic, diastolic, and mean arterial pressure), heart rate (beats/min), Lead II ECG parameters (ECG intervals and rhythm) and body temperature. Qualitative ECG analysis includes a description of waveform morphology (for P, QRS, and T waves) and any arrhythmias, and quantitative measurements include ECG interval durations (RR,PR, QRS, and QT). The QT interval should be corrected for heart rate (QTc) using an individual heart rate correction method.

The advantage of CV telemetry is that it generates a robust dataset by collecting data on every heartbeat and the arterial blood pressure is monitored continuously from at least 1-hour predose through to 22 hours postdose. Collecting beat-to-beat CV data captures pharmacodynamic effects, if present, at onset through Cmax and to recovery. The beat-to-beat data that is collected can be categorized into 1-minute means for data processing. Quantitative ECG intervals (PR, QRS, and QT) are measured for each normal heartbeat and heart rate can be expressed either as the interval between two consecutive RR intervals or from a count of each blood pressure pulse. CV telemetry studies are conducted according to GLP regulations. An optional toxicokinetic phase can be added to a stand-alone CV safety pharmacology study, in the same animals following a washout from the CV phase, as a repeat Latin square design, conducted as a dose escalation (low, mid and high) with washout between doses, or as a single dose (low, mid or high) administered once to all four animals after washout from the CV phase. The toxicokinetic study can also be performed in a separate group of animals and in this option, a single toxicokinetic data collection should be conducted as part of the CV study, for confirmatory concentration but not at Cmax to avoid disturbing the animals during CV telemetry data collection. The addition of a single toxicokinetic collection during CV monitoring can also be considered. When done, this collection is planned after Tmax to correlate exposure from the CV session with full exposure profile.

7.Respiratory System Assessment

Core respiratory studies include measures of the respiratory rate, tidal volume, and minute volume as the major study endpoints assessed because they capture direct effects on lung mechanics but also indirect respiratory effects which may originate from CNS respiratory centers, based on ICH S7A guidance 1,2 (Table 5-2). Additional endpoints that can be assessed when a respiratory liability is identified or suspected are oxygen saturation and airflow to the tracheobronchial tree (resistance, airflow, flow durations), the ease of lung motion and filling (compliance, elasticity), and alveolar gas diffusion.1,2,40 Visual observations of the respiratory rate are not sufficient to meet ICH S7A guidance critera1,2 and respiratory parameters should be quantified with fully characterized methodologies.

Most respiratory studies include evaluation of the effects of pharmaceuticals on ventilation patterns and in some cases blood oxygen saturation. When direct effects on the respiratory system are expected, advanced monitoring methodologies beyond the core battery may be considered such as pressure-volume loops of pleural pressures to quantify changes in airway resistance which can be caused by bronchoconstriction.

Rodent species (rats are used more than mice) are most often used for respiratory safety pharmacology studies. These studies use plethysmography to measure changes in volume in the airways.40 Several plethysmograph configurations can be considered for use in rodents including whole body, head-out, and nose-only. All these configurations can be used to measure the respiratory rate and tidal volume, but the whole-body plethysmography has the advantage of not requiring the animals to be restrained. Plethysmography requires an acclimation period either to the whole-body chamber or to the restraint period typically for up to 4 hours postdose for data collection. A typical study design for most small molecules is a single administration in a stand-alone respiratory study, using a parallel design with four groups of animals of at least eight animals per group which are given a single low, mid or high dose of the test substance or a control substance (Table 5-5). To reduce the use of animals, the rodent respiratory study can also be conducted using the Latin square cross over design.

Baseline data in these studies should be collected before the first dose is given and postdose data collected immediately post dose up to 4 hours later. If the assessment of the test article’s pharmacokinetics requires a longer duration, whole-body plethysmography can be used to collect data at additional timepoints up to 24 hours postdose in the rodent.

Respiratory studies in rodents can also be incorporated in repeat-dose toxicity studies whereby a subset of the study animals is assigned and a designated day during the first week of dosing (i.e., Day 3) is reserved to measure respiratory endpoints (Table 5-6). Animals could either be single-sex, with eight (8) females per group to keep from exceeding the plethysmography chamber (and 6 males used for CNS) or 5 per sex if both sexes are included due to sex differences in exposures. Safety pharmacology assessments are conducted on Day 1 for acute dosing effects or during Week 1 and last week of dosing if conducting assessments of long-acting pharmaceuticals or biologics. No assessments on the recovery animals are done. Similar to a stand-alone respiratory safety pharmacology study, a respiratory evaluation within a repeat-dose toxicity study will use plethysmography following appropriate acclimation of the study animals, with data collected pre- to 4 hours postdose. In non-rodent studies, the main study animals will be evaluated using non-invasive jacketed external telemetry equipped with respiratory inductance plethysmography (JET-RIP) bands, or in animals that have a cardio-respiratory telemetry device surgically implanted. Optional data collections can be performed at the end of dosing for repeat-dose toxicity studies and at the end of recovery period.

A key component to improve sensitivity in a rodent respiratorynsafety pharmacology study is dosing. Dosing half the cohort of animals per group in ascending order (from control to high dose) and the other half in descending order (from high dose to control) will allow all animal groups to be exposed to the technicians in the room administering the dose and have time in the plethysmography chamber to recover from the stress of being handled during dosing.

As indicated for CV studies, and for longer durations of assessment, a combined cardio-respiratory study in a large animal (i.e., non-human primates and dog, less so in minipig) is recommended. Parameters measured during the study can include breathing frequency (respiratory rate), tidal volume, minute volume (the product of respiratory rate and tidal volume and the measure of total ventilation), peak inspiratory flow, peak expiratory flow, enhanced pause, and inspiration and expiration times. Follow-up studies are established and may be performed to evaluate airway resistance, compliance, pulmonary arterial pressure, blood gases, blood pH, among others as warranted.

8.Central Nervous System Evaluation

The effects of pharmaceuticals on the CNS are usually measured using a functional observation battery (FOB)41 or modified Irwin42 assessment. CNS core safety pharmacology studies can be conducted as standalone using a rodent species consistent with the toxicology studies, or rodent CNS safety pharmacology studies are also often incorporated in repeat-dose toxicity studies (Tables 5-5 and 5-6). FOB and Irwin are good first assays to comply with ICH S7A requirements, but the main concern with these assays is their subjective and qualitative nature, as might be expected with behavioral testing. Follow-up safety pharmacology studies with quantitative CNS measures may need to be performed if there are signs or observations of adverse effects on the CNS, such as seizure liabilities, in which case, an electroencephalogram (EEG) may be necessary.

FOB and Irwin observational assessments aim to evaluate motor activity, behavioral changes, coordination, sensory-motoractivity , sensory-motor reflexes responses, and body temperature. A typical study for investigating most small molecule test articles is a single administration, parallel group design, stand-alone CNS study, with four groups of at least six rats per group (single sex; males if no exposure differences are anticipated) which are administered the control or test substance at low, mid and high doses. Animals are socially housed, with up to three animals per cage based on their dose group allocation. The trained CNS technician conducting the FOB or modified Irwin assessment is typically blinded to the treatment. Data should be collected prior to dose administration, either the day before (i.e., pretreatment) or on the day of dosing (baseline). Dosing is staggered to allow time for the same technician to conduct the FOB or modified Irwin assessments at select time points postdose.

The differences between the FOB and modified Irwin assessments are the subtleties in how the animals are handled during the observational battery. The FOB typically will have an assessment around Cmax, and 24 hours postdose, whereas the modified Irwin may have up to five observations up to and including Cmax to 5 hours postdose, or 24 hours if effects are still detected at 5 hours postdose.

The FOB and Irwin assessments also can be incorporated in repeat-dose toxicity studies to assess dose range, duration, reversibility of behavior, and physiological functions. Depending on the intended human population, rodents of different ages (neonatal, juvenile, adult) or pregnancy status can be used in the study, with six main animals (single sex) per group or five main animals per sex. If conducting these assessments as part of repeat dose toxicology studies, males are typically assigned to the CNS study while the females are assigned to the respiratory study (Tables 5-6). Again, animals are first acclimatized to the experimental room prior to being randomized and distributed in groups; this is usually done as part of the toxicity study protocol. A particular day within the toxicity study is scheduled for the FOB or Irwin assessments to be carried out, and the animals are dosed with the control substance and three dose levels of the test article at a designated baseline time point and then placed back in their home cage; the animals are then observed at selected time points up to 24 hours postdose. Dosing can be staggered during the evaluations so that a single observer can collect and record all parameters from several animals and treatment groups at each timepoint. The objective is to determine the highest dose that is tolerated with no observed adverse effect and determine the lowest dose that causes noticeable adverse effect on the behavior of the animals.

In the Irwin assessment, several observations are included:

· Socially housed home cage observations which include posture, unusual behaviors such as convulsion, shivering, vocalization, stimulation, and stereotypy;

· Handling observations, such as ease of removal, handling reactivity, body tone, salivation, lacrimation or piloerection;

·  Motor activity in open field including spontaneous motor activity, gait abnormalities, stereotypic behavior, irritability, piloerection, urination or defection; and

· Autonomic activity assessment such as touch response, startle response, righting/palpebral/ grasping reflexes, tail pinch, thermal nociception, ataxia, body temperature and pupil response.

· Clinical signs and body weights are also recorded.

Observations that can be identified during FOB assessments include:

· Ataxia;

· Reactivity;

· Catalepsy (lack of response to external stimuli);

· Passivity (lack of response or absence of struggle);

· Spontaneous motor activity (locomotor, rearing, grooming);

· Social interaction;

· Stereotypy (repetitive, purposeless behavior such as head bobbing, shaking, gnawing, licking);

· Mortality;

· Exophthalmia (protrusion of the eyeball assessed visually);

· Gait (hypotonic or hypertonic- splayed posture to muscle weakness);

· Grasping reflex (measured by wired maneuver which measures ability to grasp);

· Grip strength;

· Straub tail reaction (elevation of tail from horizontal);

· Tremor (involuntary movements caused by alternating contractions of opposing muscle groups);

· Convulsions (violent involuntary contractions of voluntary muscles which can be clonic with alternating contraction and relaxation of muscles, or tonic, which are sustained contractions, cramp-like or mixed clonic and tonic);

· Diarrhea;

· Lacrimation;

· Piloerection (ruffling of fur);

· Ptosis (partial or full eye closures due to drooping of upper eyelids, pupil size);

· Rectal temperature;

 Salivation;

· Urination;

· Corneal reflex (eye blink response);

· Righting reflex (ability to rest or the body to its normal upright position);

· Pineal reflex (response to retract or twitch the ear);

· Tail pinch; and

· Hot plate response

In ICH S7A, screening and monitoring for potential adverse CNS effects by new pharmaceutical candidates with FOB or Irwin assessments is the first step in assessing CNS safety liabilities or risk to human safety, even though these tests are dependent on the skills of the person conducting the observational battery. The conglomeration of endpoints may need to be integrated to detect potential adverse events and not just individual parameters. Some endpoints do not translate directly to CNS adverse events in humans, and complete concordance cannot be ascertained. If an adverse effect on CNS is identified during an FOB or Irwin test, during a repeat-dose toxicity study or in clinical trials, follow-up studies (e.g., cognition and learning, fear and anxiety, etc.) and seizure liability assessments using electroencephalogram (EEG) may be needed to elucidate further the nature and mechanism of action of adverse CNS effect before continuing drug development. Observations in rodents such as sterotypies (i.e., head bobbing, scratching, excessive grooming and licking) often precede convulsions, and could be clonic or tonic.

9.Safety Pharmacology for Biologics or Biotechnology-derived Pharmaceuticals

Safety pharmacology testing for biopharmaceuticals (i.e., biologics or biotechnology-derived pharmaceuticals) is covered by ICH S6(R1).

Biopharmaceuticals are known for their selectivity (like antibody binding or ligands for receptors), and they are not expected to affect the CV or respiratory systems, or the CNS. However, they may have on-target exaggerated pharmacology, or potential off-target secondary pharmacology activity that may contribute to adverse effects on the vital organs that make up safety pharmacology. For the majority of biopharmaceuticals (large molecules or biotechnology-derived products), safety pharmacology endpoints are incorporated in the repeat dose toxicology studies according to ICH S6(R1).45 If, however, biopharmaceuticals are not highly selective for receptor binding, represents a novel or first-in-class product, or both, then stand-alone or follow-on studies for more extensive safety pharmacology evaluations (i.e., as per ICH S7A) may be necessary.

For biopharmaceutical products which are active in rodents, safety pharmacological endpoints for CNS adverse effects (i.e., using FOB or modified Irwin assessments) are evaluated after a single dose or repeated dose administration; this depends on the test article’s route. of administration, dosing regimen. If the biopharmaceutical is active in non-human primates and rodents then adverse effects can be evaluated in both rodents and non-human primates, but if it is only active in the latter, then safety pharmacology endpoints are only incorporated into non-human primate repeat-dose studies.

In some cases, in vitro CNS safety studies are conducted before in vivo studies are carried out,44 guiding the selection of safety pharmacology endpoints to include in repeat-dose studies and follow-up studies as appropriate (i.e., as per ICH S6(R1) guidance).

10.Safety Pharmacology for Oncology Products

Safety pharmacology for oncology products is covered by ICH S9. For oncology products that are designed for late-stage cancers, there is a need to accelerate the clinical development of the drug candidate so they can be accessible to patients without undue delay. While no stand-alone (i.e., per ICH S7A) safety pharmacology studies are necessary, the assessment of adverse effects on the three vital organ systems (CV, respiratory, and CNS) can be incorporated in repeat-dose toxicity studies or limited to the assessment of ECGs (with scientific justification) collected from a large animal (e.g., dog, non-human primate, or minipig) toxicology study, according to ICH S9.

Repeat-dose toxicity studies evaluating the three vital organ systems from the safety pharmacology core battery should be conducted in accordance with GLP regulations and with detailed clinical observations. In rodents (usually rats), repeat-dose studies usually incorporate the FOB or Irwin assessments, with half of the main study animals in the 28-day toxicity study (e.g., five of 10 males) undergoing assessment on Day 1, as well as detailed respiratory observations (e.g., five of 10 females), with treated groups being compared with the vehicle control group. In a non-rodent repeat-dose toxicity study, ECG and hemodynamic measurements (heart rate, blood pressure, and temperature) are collected in addition to detailed observations; these are collected at baseline (i.e., predose), on Day 1 (the first day of dosing) and during the last week of dosing (i.e., Day 28 or Day 90), including interim collection mid-study if warranted. Measurements from treated groups will be compared quantitatively with the control vehicle group and significant changes will be assessed and correlated with dose levels, toxicokinetic exposures, or both. If effects are identified during evaluations on the last week of dosing, CV, respiratory and CNS evaluations will generally be added to the recovery period to assess reversibility.

The desire to accelerate the development of oncology products due to the potentially greater benefit to risk ratio is aligned with the lack of regulatory requirement for stand-alone pharmacology studies. As safety pharmacology assessments are incorporated in repeat-dose toxicology studies, patient safety can be adequately addressed while also adhering to the 3Rs of animal use according to ICH S9.

When designing follow-up safety pharmacology studies for oncology products, the pharmacology and the clinical indication need to be considered and such studies are performed on a caseto-case basis (Table 5-7). They should be streamlined to reduce the number of animals especially for biopharmaceuticals and oncology drugs according to ICH S645 and ICH S946 and supplemental tests on these vital organs as well as other organs should also be done on a case-by-case basis (Figure 5-1).

11.Comprehensive in vitro Proarrhythmia Assay

The Comprehensive in vitro Proarrhythmia Assay (CiPA)47,48 was developed in 2013 by the FDA with the goal of improving the assessment of proarrhythmic markers and to provide more relevant, reliable and comprehensive preclinical in vitro cardiac screening tools. This initiative was based around the potential for some pharmaceuticals (e.g., verapamil) to block the hERG channel however not to be proarrhythmic in humans.

As its name suggests, CiPA includes an assessment of comprehensive list of cardiac ion channels that are involved in the ventricular action potential, and as such its use supports the requirement for hERG evaluation.49,50 The first step of CiPA is to conduct patch clamp assays on hERG transfected cells for six cardiac ion channels: hERG, Nav1.5 (late sodium), Cav1.2 (calcium), KvLQT1 (slow delayed rectifying potassium current; IKs), Kir2.1 (inward rectifying potassium channel responsible for membrane potential; IK1) and Kv4.3 (fast transient outward potassium current; Ito). The data from these patch clamp assays are then used to supply computer-simulated models of the human ventricular action potential that can provide information on potential early cardiac liabilities. The algorithms driving the computerized models can predict changes in the ventricular action potential (e.g., prolongation) as well as generation of any early afterdepolarizations. Early afterdepolarizations are the early triggers of proarrhythmic risk. The in silico models, such as the O’Hara Rudy model, which is an accurate model for healthy human ventricular action potential, are capable of assessing compounds that prolong the QT interval that is not associated with proarrhythmic risk (e.g., verapamil).51 Lastly, the patch clamp data through in silico modeling are assessed for translation against induced pluripotent stem cells-derived cardiomyocytes (iPSc-CM).

CiPA also includes the use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). These cells are assessed using multi-electrode array technology to measure the extracellular field potential of compounds, helping to identify those with potentially adverse effects on CV electrophysiology. Changes in the recorded field potential waveforms underlie changes in the ventricular action potential and could indicate blockage of the hERG or other ion channels, such as the sodium channel Nav1.5, which is responsible for the upstroke of the action potential or calcium channel Cav1.2, which is responsible for the duration of the actional potential. Hence a prolonged field potential duration could result in the prolongation of ventricular action potential and could indicate a prolongation of the QT interval recorded on the ECG. Drug-induced action potential prolongation should be distinguished from prolongation arising from the variability of cell-to-cell beating with the use of positive and negative control compounds.55 As indicated above, other potassium channels in addition to hERG, like Kir2.1, Kv4.3, KCNQ1/KvLQT1_mink) are also investigated during CiPA.

12.Safety Pharmacology for Other Organs

Safety pharmacology studies for organ systems other than those included in the core battery can also be evaluated depending on the therapeutic indication and adverse findings in the toxicity studies. This includes the renal or urinary systems, the gastrointestinal system, and the autonomic nervous system, among others. Endpoints of such studies will depend on the system being tested, but may include the volume of urine excreted and electrolyte excretion, gastrointestinal transit time and gastric emptying, immunological parameters such as immune cell phenotyping, endocrine parameters such as hormone levels, and orthostatic hypotension. These parameters could be used to assess functional changes in these other organ systems.

All pharmaceutical or biopharmaceutical drug candidates should be tested for safety pharmacology of these other organ systems as necessary, and on a case-by-case basis, with a fit-forpurpose study design, either during nonclinical drug development or before Phase 2 of clinical development.

ICH E14/S7B Q&As - The Need for Nonclinical Studies for Clinical TQT Studies

The need for nonclinical studies for clinical TQT studies is covered by ICH guidance E14 and the ICH S7B Q&As. ICH S7B was implemented in 2005 to guide the conduct of nonclinical safety pharmacology studies assessing the potential of new agents to prolong ventricular repolarization (QT interval) before first-in-human studies. That same year, ICH E14 was adopted, outlining the need to conduct a clinical study to assess the effects of these new agents on the potential to delay cardiac repolarization.

The FDA has published data on the number of TQT studies conducted from 2016 through 2020 and found that 32% of studies failed to reach the E14 Q&A 5.1 threshold of 2X high clinical exposures. Additionally, another 24% of clinical studies had alternate study designs (82% of these were for oncology indications) where no TQT study could be conducted, therefore resulting in a label of “no large QTc effects.” In an effort to reduce the number of clinical TQT studies, E14/S7B Q&As were approved in February 2022, finalized in July 2022, and were implemented in August 2022.7,8 This guidance provides the opportunity to review the double negative nonclinical results (i.e., both hERG and in vivo QT/QTc studies are negative) in combination with clinical E14 Q&A 5.1 (in support of TQT waiver when clinical exposure does not reach 2x high clinical) or E14 Q&A 6.1 (to support labeling of “low likelihood of proarrhythmic effects due to delayed repolarization”).

Assuming the results from ICH S7B hERG and in vivo QT studies show no delay to ventricular repolarization, they will give confidence to regulators and clinicians that the tested compounds do not pose a risk to healthy volunteers participating in first-inhuman

studies. In order to use the nonclinical hERG and in vivo QT results (double negative) as an alternative (E14 Q&A 5.1) or as a substitute (E14 Q&A 6.1) for a clinical TQT study, there should be:

· Alignment of standard protocols;

· Experimental conditions that follow the best practices as outlined in S7B Q&As (Q&A 2.1; hERG and 3.1-3.5 in vivo QT assay); and

· Consistency of reports for regulatory review.

The in vitro hERG Q&As of S7B (Q&A 2.1 for patch clamp) provide guidance on:65

· Recording temperature (35-37 °C), voltage protocol to follow;

· Measures of recording hERG current quality;

·  Primary endpoints to measure or calculate (e.g., IC50, Hill coefficient, and confidence intervals); Concentration verification; and

· Use of positive and negative controls.

The in vivo QT assay Q&As of S7B (Q&A 3.1 to 3.4) provide guidance on:

· Species selection consistent with ICH S7B for appropriate, freely moving, non-rodent species consistent with toxicology studies;

· Exceeding anticipated therapeutic concentrations – the higher the multiple, the less reliance on assay sensitivity for QTc under E14 Q&A 6.1 (i.e., statistical measure of QTc variability; least significant difference to detect changes in QTc in milliseconds, inclusion of concentration QTc analysis, required for E14 Q&A 6.1 and recommended for Q&A 5.1);

· Use of an individual animal heart rate correction method -including QTc/RR scatter plots, assessing assay sensitivity and laboratory historical sensitivity (minimal detectable difference).

The assay sensitivity to detect changes in QTc (least significant difference) to the threshold measured in clinical studies is only required under Q&A 6.1. It is best practice to report the least significant difference for QTc for the in vivo QT assay in support of Q&A 5.1, however, there is no minimal threshold and the value is used only as a measure of variability for QTc within a study. Finally, S7B Q&A 3.5 are points to consider for reporting the results.

Thus, the nonclinical double negative data can now be advanced past first-in-human regulatory review for consideration with ICH E14 Q&As 5.1 or 6.1 as the nonclinical-clinical risk assessment for proarrhythmia. This is anticipated to increase submissions for TQT waivers and reduce the number of required clinical TQT studies. Follow-up in vitro assessments on ventricular repolarization are included in the ICH S7B best practice(Q&A 2.2-2.5) using cardiomyocytes and  proarrhythmia models (Q&A 4) (ICH E14/S7B Q&A reference).

13.Summary and Conclusion

Safety pharmacology studies has evolved over the years to keep up with the changing modalities of pharmaceuticals. Nonetheless, they have remained focused on the assessment of potential adverse pharmacological effects prior to the first human dose being given in clinical trials.

The safety pharmacology community has produced high quality functional data to assess drug safety, either as stand alone or in combination with repeat dose toxicology studies. The refinements over the past two decades have focused on best practices (e.g., using digital data collection, acclimation and refinement techniques to lower stress), and a commitment to the 3Rs principles to reduce the use of animals and social housing, individual animal heart rate correction, QTc/RR scatter plots, among others. That said, the assessment of most of these details is not standardized across the industry and are not systematically included in study reports. With the publication of the ICH S7B Q&As, those details, statistical measures and corrections and more need to be in the final reports. Towards this singular goal, core battery safety pharmacology studies have been focused on first-in-human studies, usually single dose and small sample size studies, with continuous data collection. Based on the pharmacology of the pharmaceutical or biopharmaceutical being tested, studies may be modified to accommodate other designs in follow-up studies.

The conduct of various safety pharmacology studies – in vitro hERG studies, the ability to detect a hERG block and QT prolongation in the CiPA initiative, in addition to in vivo testing– which are undertaken at the screening stage of drug candidate selection, has staved off drug attrition due to proarrhythmia at the later stages of clinical development. This necessitated integrating the in vitro hERG and in vivo QT assay nonclinical data as well as clinical data for integrated risk assessments of QT/QTc prolongation, and the potential waiver and reduction of thorough TQT clinical studies if there is no observed hERG block or QT/QTc prolongation in well-controlled nonclinical studies.

For CV assessments, stand-alone safety pharmacology studies are the gold standard rather than combination toxicology studies, but combination safety pharmacology studies will continue to be used for both small and large molecules, in keeping with the 3Rs and as long as safety pharmacology endpoints are collected in a rigorous and interpretable manner to elucidate effects of the pharmaceutical on both structural and physiological functions. For most CV studies, implanted telemetry devices with body temperature collection and respiratory capabilities are now acceptable and more prevalent than jacketed telemetry, paving the way to collect more reliable and consistent ECG and respiratory waveforms, leading to more accurate analysis that facilitate combination with toxicity studies. Currently, cell and gene therapy are not specifically addressed in the S7A and S7B/E14 guidance as they are rapidly evolving, but soon they may need to be included in the scope of the guidelines as well. There is a need to continue to assess the effects of translating the data garnered from the safety pharmacology studies to limit failures during clinical trials.

What safety pharmacology studies should be conducted for a small molecule that has nanomolar inhibition of hERG?

• Background

A small molecule (new molecular entity) is being developed as a systemic drug for neurodegenerative disease. The intention is that it will be administered as an oral tablet to be taken once daily. During nonclinical development, the molecule exhibited nanomolar inhibition of the hERG channel (IC50 <100 nM) in nonhuman primates.

• Solution

The core battery of safety pharmacology studies is required for this small molecule being developed for a neurodegenerative indication. As the molecule was being developed as a daily oral tablet, the drug development plan accounted for two CV studies to address repeat-dosing and correlate pharmacokinetics with CV measurements. In addition, in the spirit of the 3Rs, the large animal CV study was combined with respiratory collection via telemetry. For the CNS, FOB was integrated into the 28-day rodent repeat toxicology study.

The preliminary cardio-respiratory study was conducted non-GLP using an escalating dose design (at pharmacologically efficacious dose and two dose levels (2X and 6X) above the first dose using three non-naïve telemetry instrumented nonhuman primates. The pivotal GLP cardio-respiratory study was conducted using drug-naïve nonhuman primates, which were surgically implanted with a telemetry device.

A parallel group study design was used, with four groups of animals (three animals per group); animals in Group 1 were given a control (vehicle) substance and the animals in the other three groups received escalating doses of the test drug, at the lowest efficacious dose (Group 2), and at 2X and 6X above the lowest efficacious dose (Groups 3 and 4, respectively). This study design is modified from that shown in Table 5-4).

Nonhuman primates were dosed once daily, and telemetry data for CV and respiratory function were collected on Days -1, 1, 2, 4 and Day 7. On Day 8, the animals were dosed one more time to enable blood collection for pharmacokinetic analysis on Days 8 and 9 (24 hours postdose).

The results of these cardio-respiratory studies (both non-GLP and GLP) indicated no in vivo CV risk of the molecule at 6X the efficacious dose when administered once-daily for 7 days, supporting its continued development.

What safety pharmacology studies should be conducted for a small molecule that has a seizure liability?

• Background

A small molecule sodium-blocking drug that needs twice-daily oral administration is under development as an analgesic. Rat and dog studies have been used in its development but convulsions have been observed. The convulsions occurred at plasma exposures that are estimated to be higher than those likely to be seen in patients, at approximately 10X the efficacious clinical dose in dogs and 45X of the efficacious clinical dose in rats.

• Solution

Appropriate in vitro studies need to be conducted to evaluate potential mechanisms for the convulsions that have been observed.

A dog EEG study which records spontaneous electrical activity of the brain68,69 will likely be required to:

· Confirm the nature of the adverse CNS effects and establish if the convulsions were due to seizure or another toxicological effects (e.g., syncope or overt movement disorder);

· Confirm the plasma exposure when the convulsion and other CNS signs (e.g., myoclonus) are observed;

· Identify clinical signs that may be a precursor to the convulsions or seizures and that could be used as stopping criteria in clinical trials; and

· Confirm the absence of EEG abnormalities at lower doses where no convulsions were observed.

The EEG study will typically involve video-EEG68-70 and continuous recording from at least 24 hours prior to dosing until at least 24 hours after the last dose. All dogs will be screened with continuous EEG recorded and analyzed for 72 hours prior to study start to evaluate for the presence of abnormal EEG morphologies prior to assignment on study. The minimum group size is eight (typically four males and four females). The dose levels will be selected to include an EEG ‘no observed effect level’ ideally as high as possible and a higher dose level at which convulsions were observed to confirm the nature of the CNS events.

The study design may be adaptative, with dose levels for some study groups being selected based on the outcome from the results with the previous experimental groups. Although not a primary objective, the study will also confirm if convulsions are self-limiting and potentially evaluate emergency drugs (e.g., diazepam, phenytoin, or propofol) to treat any drug-induced seizures.

Toxicokinetic sample collections (including important metabolites) will be used at unscheduled timepoints when severe CNS clinical signs are observed to precisely quantify plasma exposure during those events and with concurrent analysis of EEG traces. The duration of repeat administration will be selected to exceed the duration that was required to induce convulsions in the repeated dose general toxicology study. To avoid tachyphylaxis, a 1 month wash-out period will be included.

A rat EEG study will likely not be required given the higher safety margins in this species and the data from the dog that will characterize this liability.

Chapter 6 Pharmacokinetic and Toxicokinetic Studies

The goal of preclinical development is to generate sufficient data to allow an estimation of the benefit/risk profile of a new drug product prior to its use in patients. In general,the benefit(s) are ascertained from a combination of discovery phase studies in cells (i.e., in vitro) or animal-based (i.e., in vivo) models, and from reports in the scientific literature. The risk(s) are determined through a battery of nonclinical safety assessments. Pharmacokinetic (PK) and toxicokinetic (TK) studies – often simplified as a description of ‘what the body does to the drug’– are invaluable for this process as they provide a quantitative connection between the dose administered, and the amount of drug that is subsequently present internally at various times following dosing. When paired with a specific assessment of benefit (e.g., a reduction in cholesterol) or toxicity (e.g., cardiovascular dysfunction, such as evidence of QT interval prolongation on an electrocardiogram, ECG), this information can ultimately be used to develop and justify a clinical dosing regimen that maximizes the benefits of the drug, while minimizing its risks.

This chapter will start with a brief overview of PK/TK itself, including an introduction to drug disposition and several other key background concepts. This will be followed by a discussion of the specific global regulatory recommendations/requirements pertaining to the conduct and interpretation of PK/TK analyses, largely from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use(ICH). Finally, the chapter will end with a description of how the results of PK and TK analyses should be presented in marketing and pre-marketing submissions to global regulatory bodies.