safety pharmacological technologies - classification - tier 1 and tier 2 studies - established techniques and parameters to assess

1. SAFETY PHARMACOLOGY STUDIES
L.V SAI SANTOSH
M.Pharmacy 1st year – 2nd semester
Pharmacology
Acharya Nagarjuna university

2. CONTENTS
 INTRODUCTION
 OBJECTIVES
 GENERAL CONSIDERATIONS
 SAFETY PHARMACOLOGY STUDIES
 CVS
 CNS
 RESPIRATORY
 GI SYSTEM
 RENAL SYSTEM
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3. SAFETY PHARMOCOLOGY STUDIES
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• Safety pharmacology (SP) is an essential part of the drug development process that aims
to identify and predict adverse effects prior to clinical trials.
• It identifies the “potential undesirable pharmacodynamic effects of a substance on
physiological functions in relation to exposure in the therapeutic range and above”.
AIM :
• To characterize the pharmacodynamic/pharmacokinetic (PK/PD) relationship of a
drug’s adverse effects using continuously evolving methodology.

4. OBJECTIVE OF SAFETYPHARMACOLOGY
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• SP studies are described in the international conference on harmonization (ICH) S7a
and S7b guidelines.
• According to ICH S7A:-
• To identify undesirable pharmacodynamic properties of a substances.
• To evaluate adverse pharmacodynamic and pathophysiological effect of a
substance .
• To investigate the mechanism of action of a adverse pharmacodynamic effect .

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6. GENERAL CONSIDERATIONS IN
SELECTION/DESIGN
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1) effects related to the therapeutic class of the test substance, since the mechanism of
action may suggest specific adverse effects.
2) adverse effects associated with members of the chemical or therapeutic class
3) ligand binding or enzyme assay data suggesting a potential for adverseeffects
4) results from previous safety pharmacology studies, from secondary
pharmacodynamic studies, from toxicology studies, or from human

7. USE OF IN VIVO AND IN VITRO STUDIES:
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• Ex vivo and in vitro systems can include,
• isolated organs and tissues,
• cell cultures,
• cellular fragments,
• subcellular organelles,
• receptors,
• ion channels,
• transporters and enzymes.
• In vitro systems can be used in supportive studies (e.g., To obtain a profile of the
activity of the substance or to investigate the mechanism of effects observed in
vivo).

8. SAMPLE SIZE AND USE OF CONTROLS
• The size of the groups should be sufficient to allow meaningful scientific
interpretation of the data generated.
• The number of animals or isolated preparations should be adequate to demonstrate or
rule out the presence of a biologically significant effect of the testsubstance.
• The size of the biological effect that is of concern for humans. Appropriate negative and
positive control groups should be included in the experimental design.
 ROUTE OFADMINISTRATION
• Exposure achieved similar to or greater than in humans
• If clinical use involves multiple routes, consider more than one route
• The expected clinical route of administration should be used when feasible.
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9. DOSE LEVELS OR CONCENTRATIONS
OF TEST SUBSTANCE
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In vivo studies
• Safety pharmacology studies should be designed to define the dose-response
relationship of the adverse effect observed.
• The time course (e.g., Onset and duration of response) of the adverse effect
should be investigated.
• Generally, the doses eliciting the adverse effect should be compared to the doses
eliciting the primary pharmacodynamic effect in the test species or the proposed
therapeutic effect in humans.

10. In vitro studies:
• In vitro studies should be designed to establish a concentration-effect
relationship.
• The range of concentrations used should be selected to increase the likelihood
of detecting an effect on the test system.
• The upper limit of this range may be influenced by physicochemical
properties of the test substance and other assay specific factors.
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11.  The core battery SP studies, performed according to GLP standards as per ICH
guidelines, involves the investigation of major vital organisms.
 TIER 1 – CORE BATTERY
• CVS
• CNS
• Respiratory
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SAFETY PHARMACOLOGY STUDIES
 TIER 2 – SUPPLEMENTARY STUDIES
• Renal
• Gi system
• Others

12. CARDIOVASCULAR SYSTEM
• In the last few decades, a large no of drugs have been withdrawn from market
due to adverse CVS effects, which were responsible for the 45% of post
approval withdrawals.
 PARAMETERS TO BE ASSESED
• Cardiac output
• Ventricular contractility
• Vascular resistance
• The effects of endogenous and exogenous substances
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13.  Established techniques
 In vitro –
• hERG assay
• Manual patch clamp
• Automated high-throughput patch clamp
• Isolated organ preparation
• Whole heart preparation
• Isolated purkinje fibers
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 In vivo –
• Telemetry
• Internal (surgical implant)
• External (jacketed )

14. • The electrical activity in CVS can be measured using ECG, which analyzed by dividing
the recorded trace into waves and intervals with particular focus on the QT interval
which represents cardiac repolarization.
• QT prolongation has resulted in one third of all drug withdrawals between 1990 – 2006
due to risk of developing fatal arrhythmias. [eg- TERFINADINE].
• SP tests, consisting of an in vitro assay to assess the extent of the human Ether-a-go-go
Related Gene (hERG) potassium channel, Kv11.1, blockade, in vivo telemetry and
additional in vitro/ex vivo tests were adopted to evaluate the likelihood of an NCE to
cause adverse CVS effects.
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ELECTROCARDIOGRAM

15. IN VIVO TELEMETRY
• Physiological data obtained from conscious, large mammals is accepted for
detecting any effects of an NCE on CVS functionality.
• Telemetry used for continuous measurement of
• Arterial, systemic and left ventricular BP
• Heart rate
• ECG parameters – QRS complex, QT, ST, PR
• Other factors such as changes in body temperature and plasma con of electrolytes
(e.g potassium), glucose and insulin should be taken into account when
interpreting ECG readouts.
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16. IN VITRO ISOLATED MYOCARDIAL SYSTEMS
• The effect of NCE’s on cardiac action potential can also be investigated using other
in vitro systems including isolated myocardial tissue (purkinje fibers or papillary
muscles ) or whole isolated hearts.
• For example, a functional in vitro model using isolated guinea-pig papillary muscles
can be used to evaluate direct NCE-induced effects, including the force of
contraction and refractory period, in addition to effects on the action potential.
• However, these low-throughput techniques are costly and require highly skilled
electrophysiologists.
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17. HERG ASSAY
• hERG – human eher –a-go-go related gene was first identified in late 1980’s in a
mutant fruit fly.
• hERG encodes the inward rectifying voltage gated potassium channel in the heart
(IKr) which is involved in cardiac repolarization.
• Inhibition of the hERG current causes QT interval prolongation resulting in
potentially fatal ventricular tachyarrhythmia
• In humans it is expressed widely, including in the brain, adrenal gland, thymus,
retina and in cardiac and smooth muscle tissues.
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18. STRUCTURE OF HERG
• A detailed atomic structure for hERG based on X-ray crystallography is not yet
available, but structures have recently been solved by electron microscopy.
• In the laboratory the heterologously expressed hERG potassium channel comprises 4
identical alpha subunits, which form the channel's pore through the plasma
membrane.
• Each hERG subunit consists of 6 transmembrane alpha helices, numbered S1-S6, a
pore helix situated between S5 and S6, and cytoplasmically located N- and C-termini.
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19. • The S4 helix contains a positively charged arginine or lysine amino acid residue at
every 3rd position and is thought to act as a voltage- sensitive sensor, which allows the
channel to respond to voltage changes by changing conformations between conducting
and non- conducting states (called 'gating').
• Between the S5 and S6 helices, there is an extracellular loop (known as 'the turret')
and 'the pore loop', which begins and ends extracellularly but loops into the plasma
membrane.
• The pore loop for each of the hERG subunits in one channel face into the ion-
conducting pore and are adjacent to the corresponding loops of the 3 other subunits,
and together they form the selectivity filter region of the channel pore.
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20. SCREENING OF HERG
• In the heart, hERG channels are the molecular correlate
of the IKr current which, together with other potassium
currents, is involved in action potential repolarization.
• Reduced function of hERG causes action potential
prolongation, which in rare cases can lead to the
potentially fatal ventricular tachyarrhythmia.
• In a body surface electrocardiogram (ECG), ventricular
action potential prolongation manifests itself as a
prolongation of hERG assays
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T WAVE IS DELAYED

21. MEDIUM AND HIGH THROUGHPUT ASSAY
• The ideal hERG assay provides a linear measure of channel activity under
physiologically relevant conditions.
• However, such a study is extremely laborious and only amenable to the
detailed characterization of very few selected compounds.
• It is advantageous to screen compounds for hERG activity early on in the lead
evaluation and optimization process. However, this approach requires testing
of hundreds and potentially thousands of compounds within a single drug
discovery program
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22. ELECTROPHYSIOLOGY
• The development of automated electrophysiology technologies has improved the throughput
of electrophysiological methods
• Electrophysiology can provide detailed and quantitative information on the potency and
mechanism of hERG block by a test compound.
• One of the unique advantages of such voltage clamp recordings is the ability to control
membrane potential.
• Since activation and inactivation of hERG is dependent on membrane potential, voltage
clamp recordings can differentiate between compounds.
• Limitation - the high cost of the instruments and consumables
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23. FLUX ASSAY
• An alternative to either manual or automated electrophysiology is a functional assay
that measures ion flux across cell or vesicle membranes.
• This assay offers advantage of the ability of Rubidium ion i.e. Rb+ to permeate
through hERG channels.
• Typically, cells are loaded with Rb+ overnight.
• hERG-dependent Rb+ efflux is initiated by an addition of high (50–60 mM)
extracellular potassium concentrations to depolarize the cell and open hERG channels.
• The amount of Rb+ efflux can be calculated by using 86Rb+ as a radioactive tracer or
by flame atomic absorption spectrometry (FAAS).
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24. FLUORESENCE BASED ASSAY
• The development of improved fluorescent dyes and plate readers has provided
another approach to high throughput screening of ion channel activities.
• Fluorescent dyes which are sensitive to changes in membrane potential have
proved.
• However, studying hERG by this approach presents a challenge since this channel
does not typically control a cell’s resting membrane potential.
• It has been possible, however, to select HEK-293 and CHO-K1 cell lines stably
expressing recombinant hERG channels.
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25. RADIO LIGAND BINDING ASSAY
• Radio ligand binding assays have been used extensively to screen for interaction
with the hERG channel..
• They do not provide a direct measure of IKr blockade, such binding assays can
test 50,000 to 100,000 compounds per day and are relatively inexpensive, which
is why they are commonly used in most large pharmaceutical companies.
• It can be effective for the treatment of tachycardia.
• Radio ligand binding assays are manageable to a range of assay conditions which
may impact on the binding ability of test compounds.
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26. ADVANTAGES OF hERG
• The hERG channel has been shown to be the target for class III antiarrhythmic
drugs such as amiodarone, which reduce the risk of re-entrant arrhythmias by
prolonging the action potential.
• It can be used in drug development process of new small molecule drugs with
improved cardiovascular safety profiles.
• It can also be used as a diagnostic marker in treatment of diseases like Cancer,
Epilepsy, Schizophrenic.
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28. CNS SAFETY PHARMACOLOGY
• ADR’s associated with the CNS represent a major cause for concern for
pharmaceutical companies.
• A variety of drugs exhibit CNS side effects including sedation, ataxia and nausea.
• More importantly 10% of drugs withdrawn from market between 1960 – 1999 due to
severe CNS side effects
• Effects of the test substance on the central nervous system should be assessed
appropriately.
• Motor activity, behavioral changes, Coordination
• sensory/motor reflex responses and body temperature should be evaluated.
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29.  The parameters to assess during the assessment of CNS SP-
• Behavioral pharmacology
• Learning and memory
• Ligand-specific binding
• Neurochemistry
• Electrophysiology examinations, etc.
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30.  Established techniques
• Modified Irwin's test, Functional Observation Battery (FOB)
• Photoelectric beam interruption systems
• Rotarod
• Hot plate test, Tail flick, Paw pressure
• Morris maze and passive avoidance tests
• Electrocerebral silence threshold and pentylenetetrazol seizure tests
• Electroencephalography (EEG)
• Self administration and drug discrimination lever chamber models
• Drug withdrawal: FOB, body temperature, body weight
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31. PARAMETERS TO ASSESS DURING SP
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32. IRWIN TEST
• The IRWIN TEST consists of systemic evaluation of general behavioral and
physiological observations in the rodent including arousal(state of awake), vocalization
and stereotypy.
• Drug treated animal groups are compared to a vehicle group and observational
differences between the groups are documented using a qualitative scoring system
• Although this methodology provides satisfactory assessment of gross behavioral
changes it does not encapsulate vital neuro-physiological functional assessments
outlined by the ICH
• As a result Irwin test was modified to incorporate all core functions detailed by ICH
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33. • Similarly to the modified Irwin's test, the Functional Observation Battery (FOB)
provides a more comprehensive evaluation of NCEs on the fundamental CNS
functions
• Additionally, FOBs are frequently used to carry out neurotoxicological and
neuropathological investigations.
• Drugs, such as the psychostimulant, amphetamine, and the antipsychotic,
chlorpromazine, can be used as reference compounds to validate the effect of NCEs on
neurobehavioral function.
• This type of analysis is subjective and require highly trained and experienced
observers to ensure efficient reproducibility of the experiments.
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34. RESPIRATORY PHARMACOLOGY
• Drugs of various pharmacological classes are known to have deleterious effects on
respiratory functions including life threatening conditions.
 Core battery tests
• Respiratory rate
• Tidal volume
• Hg oxygen saturation
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 Follow up studies
• Air way resistance
• Pulmonary arterial pressure
• compliance

35.  ESTABLISHED TECHNIQUES
• Plethesmography
• Head out – VT; F; VT*F; PIF/PEF/Ti/Te/fit in unrestrained animals
• Head out + pressure – above along with compliance; resistance in unrestrained
• Head – enclosed - VT; F; VT*F; PIF/PEF/Ti/Te/fit ; specific airway resistance in restrained
animals
• Barometric whole body - VT; F; VT*F; FIT; Penh
• By induction/impedance
• Telemetry (external/implanted) – VT; F; VT*F
• Invasive
• Pulmonary resistance and compliance
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36. PLETHESMOGRAPHY
• Accurate ventilatory patterns are assessed to directly monitor lung volume changes or
airflows generated by thoracic movements in conscious animals using a plethysmograph
chamber.
• Head-out, dual chamber and whole body plethysmography techniques are non-invasive
methods
• A study which compared these three plethysmography methods in rodents reported that each
system was equally sensitive.
• The whole body and head-out plethysmography provided consistent and reliable pulmonary
mechanics data, while data collected from chamber plethysmography are clearly affected by
restrainment stress in the animal
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37. • Whole body and Head out plethesmography methods in conscious rats were compared,
using theophylline as respiratory stimulant and chlordiazepoxide as a respiratory
depressant.
• The study reported that respiratory function can be accurately evaluated using head-out
plethysmography compared to whole body plethysmography.
• Another non invasive method enhanced pause (Penh), was found to be less reliable
compared to head out.
• Non-invasive head-out body plethysmography measurements for core battery respiratory
SP studies in conscious rodents are reliable, as it is simple to handle, the breathing pattern
is nearly natural (anesthesia is not required) and it allows high-throughput screening.
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38. GASTRO INTESTINAL SYSTEM
• Gastrointestinal (GI) complications are common side effects, with varying degrees of severity,
observed during and after drug development, and are associated with drug-induced morbidity
• Drug induced GI complications include nausea, emesis, constipation and may also affect the
absorption of other drugs.
• The effects of test compounds on the GI system are commonly evaluated in rodent models,
using tests assessing:
• gastric emptying
• intestinal motility
• gastric secretion
• GI injury
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39. TECHNIQUES
 ESTABLISHED TECHNIQUES
• Gastric emptying - macroscopic (ulcer index )
• Intestinal motility - histopathology
• Gastric secretion
 EMERGING TECHNIQUES
• Endoscopy - endoscopy
• Capsule – pH, pressure - capsules
• Radiotelemetry - biomarkers – Citrulline; miR- 194
• Strain gauges for contraction, EMG
• In-silico (PBPK modelling)
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40. GASTRIC SECRETION
• Gastric screening is evaluated by the parenteral administration of the test drug
following pylorus ligation and stomach contents act as screen for changes
such as volume, pH, total acidity and acid output over tiem.
• Agonists of opioids, dopamine receptors, beta adrenoceptors reduce gastric
emptying where as uscarinic receptor agonists increase.
• Anticancer compounds have shown greater GI complications hence it would be
beneficial to include GI testing as part of the routine safety pharmacology
studies for this class of compounds.
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41. RENAL SYSTEM
• Based on the data available from preclinical testing and clinical trials, it can be inferred
that drug-induced changes in kidney function, including nephrotoxicity, may be
underestimated.
• There is a growing need to integrate routine evaluation of renal functions into SP testing,
which can be grouped into,
• Altered renal functions (diuresis or anti diuresis)
• Organ damage
• Acute kidney injury
• Localized injury to glomerulus, renal papillae, or different regions
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42. PARAMETERS TO BE ASSESED
 CORE BATTERY TESTS
• Clearance rate
• Glomerular filtration rate
• Urinary volume
• Osmolarity
• pH, Na+, Cl-, K+
• Creatinine
• Urea
• Serum Na+, Cl-, K+, creatinine
• Blood urea nitrogen
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44. KIDNEY INJURY MARKERS
• Kidney injuries are being assessed using two types of markers.
1. FUNCTIONAL MARKERS –
• urinary glucose, protein, albumin and calciumor, indeed, any other molecule known to be
transported in a certain region of the kidney
2. LEAKAGE MARKERS –
• Urinary excretion of aspartate aminotransferase (AST), alanine amino transferase (ALT), lactate
dehydrogenase (LDH), γ-glutamyl transferase (GGT), alkaline phosphatase (ALP) and N-acetyl-
β-D-glucosaminidase (β-NAG) are used as leakage markers for kidney injury measurement by
clinical chemistry
• Further leakage markers like kidney injury molecule-1 (KIM-1) and clusterin (CLU) can be
measured with different techniques based on antibody detection.
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46. REFERENCE
 REVIEW: FRONTIERS IN PHARMACOLOGY Principles of Safety
Pharmacology MK Pugsley1, S Authier2 and MJ Curtis3
 Hamdam, J., et al., Safety pharmacology — Current and emerging concepts,
Toxicol. Appl. Pharmacol. (2013),
 Toxicology and Applied Pharmacology
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47. THANK YOU
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