Toxicology ## screening Models.

2. MAHRASHI DAYANAND UNIVERSITY, ROHTAK
DEPARTMENT OF PHARMACEUTICAL SCIENCE
PHARMACOLOGICAL & TOXICOLOGICAL SCREENING METHODS-II.
TIER-1: CVS, CNS & RESPIRATORY SAFETY PHARMACOLOGY
SUBMITTED BY: UNDER THE GUIDANCE OF:
TARANJUM KHAN DR.ABHILASAAHLAWAT
M.PHARMACY IST YEAR ASSISTANT PROFESSOR
PHARMACOLOGY MDU, ROHTAK.
ROLLNO: 1803.

3. OUTLINES:
 Basic Introduction
 Principle ,Objective and categories.
 Safety pharmacology ,Experimental Design
 General consideration
 Tier- 1: Safety core, Follow-up Studies, Evaluation Methods
 CNS Safety pharmacology
 CVS Safety pharmacology
 Respiratory safety pharmacology
 References

4. BASIC INTRODUCTION
 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.

5. Basic objectives & Scope of Safety Pharmacology.
 To help protect clinical trial participants and patients receiving marketed products from
potential adverse effects of pharmaceuticals
 Avoiding unnecessary use of animals and other resources.
Scope:
 Applies to new chemical entities and biotechnology-derived products
 Applied to marketed pharmaceuticals when appropriate(e.g., when adverse clinical events, a
new patient population, or a new route of administration raises concerns not previously
addressed).3

7. Principle
 The specific studies that should be conducted and their design will vary based on the individual
properties and intended uses of the pharmaceuticals.Scientifically valid methods should be
used (internationally recognized)
 The use of new technologies and methodologies with sound scientific principles is encouraged.
 Safety pharmacology endpoints can be incorporated in the design of toxicology, kinetic,
clinical studies, etc.
 Should be evaluated in specific safety pharmacology studies.
 Although adverse effects of a substance may be detectable at exposures that fall within the
therapeutic range in appropriately designed safety pharmacology studies, they may not be
evident from observations and measurements used to detect toxicity in conventional animal
toxicity studies.

8. Safety Pharmacology & their Categories
 The studies that investigate the potential undesirable pharmacodynamic
effects of a substance on Safety Pharmacology Studies for Human
Pharmaceuticals physiological functions in relation to exposure in the
therapeutic range.
 Three categories:
 Primary pharmacodynamic
 Secondary pharmacodynamic
 Safety pharmacology studies.

9. General Consideration In Selection
 Mechanism of action may suggest specific adverse effects e.g.,
proarrhythmia is a common feature of antiarrhythmic agents.
 Primary pharmacodynamics effects e.g., anti-psychotics and QT
prolongation.
 Ligand binding or enzyme assay data suggesting a potential for adverse
effects.
 Results from previous safety pharmacology studies, from secondary
pharmacodynamics studies, from toxicology studies.

10. Test System
 General consideration on selection of animal model should include:-
 pharmacokinetic profile
 Species
 Strain
 gender, Age.
 the susceptibility sensitivity
 reproducibility of the test system and available background data on the substance.
 The time point for the measurement of human should be based on :
 Pharmacokinetic factor
 Pharmacodynamics factor

11. Experimental Designs
 Sample size & Use of Controls
 Size of the groups should be sufficient to allow meaningful scientific interpretation.No.
of animal should be adequate.
 Negative and positive control groups should be included;positive group may not be
necessary.Exclusion of controls from studies should be justified.
 Route of Administration :-
 Clinical route of administration should be used.
 Exposure to the parent substance and its major metabolites should be similar to that
achieved in humans.
 If clinical use involves multiple routes, consider more than one route.

12.  Dose Levels or Concentrations of Test Substance:
 (A) In Vivo Studies-
 Toxic range (e.g., tremors or fasciculation during ECG recording) may confound the
interpretation of the results and may also limit dose levels.
 Dose that produce moderate adverse effects in this study similar route & duration.
 These adverse effects can include dose-limiting pharmacodynamics effects or other
toxicity.
 Testing of a single group may be sufficient.
 (B) In Vitro Studies-
 To establish a Concentration-effect relationship.The range of concentrations selected
detect an effect on the test system.
 This range may influence physico- chemical properties of the test substance and other
assay specific factors.In the absence of an effect, the range of concentrations selected
should be justified.

13. Safety pharmacology core battery: Tier-1
 Safety pharmacology core battery is to investigate the effects of the test
substance on vital functions.
 Central Nervous System
 Cardiovascular System
 Respiratory System
 Follow-up Studies For Safety Pharmacology Core Battery
 These are meant to provide a greater depth of understanding than, or
additional knowledge to, that provided by the core battery on vital
functions for potential adverse pharmacodynamic effects

14. 1.CENTRAL NERVOUS SYSTEM:
 In core battery In Follow-up Studies:
 Motor activity Learning and Memory
 Behavioural changes Ligand specific binding
 CoordinationSensory/motor Neurochemistry.
reflex response examination visual and Auditory
 Body temperature

15. Evaluation Methods
 Established techniques-
 Rota rod
 Hot plate test
 Tail flick, paw pressure
 photoelectric beam interruption techniques
 passive avoidance tests
 Pentylenetetrazol (PTZ) seizure tests
 Electroencephalography (EEG)
 Emerging techniques
 Automated video systems
 Integrated video and EEG systems

16. Parameters to be assesed

17. Irwin Test
 The IRWIN TEST consists of systemic evaluation of general behaviol 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 ICHAs a result Irwin test was
modified to incorporate all core functions detailed by ICH
 Similarly to the modified Irwin's test, the Functional Observation Batteryprovides a more comprehensive
evaluation of NCES on the fundamental CNS functionsAdditionally, 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.

18. 2. CARDIOVASCULAR SYSTEM
 Core Battery
 Heart Rate.
 Blood pressure
 Follow up studies
 Cardiac output
 Vascular contractility
 Vascular resistance
 Endogenous and Exogenous
substances

19. Evaluation Methods
 Electrocardiogram (ECG)
 Established techniques
 (In vitro hERG assay)
 Manual patch clamp
 Automated high-throughput patch clamp
 Isolated organ preparation
 Whole heart preparation
 Isolated purkinje fibres

20. Electrocardiogram
 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- TERFINADINEJ.
 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 toause
adverse CVS effects.14

21. 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 ventricularBP
 Heart rates
 ECGparameters-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.

22. HERG ASSAY

23. Basic Introduction and screening of HERG System.
 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.
 nhibition 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.
 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

24. 3. RESPIRATORY SYSTEM
 Core Battery
 Respiratory Rate.
 Tidal volume
 Hemoglobin
 Follow up studies
 Airway resistance
 compliance
 oxygen saturation
 pulmonary arterial pressure
 Blood gases

25. Evaluation :
 Plethysmography
 Head out Plethysmography
 whole body plethysmography
 Respiratory parameters-

26. PLESTHYSMOGRAPHY
 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.
 Whole body and Head out plethesmography methods in conscious rats were comp 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

29. References
 ICH Harmonized Tripartite Guideline (S6) "Preclinical Safety Evaluation of Biotechnology-derived
Pharmaceuticals" (1997).
 Mattsson, J. L., Spencer, P. J. and Albee, R. R.: A performance standard for clinical and Functional
Observational Battery examinations of rats. J. Am. Coll. Toxicol. 15, 239 (1996).
 Irwin, S.: Comprehensive observational assessment: la. A systematic, quantitative procedure for
assessing the behavioural and physiologic state of the mouse. Psychopharmacologia (Berl.) 13, 222-
257(1968).
 Safety Pharmacology Studies for Human Pharmaceuticals S7A, Current Step 4 version, dated 8
November 2000.
 Toxicology and Applied Pharmacology, Safety pharmacology and emerging concepts, YTAAP-
12785; No. of pages: 10; 4C: 3
 Currenthttps://www.cyprotex.com/toxicology/cardiotoxicity/hergsafety