Safety pharmacology studies

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 1 st year – 2 nd semester Pharmacology Acharya Nagarjuna university

2. CONTENTS INTRODUCTION OBJECTIVES GENERAL CONSIDERATIONS SAFETY PHARMACOLOGY STUDIES CVS CNS RESPIRATORY GI SYSTEM RENAL SYSTEM 2

3. SAFETY PHARMOCOLOGY STUDIES 3 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 SAFETY PHARMACOLOGY 4 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 .

5. 5

6. GENERAL CONSIDERATIONS IN SELECTION/DESIGN 6 effects related to the therapeutic class of the test substance, since the mechanism of action may suggest specific adverse effects . adverse effects associated with members of the chemical or therapeutic class ligand binding or enzyme assay data suggesting a potential for adverse effects 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: 7 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. T he number of animals or isolated preparations should be adequate to demonstrate or rule out the presence of a biologically significant effect of the test substance. 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 OF ADMINISTRATION 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. 8

9. DOSE LEVELS OR CONCENTRATIONS OF TEST SUBSTANCE 9 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 upp e r l im it o f th i s r a n g e m ay b e in f lu e nc e d b y p h y s i c o c he m i c al pr o p e rti e s o f t he t e s t substance and other assay specific factors. 10

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 11 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 12

13. Established techniques In vitro – hERG assay Manual patch clamp Automated high-throughput patch clamp Isolated organ preparation Whole heart preparation Isolated purkinje fibers 13 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. 14 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. 15

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. 16

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. 17

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. 18

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. 19

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 20 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 21

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 22

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). 23

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. 24

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. 25

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. 26

27. 27

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. 28

29. The parameters to assess during the assessment of CNS SP- Behavioral pharmacology Learning and memory Ligand-specific binding Neurochemistry Electrophysiology examinations, etc. 29

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 30

31. PARAMETERS TO ASSESS DURING SP 31

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 32

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. 33

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 34 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 35

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 36

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. 37

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 38

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) 39

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. 40

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 41

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 42

43. 43

44. KIDNEY INJURY MARKERS Kidney injuries are being assessed using two types of markers. FUNCTIONAL MARKERS – urinary glucose, protein, albumin and calciumor , indeed, any other molecule known to be transported in a certain region of the kidney 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. 44

45. 45

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 46

47. THANK YOU 47