Safety pharmacology — current and emerging concepts

YTAAP-12785; No. of pages: 10; 4C: 3 J. Hamdam et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx Contents lists available at Toxicology and Applied Pharmacology Invited Review Article Safety pharmacology — Current and emerging concepts Junnat Hamdam , Swaminathan Sethu , Trevor Smith , Ana Alfirevic Mohammad Alhaidari , Jeffrey Atkinson , Mimieveshiofou Ayala Helen Box Michael Cross Annie Delaunois Ailsa Dermody , Karthik Govindappa Jean-Michel Guillon , Rosalind Jenkins , Gerry Kenna , Björn Lemmer , Ken Meecham , Adedamola Olayanju Sabine Pestel , Andreas Rothfuss , James Sidaway , Rowena Sison-Young Emma Smith , Richard Stebbings Yulia Tingle , Jean-Pierre Valentin Awel Williams , Dominic Williams ,, Kevin Park , Christopher Goldring , OOF a MRC Centre for Drug Safety Science, University of Liverpool, UK b Lorraine University Pharmacolor Consultants Nancy PCN, France c UCB Pharma, Belgium d Sanofi-aventis, France e Astra-Zeneca, UK f Ruprecht-Karls-Universität Heidelberg, Germany g Huntingdon Life Sciences, UK h Boehringer-Ingelheim, Germany i Roche, Switzerland j National Institute for Biological Standards and Control, UK Safety pharmacology (SP) is an essential part of the drug development process that aims to identify and pre- 43 Received 17 January 2013 dict adverse effects prior to clinical trials. SP studies are described in the International Conference on 44 Revised 31 March 2013 Harmonisation (ICH) S7A and S7B guidelines. The core battery and supplemental SP studies evaluate effects 45 Accepted 15 April 2013 of a new chemical entity (NCE) at both anticipated therapeutic and supra-therapeutic exposures on major 46 Available online xxxx organ systems, including cardiovascular, central nervous, respiratory, renal and gastrointestinal. This review 47 outlines the current practices and emerging concepts in SP studies including frontloading, parallel assess- 48 Safety pharmacology ment of core battery studies, use of non-standard species, biomarkers, and combining toxicology and SP as- 49 International Conference on Harmonisation sessments. Integration of the newer approaches to routine SP studies may significantly enhance the scope of 50 SP by refining and providing mechanistic insight to potential adverse effects associated with test compounds. 51 Central nervous system 2013 Published by Elsevier Inc. 52 Abbreviations: ADR, Adverse Drug Reaction; ALP, alkaline phosphatase; AKI, acute kidney injury; ALT, alanine aminotransferase; AP, action potential; AST, aspartate aminotrans- ferase; BP, blood pressure; BUN, blood urea nitrogen; CLU, clusterin; CNS, Central Nervous System; CVS, Cardiovascular System; ECG, Electrocardiogram; EEG, electroencephalog-raphy; EMA, European Medicines Agency; FDA, Food and Drug Administration; FOB, Functional Observation Battery; GFR, Glomerular Filtration Rate; GGT, γ-glutamyl transferase;GI, Gastrointestinal; GST, glutathione S transferase; hERG, human Ether-a-go-go related gene; hESC, human embryonic stem cells; HR, heart rate; ICH, International Conference onHarmonisation; KIM-1, kidney injury molecule-1; LDH, lactate dehydrogenase; miR, microRNA; β-NAG, N-acetyl-β-D-glucosaminidase; NCE, New Chemical Entity; NGAL, Neutro-phil gelatinase-associated lipocalin; NMR, Nuclear Magnetic Resonance; PBPK, physiologically based pharmacokinetics; PEB, photoelectric beam interruption technique; RPA-1,renal papillary antigen-1; SP, Safety Pharmacology; TFF3, trefoil factor 3; VQM, Ventilation (V)/perfusion (Q) mismatch (M).
⁎ Correspondence to: D. Williams, MRC Centre for Drug Safety Science and Institute of Translational Medicine, Department of Molecular & Clinical Pharmacology, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool L69 3GE, UK. Fax: +44 151 7945540.
⁎⁎ Correspondence to: C. Goldring, MRC Centre for Drug Safety Science and Institute of Translational Medicine, Department of Molecular & Clinical Pharmacology, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool L69 3GE, UK. Fax: +44 151 7945540.
E-mail addresses: (D. Williams), (C. Goldring).
0041-008X/$ – see front matter 2013 Published by Elsevier Inc.
Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013), J. Hamdam et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx Core battery organ systems and studies Cardiovascular system Central nervous system Locomotor activity and motor co-ordination Sensorimotor reflexes and pain perception assessment . . . . . . . . . . . . . . . . . .
CNS follow-up studies Drug seizure liability Drug abuse and dependence liability Respiratory system Invasive plethysmography Supplemental organ systems and studies Gastrointestinal system Gastric emptying and intestinal motility . . . . . . . . . . . . . . . . . . . . .
Kidney injury markers Recent and emerging concepts Integrated core battery assessment Integrating safety pharmacology end points into toxicology studies Drug–drug interactions SP studies were generally performed during the drug development 123 stage on the selected candidate drug prior to FiH trials. Currently, the 124 Non-clinical pharmacological studies, including primary pharmacol- onset of SP studies has shifted towards the early drug discovery process 125 ogy, secondary pharmacology and safety pharmacology (SP), are an es- (). Thus, SP studies in addition to assessing and mitigating risks as- 126 sential element of the drug discovery and development process. Unlike sociated with the selected candidate drug can now facilitate lead candi- 127 primary and secondary pharmacology studies that explore the mode of date selection by hazard identification and elimination of new chemical 128 action of the candidate drug and its effects related or unrelated to the entities (NCE) with safety liabilities The purpose 129 therapeutic target, respectively, SP identifies the "potential undesirable of this review is to provide a combined and comprehensive overview of 130 pharmacodynamic effects of a substance on physiological functions in both current practices and newer technologies, followed by the emerg- 131 relation to exposure in the therapeutic range and above" ing concepts in SP studies: frontloading, alternate models, integrated 132 which are not identified by standard non-clinical toxicological studies.
core battery assessments, integration of SP endpoints into regulatory 133 SP studies are, therefore, performed to ensure the safety of clinical par- toxicology studies, drug–drug interactions and translational SP.
ticipants in first in human (FiH) trials (through im- proved decision-making in the selection of lead candidate drugs. Efforts Core battery organ systems and studies to standardize SP studies resulted in multiple guidelines from the Inter- national Conference on Harmonisation (ICH) including ICH S7A and S7B Cardiovascular system (). The core battery SP studies, performed according to good laboratory practice (GLP) standards as per the ICH guidelines, in- In the last few decades, a large number of drugs have been with- 137 volves the investigation of the major vital organ systems including the drawn from the market due to adverse cardiovascular system (CVS) 138 cardiovascular system (CVS), central nervous system (CNS) and respira- effects, which were responsible for 45% of post-approval withdrawals 139 tory system. In addition, supplemental studies investigating the renal (The electrical activity in the CVS can be mea- 140 and gastrointestinal (GI) systems and other organ specific follow-up in- sured using electrocardiogram (ECG), which is analysed by dividing 141 vestigations may compliment the core battery studies. However, these the recorded trace into waves and intervals with particular focus on 142 are optional and their conduct is determined by the nature of the lead the QT interval which represents cardiac repolarisation. It is impor- 143 candidate drugs being tested and the type of adverse events anticipated.
tant to note that QT prolongation has resulted in one third of all 144 Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013),

J. Hamdam et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx Fig. 1. Safety pharmacology study approaches. Initially, SP studies were conducted after lead candidate identification to profile safety risks in humans according to GLP compliance.
In addition, more recent strategy is to initiate SP studies (non-GLP) much earlier in the drug discovery process aims to identify hazardous NCEs facilitating lead candidate selection.
This ensures the reduction of risks in humans and lead candidate attrition. FiH — first in human, GLP — good laboratory practice.
drug withdrawals between 1990 and 2006 due to the primates or guinea-pigs, depending upon the experimental conditions. 193 risk of developing fatal arrhythmias. An example of a drug that caused However, due to significant inter-individual variation 194 numerous fatalities due to QT prolongation is terfenadine ( ), an individual correction formula that utilises a complex model 195 ), this led to the implementation of the ICH S7B guidance of linear regression is applied; however, it requires a large number of 196 that describes a "non-clinical testing strategy for assessing the poten- HR measurements to obtain). Finally, other fac OOF an acceptable level of accuracy (197 tial of a test substance to delay ventricular repolarisation" tors such as changes in body temperature 198 ). Consequently, a core battery of SP tests, consisting of an in and plasma concentrations of electrolytes (e.g. potassium), glucose and 199 vitro assay to assess the extent of the human Ether-a-go-go Related insulin, should be taken into account when interpreting ECG readouts.
Gene (hERG) potassium channel, Kv11.1, blockade, in vivo telemetry and additional in vitro/ex vivo tests were adopted to evaluate the In vitro isolated myocardial systems likelihood of an NCE to cause adverse CVS effects ().
The effects of NCEs on the cardiac AP can also be investigated 202 using other in vitro systems including isolated myocardial tissue 203 In vitro hERG assay (purkinje fibres or papillary muscles) or whole isolated hearts. For ex- 204 There is considerable focus on the promiscuous hERG channel, ample, a functional in vitro model using isolated guinea-pig papillary 205 which mediates an inward current, that, when blocked, slows myocar- muscles can be used to evaluate direct NCE-induced effects, including 206 dial repolarisation associated with prolongation of the QT interval in the force of contraction and refractory period, in addition to effects on 207 the ECG. This prolongation lengthens the duration of the cardiac action the AP (However, these low-throughput tech- 208 potential (AP) (, which appears to be a critical con- niques are costly and require highly skilled electrophysiologists.
tributing factor in the development of a fatal arrthymia: Torsades de Pointes ). The effects of an NCE on the hERG channel can be detected using screening methodologies such as radio-labelled Tests and parameters available to assess CVS safety pharmacology. The table outlines the t1:2 ligand binding and automated voltage clamp assays. Alternatively, the core and follow-up CVS associated parameters in SP testing. It also lists out the established t1:3 manual in vitro electrophysiology patch clamp assay is used to quantify and emerging techniques associated with these investigations. hERG — human t1:4ether-à-go-go-related gene; IC50 — half maximal inhibitory concentration; HR — heart t1:5 NCE-induced hERG inhibition with a strong accuracy rate for predicting rate; BP — blood pressure.
in vivo CVS toxicity ). However, this in vitro assay is not without limitations, since the hERG channel may be functionally Cardiovascular system (CVS) assessment compromised through related, poorly understood molecular mecha- In vitro hERG assay (hERG IC50) In vitro isolated organ preparation Telemetry (HR, BP) In vivo telemetry In general, physiological data obtained from conscious, large mam- Established techniques mals (e.g. dogs, minipigs and non-human primates) is accepted as the gold standard for detecting any effects of an NCE on CVS functionality.
Manual patch clamp Telemetry is efficiently utilised in SP to produce reliable data sets Automated high-throughput patch clamp while using as few animals as possible Further- Isolated organ preparation more, it allows the measurement of CVS parameters in conscious freely Whole heart preparation moving animals with UNCORRECTED PR minimal stress. Telemetry can be divided into two Isolated purkinje fibres distinct techniques: 1) Jacketed (or External), a non-invasive technique which records ECG parameters and 2) Implanted (or Internal), an inva- Internal (surgical implant) sive technique requiring surgery, which can simultaneously measure External (jacketed) ECG, haemodynamic parameters, such as blood pressure (BP) and con- Emerging techniques tractility, and body temperature. Additionally, telemetry can be used for the simultaneous measurement of other core organ system parameters.
Assays for other ion channels Telemetric devices are used for the continuous measurement of arte- Automated high-throughput patch clamp rial, systemic and left ventricular BP, heart rate (HR) and ECG parame- Human embryonic stem cell derived cardiomyocytes Human induced pluripotent stem cell derived cardiomyocytes ters: the QRS complex and the QT, ST and PR intervals. Since the PR and QT intervals are influenced by the HR, they should be corrected using the relevant formula, determined by the study design and species Femoral artery cannula used. In general, van de Water's correction is used for dogs and minipigs, High definition oscillometry while Fridericia's or Bazett's corrections are used in either non-human Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013), Technological advancements have led to the improvement of auto- Tests and parameters available to assess CNS safety pharmacology. Table outlines the t2:2 core and follow-up CNS associated parameters in SP testing. It also lists out the t2:3 mated patch clamp assays and this has been beneficial for in vitro CVS established and emerging techniques associated with these investigations.
studies by facilitating lead candidate optimisation during the drug dis- covery and development process. There are now a number of commer- Central nervous system (CNS) assessment cially available high-throughput automated patch clamp platforms that utilise planar array technology, which can rapidly quantify the degree of an NCE's hERG blockade (While the benefit of Higher cognitive function Locomotor activity Seizure liability being able to screen large numbers of NCEs rapidly is alluring, it is diffi- Motor co-ordination cult to obtain accurate test concentrations during the screening process.
Sensorimotor reflexes: nociception Therefore, this platform should be used in conjunction with other meth- Established techniques Modified Irwin's test, Functional Observation Battery (FOB) In addition to hERG, the cardiac AP is also regulated by the activity Photoelectric beam interruption systems of other ion channels, many of which may also be part of a vulnerable cellular pathway. Some of the following channels have been implicat- Hot plate test, Tail flick, paw pressure ed in other cardiac arrthymias: the slow delayed rectifier potassium Morris maze and passive avoidance tests Electrocerebral silence threshold and pentylenetetrazol seizure tests v7.1/hKCNQ1/hminK); voltage gated potassium chan- nel (hKv1.5); voltage gated sodium-permeable channel (hNav1.5); Self administration and drug discrimination lever chamber models hyperpolarisation-activated cyclic nucleotide-gated channel (hHCN4); Drug withdrawal: FOB, body temperature, body weight potassium-permeable outward voltage gated potassium channel (hKv4.3/ Emerging techniques hKChIP2); L-Type calcium channel (hCav1.2) and inwardly rectifying Automated video systems potassium channel (hKir2.1) ).
Integrated video and EEG systems Electrophysiological investigations of these ion channel subunits can In vitro hippocampal brain slice assay also be conducted using the above mentioned electrophysiological techniques . This data can provide more informa- tive SP profiles for NCEs for lead candidate development.
Previously, implanted telemetry was required to record CVS parame- ). The Irwin test consists of the systematic evaluation of a battery 273 ters, but recently, jacketed ECG telemetry in combination with novel of general behavioural and physiological observations in the rodent 274 high definition oscillometry methodologies for BP recordings is used as including arousal, vocalisation and stereotypy. Drug treated animal 275 an alternative. Although high definition oscillometry is non-invasive groups are compared to a vehicle group and observational differences 276 and cheaper than implanted telemetry ), there are between the groups are documented using a qualitative scoring system 277 short-comings that include: 1) lower signal to noise ratio; 2) shorter du- (). Although this methodology provides satisfactory 278 ration of recordings; and 3) lack of in-depth pharmacological validation.
assessment of gross behavioural changes it does not encapsulate other 279 However, there are now BP measurement techniques that only require a vital neuro-physiological functional assessments outlined by the ICH. 280 small transducer to be inserted into the femoral artery As a result the Irwin test has been differentially modified by various 281 ). Finally, it is important to monitor circadian rhythms, particularly drug companies to incorporate all core battery functions detailed in 282 in rodents as blood pressure peaks during the night when activity is the ICH guidelines Similarly to the modified 283 Irwin's test, the Functional Observation Battery (FOB) provides a more 284comprehensive evaluation of NCEs on the fundamental CNS functions 285 Central nervous system (Additionally, FOBs are frequently used to carry out neuro- 286toxicological and neuropathological investigations (). 287 Adverse drug reactions (ADRs) associated with the central nervous Drugs, such as the psychostimulant, amphetamine, and the antipsy- 288 system (CNS) represent a major cause for concern for pharmaceutical chotic, chlorpromazine, can be used as reference compounds to validate 289 companies. A variety of clinically used drugs such as anti-histamines the effect of NCEs on neurobehavioural function (). 290 (e.g. diphenhydramine) and benzodiazepines (e.g. diazepam) exhibit The aforementioned behavioural assessments are not without their 291 common CNS side effects including sedation, ataxia and nausea limitations, however, as this type of analysis is subjective and requires 292 (). More importantly, however, 10% of all drugs with- highly trained and experienced observers to ensure efficient reproduc- 293 drawn from the market between1960 and 1999 were due to severe CNS ibility of experiments. Nonetheless, the simultaneous assessment of 294 adverse effects (). Therefore, it is beneficial for the phar- behaviour, locomotor activity, motor coordination and sensorimotor 295 maceutical industry to detect these ADRs early in the drug discovery reflexes including nociception which are discussed below can be incor- 296 and development process in order to save time and reduce costs, ulti- porated into a modified FOB ).
mately leading to the design of clinically safer compounds eason, the CNS has been included in the regulatory Locomotor activity and motor co-ordination guideline ICH S7A ). The effects of NCEs on the CNS are evalu- Procedures assessing locomotor activity generally rely on photo- 299 ated using a variety of core battery SP studies as outlined by the ICH to de- electric beam interruption techniques using commercially available 300 tect potential undesirable pharmacodynamic effects on various neuro- automated test systems, such as the Actimeter ). Al- 301 physiological functions such as "motor activity, behavioural changes, though this methodology measures locomotion exclusively, assessment 302 coordination, sensory/motor reflex responses and body temperature" in conjunction with direct observational tests (e.g. modified Irwin test), 303 (Unlike CVS SP assessments, CNS core battery studies are gen- can effectively determine whether a candidate drug has a sedative or 304 erally performed using unanaesthetised animals, primarily rodent models psychostimulant effect by measuring the total distance covered in the 305 (). The various established and emerging techniques cage ). Unlike behavioural experiments, these auto- 306 used to assess neurological functions in CNS SP are depicted in mated techniques are less labour intensive and allow the simultaneous 307investigation of an array of tests within a larger animal group 308 Therefore, data obtained from such techniques tend to be 309 Procedures for assessing the effect of NCEs on behaviour and physi- more statistically significant in comparison to data obtained by the sub- 310 ological state were first described by Irwin in the late 1960s ( jective modified Irwin's test Motor coordination 311 Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013), J. Hamdam et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx the vehicle is determined (). An increase and a 358 Parameters assessed during safety pharmacology assessment of CNS. Table lists the decrease in seizure threshold are associated with anticonvulsive (as 359 various parameters assessed as part of the modified Irwin's test and the Functional Ob- observed with phenobarbitol) and proconvulsive activity (as observed 360 servation Battery (FOB) during CNS functional examination.
with D-amphetamine), respectively The ECS fied from .
threshold test fails to deduce anticonvulsive activity, however, the PTZ 362 Autonomic nervous seizure test can deduce both pro- and anticonvulsive activities (363 ). Nonetheless, it is important to note that both ECS and PTZ 364 Approach response tests should be performed for full seizure liability assessment as discrep- 365 ancies in both models have been documented Handling reactivity A more comprehensive method for assessing drug seizure liability is Excessive urination Tail pinch/tail flick via electroencephalography (EEG), whereby implanted telemetric de- 368 Palpebral reflex Bizarre behaviour vices or electrodes fixed onto the brain surface measure brain electrical 369 activity ). This method is extremely sensitive in illus- 370 trating the proconvulsant activity of lead compounds where no overt Rectal temperature Landing foot splay (Un)supported rears convulsions are detected using the more traditional assessments. EEG 372can also assess drug induced convulsive effects on various regions 373of the brain. Seizure liability has been assessed via EEG in a variety of 374 function is most frequently assessed by the RotaRod method. Animals species, such as, non-human primates, dogs and rodents 375 are trained on a rotating rod for a number of days prior to the first Despite this, the 376 test session hence extensive training implemented by competent inves- EEG fails to provide mechanistic information on drug induced modula- 377 tigators is mandatory to ensure accuracy in assessments ( tion of sensory receptors anillustrating a requirement foOOF d their respective sensory motor pathways, 378 This method directly investigates the effect of lead compounds r extensive in vitro molecular evaluation of 379 on neuromuscular coordination, and thus, should be used in combina- targeted neuronal receptors ( tion with other locomotor investigations to assess the overall effect on all aspects of motor function ).
Drug abuse and dependence liability Commonly prescribed drugs, such as anxiolytic benzodiazepines 382 Sensorimotor reflexes and pain perception assessment (e.g. diazepam) and opioid painkillers (e.g. morphine), are frequently 383 Identification of drug-induced gross defects in sensorimotor func- abused, due to their desirable psychotropic effects 384 tion is determined via manipulative neurological reflex examinations . Such drugs can also induce physical and psychological 385 including pupil response, startle reflex and tail pinch, as illustrated in side effects upon treatment cessation and thus are associated with 386 These functional investigations are performed in a modified human drug dependence (). Hence, preclinical 387 Irwin's test or FOB. In addition, using thermal and mechanical stimuli, evaluation of drug abuse and dependence liability of lead compound 388 nociception is assessed using a variety of basic techniques, such as, the has become increasingly important in SP, with its inclusion in the regu- 389 hot plate, tail flick, paw pressure and plantar tests, which primarily re- latory guidelines by the European Medicines Authority () 390 cord the latency of the nocifensive reflex response and the Food and Drug Administration ). This methodology is advantageous in its capacity to delineate an- Many initial in vitro and subsequent in vivo studies have been 392 algesic properties of drugs as exemplified by morphine, which increases employed by pharmaceutical companies to evaluate the drug abuse 393 the time taken for the animal to react to noxious stimuli. Furthermore, and dependence liabilities of NCEs. The EMA and FDA have advocated 394 this test can also be used to decipher whether a drug induces hyper- a two-step evaluation of such studies. The initial tier relies on the 395 responsiveness to nocifensive stimuli comparison of lead compounds with established reference com- 396pounds of abuse, such as cocaine, using in vitro ligand binding, bio- 397 CNS follow-up studies genic amine reuptake and synaptosomal dopamine release assays 398 Along with these core battery studies, the ICH has suggested non- Positive results from these studies are indicative 399 mandatory additional studies to be performed during drug development of the NCE's risk abuse potential, and thus, must be confirmed in the 400 These investigations relate to higher cognitive function second tier of in vivo drug abuse and dependence studies 401 such as ‘behavioural pharmacology, learning and memory, ligand- ). These include investigations into the reinforcing properties of 402 specific binding, neurochemistry, visual, auditory, and/or electrophysiol- the drug (self-administration), the similarities of the psychotropic ef- 403 ogy examinations' ). Learning/memory paradigms used to fects of the drug with known psychoactive compounds of abuse (drug 404 assess cognition include the Morris maze and passive avoidance tests.
discrimination) and its ability to cause unwanted physical/psychological 405 These particular studies have been reviewed elsewhere ( effects upon drug withdrawal (i.e. drug dependence potential). Self- 406 ). There is growing support for the requirement to perform more administration, drug discrimination and drug withdrawal tests are 407 comprehensive CNS testing prior to FiH trials, including follow-up stud- generally carried out in rodents, however, it has been debated that 408 ies in proconvulsive activity and, more recently, drug abuse and depen- non-human primate models should also be used due to species dif- 409 dence liability ( ferences in receptor profiles between rodent and humans (410).
Drug seizure liability During self-administration tests, rodents are trained to press a lever 412 It is beneficial to investigate the proconvulsive activity associated in order to self-administer an i.v. infusion of a known reference com- 413 with candidate drugs earlier in the drug development process in pound of abuse, such as cocaine In a reinforcement 414 order to avoid future termination due to fatal drug-induced seizures, schedule, the animal must execute a fixed number of operant responses 415 a major concern for the pharmaceutical industry. Drug seizure liabil- in order to receive infusion of the positive ‘rewarding' substance of 416 ity is generally assessed in rodent models, where convulsions are abuse, also known as the fixed ratio ). Subsequently, 417 induced in the animal either by electrical stimulation across the cere- the reference compound is replaced with the test compound and the 418 brum (electrocerebral silence (ECS) threshold test) or injection with frequency at which the animal emits operant responses to receive the 419 the validated proconvulsant, pentylenetetrazol (PTZ seizure test) i.v. infusion of the test drug is indicative of its drug reinforcing proper- 420 Candidate drugs are administered prior to ties and thus drug abuse potential It is important 421 proconvulsive stimuli and their convulsive threshold with respect to to note that the sensitivity of this test is highly dependent upon the 422 Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013), J. Hamdam et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx choice of training substance, thus validation with a variety of training studies involving the vital organ systems (The guidelines 486 substances should be implemented for greater accuracy of results. Un- indicate to carry out the two sets of studies, the core battery tests and 487 like self-administration, drug discrimination procedures test the ability follow-up studies. The core tests include the assessment of respiratory 488 of the animal to distinguish between the subjective effects of a training rate, tidal volume and haemoglobin oxygen saturation. Follow-up stud- 489 drug of abuse to that of the vehicle (i.e. saline) using a two lever cham- ies that are meant to provide greater depth of understanding of the core 490 ber Drug discrimination is also highly specific in test observations include the assessment of airway resistance, com- 491 that the training drug must have a similar mechanism of action to the pliance, pulmonary arterial pressure, blood gases and blood pH. The 492 test compound ).
species used for routine testing based on the test compound and the 493 Unlike drug abuse, drug dependence is typified by observed phys- study design include rodents, dogs and primates (). 494 ical and psychological withdrawal symptoms on drug treatment ces- However, special considerations on experimental design should be 495 sation, thus animal training is not required. Although many abused taken into account during species selection for respiratory safety testing 496 drugs are linked with drug dependence, such as morphine, heroin which would improve the predictability of potential respiratory adverse 497 ), this does not necessarily mean that both drug abuse and dependence coincide with one another. Generally, rodents are chronically treated with the test drug over a 2–3 week period and The SP approach for assessing respiratory system involvement 500 withdrawal symptoms are evaluated over a week post drug treatment includes the assessment of pumping efficiency and gaseous exchange 501 cessation (The EMA has listed the following as using a variety of measuring apparatus to assess these parameters 502 drug withdrawal endpoints: changes in behaviour, body temperature, (Accurate ventilatory patterns are assessed to directly monitor 503 body weight and food intake. Furthermore, it is suggested that multi- lung volume changes or airflows generated by thoracic movements 504 ple endpoints should be investigated to assess dependence liability, in conscious animals using a plethysmograph chamber 505 as no single measure is sufficient for complete evaluation. Additionally, ). Head-out, dual chamber 506 the EMA recommends that observations should be made continually, and whole body plethysmography techniques are non-invasive methods 507 over a long period of time ).
that are currently used to evaluate typical parameters of respiration 508 An important point to consider when determining abuse and de- including tidal volume, minute volume, mid-expiratory flow, and respi- 509 pendence liability, is the choice of species utilised ( ratory rate ). Industry opinion varies regarding the 510 Preferential use of non-human primates over rodents has preferred method for preclinical safety assessment of respiratory func- 511 been suggested for specific assessment of the aforementioned param- tion in the rat. A study which compared these three plethysmography 512 eters due to similarities in diurnality, drug metabolism and neurolog- methods in rodents reported that each system was equally sensitive. 513 ical receptor expression with humans ( The whole body and head-out plethysmography provided consistent 514and reliable pulmonary mechanics data, while data collected from dual 515 chamber plethysmography are clearly affected by restrainment stress 516 New video automated testing systems, have been developed to eval- in the animal Recently, whole body and head-out 517 uate visceral pain in rodents by quantifying licking behaviours in the plethysmography methods in conscious rats were compared, using 518 rodent in response to a noxious stimuli (). The neurokinin-1 receptor antagonist GR205171A was shown to potentiate licking responses associated with capsaicin administration Tests and parameters available to assess respiratory function in safety pharmacology t4:2 ). This automated method is high throughput and allows the studies. Table outlines the core and follow-up respiration associated parameters in SP t4:3 quantification of licking behaviour over long periods of time. The emer- testing. Also lists out the established and newer techniques associated with these in- t4:4vestigations. PIF — Peak inspiratory flow; PEF — Peak expiratory flow; Ti — inspiratory t4:5 gence of integrated video EEG and computerized analysis has facilitated time; Te — expiratory time; FIT — fractional inspiratory time; Penh — Enhanced pause. t4:6 the simultaneous assessment of new compounds on behaviour (via video), seizure liability and disruption of sleep patterns (via EEG) in Respiratory function assessment non-human primates . Therefore, continuous mea- surement with less interference is possible, giving an indication of Airway resistance long-term effects of the drug.
Pulmonary arterial pressure More recently, telemetry has been used in the continual assess- Haemoglobin oxygen saturation ment of withdrawal symptoms associated with morphine and chlor- diazepoxide drug discontinuation in rats Established techniques It is worth noting that marked hypothermia and decreases in arterial blood pressure were observed in mice, 12 h after morphine Tidal volume (VT); breathing rate (f); minute volume (VTxf); PIF/PEF/Ti/Te/FIT — discontinuation, during their nocturnal phase, thus highlighting the in unrestrained animals need for such automated technology in assessing drug dependence.
Head out + pressure Tidal volume; breathing rate; minute volume; PIF/PEF/Ti/Te/FIT; compliance; resistance — in unrestrained animals Respiratory system Tidal volume; breathing rate; minute volume; Drugs of various pharmacological classes are known to have delete- PIF/PEF/Ti/Te/FIT; specific airway resistance — in restrained animals rious effects on respiratory functions including life threatening con- Barometric whole body ditions (More recently, drugs which had serious Tidal volume; breathing rate; minute volume; FIT; Penh By induction/impedance respiratory implications include Duragesic Patch and Advair. Prozac Telemetry (external/Implanted) — tidal volume; breathing rate; was another drug which increased the risk of pulmonary hypertension of the newborn in infants delivered by women who used Prozac during the third trimester of their pregnancy. Hence, a mandatory and detailed Pulmonary resistance and compliance preclinical testing assessing the effects of new compounds on respirato- Emerging techniques ry function was required. Therefore, as per the ICH recommendations, Unrestrained video-assisted plethysmography the SP assessment of the potential adverse reactions of new drugs re- Biomarkers: VQM — Ventilation (V)/perfusion (Q) mismatch (M) quires evaluation of respiratory function as part of the core battery Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013), J. Hamdam et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx theophylline as a respiratory stimulant and chlordiazepoxide as a respira- dogs which provides new opportunities to characterise respiratory status 583 tory depressant. The study reported that respiratory function can be accu- Unrestrained video-assisted plethysmography is 584 rately evaluated using head-out plethysmography compared to whole an emerging approach which can be performed in small animals, such 585 body plethysmography. The authors also addressed the demand for addi- as rodents, to assess specific airway resistance and the breathing pattern, 586 tional invasive methods to evaluate ventilator parameters such as mid- accurately, in a non-invasive fashion ().
expiratory flow (Another non-invasive respiratory Ventilation (V)/perfusion (Q) mismatch (VQM) is the main cause of 588 function assessment is the use of the variable, enhanced pause (Penh), gas-exchange abnormalities observed in various pulmonary diseases. It 589 which is measured by whole body plethysmography (barometric) in un- can be exacerbated by certain pharmacological agents resulting in 590 restrained animals. Despite being a simple procedure, it was found that unwanted effects on the respiratory system, including hypoxemia. A re- 591 Penh was less reliable compared to head-out plethysmography method cent report has addressed the relevance, techniques to assess VQM, and 592 in its correlation with other pulmonary parameters such as resistance, the potential use of VQM as a safety biomarker during drug develop- 593 hence is not used extensively as part of respiratory SP core battery studies ment (). With further validation, VQM can be used 594 Thus, non-invasive whole body or head-out plethys- in respiratory SP based on the pharmacological properties of the NCE 595 mography is the most common system used to evaluate the ventilatory being explored for development.
function in conscious animals in the laboratory. Non-invasive head-out body plethysmography measurements for core battery respiratory SP Supplemental organ systems and studies studies in conscious rodents are reliable, as it is simple to handle, the breathing pattern is nearly natural (anaesthesia is not required) and it Gastrointestinal system allows high-throughput screening. Training the animals in the chamber prior to experimentation will reduce the animal stress induced variation Gastrointestinal (GI) complications are common side effects, with 599 in the assessments. However, lung resistance and compliance assess- varying degrees of severity,ment, and are associated wOOF observed during and after drug develop- 600 ments to refine respiratory SP profile cannot be obtained using head- ith drug-induced morbidity 601 out or whole body plethysmography.
). Drug induced GI complications include nausea, emesis, 602constipation and may also affect the absorption of other drugs. There- 603 Invasive plethysmography fore, it is important to study the effect of the test drug on the GI system 604 Follow-up respiratory SP studies using invasive plethysmography ), routinely, to improve the safety and efficacy for 605 methods are performed to further investigate any unwanted potentially NCE development. According to ICH S7A recommendations, the effect of 606 deleterious effects on respiratory functions observed during core bat- test compounds ought to be assessed using gastric emptying, intestinal 607 tery studies, or any potential adverse effects that may be suspected motility and gastric secretion in appropriate animal models. Evaluation 608 due to the inherent pharmacological properties of the test compound.
of GI function is supplementary and, therefore, is indicated based on the 609 These studies involve the assessment of changes in the mechanical knowledge of the NCE being tested ). 610 properties of lungs such as pulmonary resistance and compliance The commonly altered GI physiological functions include motility and 611 for the identification of bronchoconstriction and obstruction. Invasive ulcerations, but also gastric mucus production, hydrochloric acid and bi- 612 procedures designed to assess these parameters accurately involves carbonate secretion, which are commonly seen with prostaglandin E1 613 orotracheal intubation, pulmonary manoeuvres and surgical implanta- analogues and some non-steroidal anti-inflammatory drugs (NSAIDs). 614 tion of pleural pressure sensors for chronic resistance recording or The effects of test compounds on the GI system are commonly evaluat- 615 tracheotomised, intubated animals ). The advantages ed in rodent models, using tests assessing: gastric emptying, intestinal 616 of these techniques are that they do not factor restrainment stress of an- motility, gastric secretion and GI injury (The SP 617 imals in the measurements and are accepted as the gold standard for ac- tests available to assess drug-induced GI changes are shown in . 618 curate assessment of resistance and compliance. The major drawbacks include the use of anaesthesia which decreases the breathing frequency Gastric emptying and intestinal motility and the requirement of experienced and specially trained personnel.
Gastric emptying and intestinal motility is evaluated by feeding 620 the animals with barium sulphate (BaSO4) or a charcoal test meal 621 subsequent to test compound administration. The test meals may be 622 Similar to the other SP vital organ studies, telemetry can also be used either as an indicator for liquid transport (phenol red) or for 623 used effectively in respiratory safety assessment ( transport of solids (BaSO4, charcoal). At the desired time point, ideally 624 ). The Kearney group has evaluated a novel surgical implanted close to Cmax, the stomach is extracted and weighed, since the weight 625 telemetry method incorporated with an impedance sensor for chronic evaluation of respiratory parameters (They validated the use of such implantable telemetry via successful com- Tests and parameters available to assess gastrointestinal function and integrity in safety t5:2 parison with pneumotachograph recorded values in conscious Beagle pharmacology studies. Table lists both established and newer techniques in gastrointesti- t5:3 dogs following i.v. administration of doxapram. This type of tech- nal SP studies. EMG — electromyograph; miR — microRNA; PBPK — physiologically based t5:4 been validated in non-human primates allowing the simultaneous evaluation of both CVS and respiratory function Gastrointestinal toxicity assessment Another variant in this technology is the use of respiratory inductive plethysmography (RIP) with telemetry which allows the continuous monitoring of respiratory parameters in non- restrained large animals for extended periods of time including awake Macroscopic (ulcer index) Intestinal motility and sleep states ). All these experimental approaches Gastric secretion are dedicated to ventilatory machinery (the pumping apparatus) rather than to a true evaluation of respiration efficiency. In this respect, blood gas analysis and haemoglobin saturation should not be neglected.
Capsule — pH, pressure Newer and emerging approaches for respiratory SP include modifications in plethysmography, telemetry and potential biomarkers for specific re- Strain gauges for contraction, EMG spiratory disorder. Barometric, whole-body plethysmography is a safe, In-silico (PBPK modelling) non-invasive and reliable technique for investigation of lung function in Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013), J. Hamdam et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx of the stomach is directly correlated to the weight of the gastric con- part of SP, is supplementary or is indicated based on the knowledge 688 tent. Weighing of the stomach when full and empty for stomach con- obtained about the NCE under test ).
tent weight is necessary to obtain more reliable results. Changes in Routinely, clinical chemistry-based evaluations, using urine and 690 the weight between the test groups indicate altered gastric emptying.
serum samples, are used to assess drug-induced renal impairment 691 Regarding intestinal motility measurements, intestines from the duo- () and isolated organ prep- 692 denum (to either ileum or rectum) are prepared, and the length of the arations are carried out for additional mechanistic studies. sum- 693 intestine filled with BaSO4 or charcoal from the test meal in relation marizes the various approaches and parameters in the renal SP testing. 694 to the length of the whole gut is determined by visual inspection.
A report can be referred for objective analysis of renal assessment strat- 695 Any difference in the BaSO4/charcoal transit length between the test egies in SP (The battery of tests includes measure- 696 groups and the controls infer alteration in the intestinal motility.
ment clearance rate, glomerular filtration rate (GFR), urinary volume, 697 When phenol red is used, any change in the spectral absorbance in osmolality, pH, Na+, Cl−, K+, creatinine and urea, along with serum 698 specific parts of the gut (normally collected in ten sub segments) in- Na+, Cl−, K+, creatinine and BUN (blood urea nitrogen) for assessment 699 dicates altered intestinal transit.
of kidney function.
Gastric secretion Renal function assessments Gastric secretion is evaluated by the parenteral administration of the GFR, a main parameter for assessing renal function is calculated 702 test drug following pylorus ligation and the stomach contents act as using both urine and serum samples obtained from the animals. Multi- 703 screen for changes, which only occur locally, in volume, pH, total acidity ple serum collections should not be taken before/during urine sampling, 704 and acid output over time. Gastric secretion tests are typically performed as blood sampling will affect urinary volume ). Neverthe- 705 following changes in gastric emptying. Agonists of opioid, dopamine re- less, it would be useful to have samples from multiple time points, since 706 ceptors, and beta-adrenoceptors markedly reduce gastric emptying and knowledge of kinetics is necessary to understand the function. Hence, 707 intestinal motility. However, muscarinic receptor agonists tend to in- limiting to three samples wit OOF hin 24 h would prove beneficial without 708 crease gastric emptying, intestinal motility, and gastric secretion, where- causing any other interference Therefore, mathe- 709 as antagonists have the opposite effects. Unpublished data from Dr matical modelling is used to extrapolate the data obtained to calculate 710 Sabine Pestel (Boehringer-Ingelheim Pharma GmbH & Co) on 59 test com- GFR and improve the reliability of the data, while using fewer animals 711 pounds evaluated between 2009 and 2001 showed a greater incidence (. If the study design requires samples from the 712 and severity on gastric emptying (85% vs. 45%) and intestinal transit same animal, larger animals, such as dogs, can be used 713 (70% vs. 25%) of compounds derived from oncology vs. non-oncology ). However, an integrated pharmacology testing system in surgi- 714 projects. Those effects were detected at lower margins for oncology vs.
cally prepared rats has been recently developed for simultaneous mea- 715 non-oncology projects ( 2–5 vs 10–30-fold on a dose basis). It is im- surements of GFR and renal plasma flow. This system successfully 716 portant to note that anticancer compounds have shown greater GI com- combined BASi Culex® automated blood sampling, radiotelemetry, 717 plications hence it would be beneficial to include GI testing as part of the quantitative urinalysis, and nephron site-specific urinary biomarkers 718 routine safety pharmacology studies for this class of compounds.
of injury into one model testing system 719). Renal toxicity can be predicted using clinical chemistry 720 following a single administration of the test drug (), 721 GI injury assessments are usually performed following lead candi- date drug administration and are preformed through visual examina- tion of the stomach and intestinal tract and ulceration index scores. A Parameters to assess renal function and integrity of the kidney in safety pharmacology in- t6:2 recent advance in SP for GI assessment is the use of biomarkers for vestigations. Table lists both established and emerging parameters in renal safety pharma- t6:3 cology studies. ALP — alkaline phosphatase; AST — aspartate aminotransferase; ALT — t6:4 GI injury. Biomarkers specific for GI injury, such as blood citrulline, alanine aminotransferase; BUN — blood urea nitrogen; CLU — clusterin; GGT — γ-glutamyl t6:5 faecal miR-194 and calprotectin, are being explored and hold promise transferase; GFR — glomerular filtration rate; GST — glutathione S transferase; KIM-1 — kid- t6:6 in safety assessments (). However, further ney injury molecule-1; LDH — lactate dehydrogenase; β-NAG — N-acetyl-β-D- t6:7 validation and consensus are needed prior to their implementation glucosaminidase; NGAL — neutrophil gelatinase-associated lipocalin; RPA-1 — renal papil- t6:8 in routine SP assessments. In addition, the use of the wireless capsule, lary antigen-1; TFF3 — trefoil factor 3.
radiotelemetry and in-silico (PBPK modelling) in the assessment and Renal injury markers prediction of gastric emptying, intestinal motility and GI injury to Qualified (known) Qualified New leakage/ reduce undue stress to the animals and to reduce animal numbers inducible markers Functional makers are also being explored.
under investigation Based on the data available from preclinical testing and clinical trials, it can be inferred that d rug-induced changes in kidney function, includ- ing nephrotoxicity, may be underestimated ). In addition, unpublished data from Dr Sabine Pestel (Boehringer-Ingelheim Pharma GmbH & Co) on 99 test compounds evaluated between 2004 and 2011 showed that nearly 70% of all test compounds demonstrated effects on renal function, and close to 50% were indicative of kidney injury based on changes in the biomarkers. Therefore, there is a growing need to integrate routine eval- uation of the renal system into SP testing, which can be grouped into al- tered renal functions (diuresis or anti-diuresis) and organ damage, such as acute kidney injury (AKI), this can include localized injury to glomer- uli, renal papillae and/or different regions of the tubules According to ICH recommendations, testing of renal function by measuring urine volume and electrolyte excretion in rats or dogs, as Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013), J. Hamdam et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx but the sensitivity is rather low when compared to NMR-based the drug discovery and development process. This requirement is 782 metabolomics methods (). However, with addressed by the practice of "frontloading" in SP studies. "Frontloading" 783 newer evaluation tools and semi-automatic approaches, sensitivity is defined as "safety studies conducted during lead optimisation of com- 784 could be considerably increased.
pounds before selection of a candidate drug for development and regu- 785latory studies are performed (Understanding 786 Kidney injury markers more about the propensity of molecules to cause adverse effects prior 787 Kidney injuries are also being assessed using functional and leakage to initiation of in vivo studies is becoming increasingly important to re- 788 markers. Functional markers suggesting kidney injury may include uri- duce the likelihood of termination at later stages of drug development. 789 nary glucose, protein, albumin and calciumor, indeed, any other mole- Unlike the core battery assessments, frontloading SP studies are not 790 cule known to be transported in a certain region of the kidney.
performed according to GLP compliance . The cur- 791 Urinary excretion of aspartate aminotransferase (AST), alanine amino- rent practice and perspective of frontloading in major organ system SP 792 transferase (ALT), lactate dehydrogenase (LDH), γ-glutamyl transferase assessment have been discussed elsewhere (GGT), alkaline phosphatase (ALP) and N-acetyl-β-D-glucosaminidase With regard to the CVS, this challenge can be tackled by performing 794 (β-NAG) are used as leakage markers for kidney injury measured in vitro assays, similar to the hERG assay, for many of the ion channels 795 by clinical chemistry. Further leakage markers like kidney injury previously mentioned. Furthermore, telemetry studies can also be used 796 molecule-1 (KIM-1) and clusterin (CLU) can be measured with different to provide in vivo assessment for numerous NCEs' effects on the CVS, 797 techniques based on antibody detection. Acute kidney injury (AKI) pre- prior to pre-clinical trials. From a CNS safety pharmacology perspective, 798 dominantly includes proximal tubule toxicity due to the high concen- in vitro receptor ligand binding assays are used to assess potential 799 tration of test drug in the loop of Henle and renal papillae, injuries NCE-induced effects on a variety of neuronal targets including gamma- 800 here are more commonly associated with drug-induced nephrotoxicity aminobutyric (GABA), N-Methyl-D-aspartic acid (NMDA) and dopamine 801 ). These kidney injuries are assessed primarily using histol- receptors which have bee). Frontloading c OOF n extensively reviewed elsewhere (802 ogy and approved biomarkers. In rats, drug toxicity has been shown to an also be applied to assess seizure liability 803 vary with circadian rhythm application since kidney through in vitro assays, such as the semi-automated Slicemaster system, 804 functions are shown to be influenced significantly by time of day that only requires minute concentrations of the NCE and can measure 805 The various parameters both electrophysiological recordings in up to eight rodent hippocampal 806 established and emerging in renal SP studies are shown in brain slices ). However, they can only assess pro- 807convulsive activity in specific brain regions and since seizures have a 808 complex mechanism these assays should be complemented with in 809 One of the recent advances in SP which can increase the depth and vivo assessment. Frontloading in respiratory SP studies include selectiv- 810 breadth of renal toxicity (functional & injury) assessments, is the use ity binding screens, rodent plethysmography and arterial blood gas mea- 811 of molecular biomarkers. The use of molecular biomarkers improves surement which are the common techniques used, whereas isolated 812 the predictability of renal toxicity as histological examination can con- organs/tissues/cells and anaesthetized animals are used if there is a 813 tribute to false negative findings, due to the time taken for histopatho- need to assess lung mechanics as part of frontloading 814 logical manifestation following insult, and region of section used for the ). For the renal system, routine practice of frontloading is relatively 815 examination (regional bias). Therefore, there is a need for molecular low ; the same holds true of 816 biomarkers to detect and predict region specific nephrotoxicity more the GI system.
effectively Recently, newer kidney injury Taken together, the frontloading concept not only facilitates the 818 biomarkers qualified for preclinical testing include KIM-1, CLU, albu- early identification of potentially hazardous substances, thus contrib- 819 min, total protein, β2-microglobulin, cystatin C and trefoil factor 3 uting to better decision making for the selection of safer candidate 820 (TFF3) in urine ). Some of these biomarkers can drugs administered in FiH trials, but also reduces the number of in 821 provide key information on the region of injury as indicated in vivo safety studies required to decipher the toxicity of such NCEs as 822 Owing to the potential of molecular biomarkers in contributing a result of early termination of potentially unsafe candidates.
to false positive findings, a positive association in predicting renal toxic- ity should be based on information obtained collectively from renal function assessment, histology and molecular biomarker readout. Re- cently, metabolomics approaches involving the use of NMR and mass The zebrafish is a well-established model organism for use in de- 825 spectroscopy to identify known nephrotoxic biomarkers are being ex- velopmental biology and more recently in toxicology and disease 826 The zebrafish model in CNS 827studies has been validated, offering a ‘sufficient', 72% predictability 828 Recent and emerging concepts of proconvulsive activity through the use of validated anticonvulsant 829and proconvulsant compounds in assessing seizure liability, via auto- 830 SP is continuously evolving and some recent trends to enhance mated measurements of locomotor activity (). Sim- 831 and refine the scope UNCORRECTED PR include focus towards frontloading, exploration ilarly to the in vitro hippocampal brain slice assay, relatively small 832 of alternate models, combining core battery tests, integration of SP amounts of NCEs are required to perform the screen. Many other be- 833 endpoints into regulatory toxicology endpoints and correlation be- havioural paradigms, such as addiction, memory and anxiety can be 834 tween non-clinical safety endpoints and clinical outcomes. As tech- assessed using the zebrafish model ). 835 niques and methodologies continue to improve, SP has adapted to There is a great potential for this model to be used in early drug fail 836 contribute to improved decision making in lead candidate selection fast strategies, especially for CNS targeted NCEs. Renal safety assess- 837 during drug discovery and development.
ment studies conducted in simpler animal models and/or simple or- 838gans, such as teleost pronephros systems in zebrafish, can render 839 renal safety testing routine (840). This model can be explored as one of the more viable options, 841 There is a clear need for the implementation of safety assessments in without compromising on the predictability of adverse events, since, its 842 the initial stages of drug discovery and development which would facil- gentamicin-induced patho-phenotype was similar to that of those 843 itate ranking of NCEs leading to the improved identification of lead observed in the mammalian renal system. However, the use of in vivo 844 candidates, ultimately reducing valuable time and costs involved in zebrafish models as early screening methods in SP is a matter of debate 845 Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013), J. Hamdam et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx (as the use of this model is yet to be recognised by exposure to the compound in the therapeutic range and above, before 908 the regulatory bodies. Nonetheless, it is amenable to the early phases evaluating and investigating the cause of these effects through toxico- 909 of drug discovery in terms of turnaround time, cost, NCE requirement logical and/or clinical studies On the 910 and throughput, thus having the potential to facilitate early screening other hand, toxicological studies focus on exploring the adverse phar- 911 methods in screening for safety liabilities ).
macodynamic effects of compounds up to the maximum tolerated 912 The exploitation of human embryonic stem cell derived cardio- dose level. In particular, they centre on addressing general safety and 913 myocytes (hESC-CM) and human inducible pluripotent stem cell derived are designed to include high doses at which overt toxicity may be ob- 914 cardiomyocytes (hiPS-CM) as models of in vitro high throughput drug served Integration of SP and toxicology studies will 915 screening and CVS safety assessment have the potential to significantly improve the resolution of the safety profile and risk factor identification 916 refine CVS studies due to their biological relevance and mass production more effectively (). When 917 capacity ). Unlike mammalian cell lines, these cells integrating SP and toxicological studies, consideration needs to be given 918 inherently express the hERG channel and other ion channels which con- to various factors: the selection of species, number of animals, study de- 919 tribute to the AP as well. As heart complications are not simply due to signs, reduction of cost and timelines to the endpoints that can be inte- 920 hERG blockade alone, these cell lines can facilitate the measurement of grated ). SP studies are 921 a multitude of target ion channels, thus enhancing the SP profile of typically single-dose studies in which a given effect can be measured 922 NCEs (hESC-CMs, have shown to be more sensi- over time, while in toxicological studies, data may be collected sequen- 923 tive compared to current in vitro isolated tissue preparations in CVS safe- tially over days or weeks of treatment, especially for substances that 924 ty assessments Importantly, iPS-CMs derived from may chronically accumulate in the body ). Personnel 925 patients with long QT syndrome emulated the electrophysiological fea- training is essential for effective integration of SP and toxicological 926 tures of the disorder revealing the irrefutable potential of the use of end point assessments ). Additionally, 927 iPS-CMs derived from human patients for utilisation of drug screening animals have to be trained in order to reduce stress level during routine 928 in appropriate disease models ). Therefore, this sample collection and care sho OOF uld be also be taken to avoid disturbances 929 approach will offer the opportunity to screen not only NCEs in normal to the animals which may disrupt physiological functions and SP read 930 tissue but also hiPS-CM originating from patients suffering various dis- outs Sometimes there is no viable solution and 931 ease(s), offering a disease-model approach focused on the anticipated multiple experiments do need to be performed, but with careful plan- 932 target population. Although these models are promising, they are not ning and compromise, this can normally be accomplished, as a well- 933 without their shortcomings as issues with stability of cardiomyocyte designed SP study could allow for multiple administrations of a com- 934 phenotype, genetic variation and reproducibility of differentiation re- pound ). With regards combining SP and toxicology 935 main a concern. Thus, these models require further validation and in CNS, behavioural tests such as the modified Irwin test or FOB can 936 standardisation in order to be fully implemented in CVS SP studies be easily integrated into toxicology studies with minimal or no impact 937 on histological data obtained (). The main disadvan- 938tage when combining behavioural assessments and toxicology studies 939 Integrated core battery assessment is that the data received can be highly influenced by the experience 940and training of the individuals which perform and interpret the assess- 941 As mentioned previously, telemetry, an increasingly popular tech- ments as indicated earlier. Another important issue when combining 942 nique, is evolving to provide relevant and reliable in vivo data from a these endpoints is that the behavioural assessments need to be con- 943 variety of physiological systems that are examined as part of SP stud- ducted when other parameters, such as blood sampling are not being 944 ies. This revolutionary technique has changed SP so that many core measured; this avoids the possibility of sampling affecting the other 945 battery safety studies, which are traditionally investigated separately, parameters. However, in long-term toxicology studies this should not 946 can now be measured simultaneously in conscious animals across a be an issue as long as there is good communication between the person- 947 variety of species ).
nel performing the behavioural assessments and toxicology studies. 948 This not only reduces the number of animals used per study, but Currently, there are guidelines (ICH S6, ICH S7A and ICH S9) that relate 949 also enhances the statistical power of the results as the animals can to the integration of SP endpoints in toxicology studies and this will 950 be used as their own respective vehicle control ( become more prevalent in the future.
A prime example of this is the use of integrated video teleme- try in assessing the neurobehavioural (via video recordings) and car- Drug–drug interactions diovascular (via telemetric devices) effects of candidate drugs in canine and non-human primate models As mentioned earlier in this review, drug–drug interactions can 953 Combining video recording with telemetry cause adverse side effects that can lead to attrition of lead candidates 954 allows integrated CNS and CVS observations over extended periods or drugs. There are a number of assays available to assess the binding 955 of time with minimal stress caused to experimental animals. The properties of an NCE (and these include the extent 956 combination of respiratory SP studies using radio telemetry and auto- of cytochrome P450 inhibition () and 957 mated blood sampling UNCORRECTED PR offers an integrative pharmacological and tox- P-glycoprotein interactions (In vitro binding affini- 958 icological approach inevitably decreasing the number of animals ties should be used cautiously when extrapolating in vivo data; howev- 959 without compromising, the credibility of the data obtained and the er, with well-designed experiments these assays can provide benefits 960 predictive ability of the studies (The use of with regard to compound design and the prediction of potential 961 emerging technologies will aid in the integration of GI toxicity screen- unwanted interactions. Given the low cost of these assays, it would be 962 ing as part of the other mandatory core testing, since methods like beneficial to include these preliminary screens and this is supported 963 capsule endoscopy and radio-telemetry are non or less invasive and by the recent ICH draft guidance ( can be used simultaneously alongside cardiovascular and respiratory assessments ().
Translational safety pharmacology Integrating safety pharmacology end points into toxicology studies SP is evolving to keep pace, adapt, to incorporate the latest scien- 966 tific knowledge and novel technologies for the safety evaluation of 967 SP can be referred to as studies that investigate the possible undesir- compounds in non-clinical assays, and to identify the effects that 968 able pharmacodynamic effects on physiological functions as a result of may pose a risk to human volunteers and patients. There are recent 969 Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013),

J. Hamdam et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx Fig. 2. Established and emerging parameters and techniques in safety pharmacology studies. Illustration established and emerging parameters/techniques investigated in five importantorgan systems to assess lead compounds in safety pharmacology studies. AP — action potential; ALP — alkaline phosphatase; AST — aspartate aminotransferase; ALT — alanine aminotrans-ferase; BP — blood pressure; BUN — blood urea nitrogen; CLU — clusterin; CM — cardiomyocyte; EEG — electroencephalography; ECG — electrocardiogram; FOB — Functional ObservationBattery; GGT — γ-glutamyl transferase; GFR — glomerular filtration rate; GST — glutathione S transferase; HDO — high definition oscillometry; hESC-CM — human embryonic stem cellderived cardiomyocytes; hiPS-CM — human inducible pluripotent stem cell derived cardiomycotes; HR — heart rate; KIM-1 — kidney injury molecule-1; LDH — lactate dehydrogenase;miR — microRNA; β-NAG — N-acetyl-β-D-glucosaminidase; NGAL — neutrophil gelatinase-associated lipocalin; PBPK — physiologically based pharmacokinetics; PEB — photoelectricbeam interruption technique; RPA-1 — renal papillary antigen-1; TFF3 — trefoil factor 3; VQM — ventilation (V)/perfusion (Q) mismatch (M).
examples of promising future areas for the development of SP that il- the zebrafish lends real potential as a fast means for early compound 997 lustrate the challenges, as reviewed in ( screening in all aspects of frontloading Further vali- 998 ). However, a more sophisticated translation of dation of this model in a variety of studies may result in their regular use 999 human outcomes to preclinical animal models and vice versa still re- as a frontloading model in the future. The incorporation of the emerging 1000 mains an essential goal. Several individual organisations or consortia concepts, such as biomarkers and common SP-toxicological endpoints, 1001 efforts are trying to address this issue by conducting retrospective should be carried out alongside mandatory SP protocols to validate the 1002 accuracy and reproducibility of these tests, which will ultimately aug- 1003 or prospective studies (It ment SP studies and predictive end points for safer therapeutics.
is clear that the confidence in the translational SP models will im- prove as the number of NCEs that progress through SP model and Conflict of interest statement subsequent human trials increases. This will enhance the validity of the non-clinical safety assessment models that are used ultimately fa- The authors declare that there are no conflicts of interest.
cilitating better decision making at all stages of drug discovery and We would like to thank Michael Hoffmann (Bayer Pharma, Germany), 1008 Brian Guth (Boehringer-Ingelheim, Germany), Andrea Parenti (Merck 1009 Over the last decade, SP has made tremendous progress in both the Serono Research, Merck KGaA, Germany), Herbert Himmel (Bayer 1010 regulatory requirements and the knowledge gained while developing Pharma, Germany), Julia Schlichtiger (Boehringer-Ingelheim, Germany), 1011 NCEs ). A schematic summation of the current and Christian Friechel (Roche, Switzerland) and Andrea Greiter-Wilke 1012 emerging trends in SP studies is represented in It has become in- (Roche, Switzerland) for their input and guidance in the preparation 1013 creasingly evident that more suitable high throughput in vitro screening of this manuscript.
methods are required to be implemented at the earliest stages of drug discovery to obtain information about compounds prior to the initiation of clinical trials. Fail fast drug strategies at this stage would prevent the Adler, A., Cieslewicz, G., Irvin, C.G., 2004. 1016 progression of potential unsafe NCEs into later discovery, thus saving valuable time and costs for the pharmaceutical industry. It is also worth Ali, S., Champagne, D.L., Spaink, H.P., Richardson, M.K., 2011. 1019 exploring the value of using other models to answer various SP questions.
Although the emergence of the zebrafish model is still a matter of debate, Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013), J. Hamdam et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx 1022 Amen, E.M., Becker, E.M., Truebel, H., 2011. FDA, 2010. Guidance for Industry: Assessment of Abuse Potential of Drugs. 1108 1024 Ator, N.A., Griffiths, R.R., 2003. Froger-Colleaux, C., Rompion, S., Guillaume, P., Porsolt, R.D., Castagne, V., Moser, P., 1111 1026 Authier, S., Legaspi, M., Gauvin, D., Chaurand, F., Fournier, S., Troncy, E., 2008. Fuchs, T.C., Hewitt, P., 2011. 1114 1029 Authier, S., Paquette, D., Gauvin, D., Sammut, V., Fournier, S., Chaurand, F., Troncy, E., Fung, M., T., Anna, Mybeck, Kathy, Hsiao-Hui, Jasmanda, Hornbuckle, Ken, Muniz, Edmundo, 1116 1033 Authier, S., Haefner, P., Fournier, S., Troncy, E., Moon, L.B., 2010. Gacsalyi, U., Zabielski, R., Pierzynowski, S.G., 2000. 1119 Gauvin, D.V., Yoder, J.D., Dalton, J.A., Baird, T.J., 2010. 1121 1036 Bankstahl, M., Bankstahl, J.P., Bloms-Funke, P., Loscher, W., 2012. Glennon, R.A., 1999. 1124 1039 Barros, T.P., Alderton, W.K., Reynolds, H.M., Roach, A.G., Berghmans, S., 2008. Globig, S., Witte, K., Lemmer, B., 1999. 1126 1042 Bass, A.S., Vargas, H.M., Kinter, L.B., 2004. Goineau, S., Rompion, S., Guillaume, P., Picard, S., 2010. 1129 1044 Bass, A.S., Vargas, H.M., Valentin, J.P., Kinter, L.B., Hammond, T., Wallis, R., Siegl, P.K., Yamamoto, K., 2011. Grant, A.O., 2009. Guth, B.D., Rast, G., 2010. 1133 1049 Bates, J.H., Thompson-Figueroa, J., Lundblad, L.K., Irvin, C.G., 2008. Guth, B.D., Bass, A.S., Briscoe, R., Chiv ers, S., Markert, M., Siegl, P.K., Valentin, J.P., 2009. 1135 1052 Boudonck, K.J., Rose, D.J., Karoly, E.D., Lee, D.P., Lawton, K.A., Lapinskas, P.J., 2009.
Hancox, J.C., McPate, M.J., El Harchi, A., Zhang, Y.H., 2008. 1139 1055 Bowes, J., Brown, A.J., Hamon, J., Jarolimek, W., Sridhar, A., Waldron, G., Whitebread, S., Harrison, A.P., Erlwanger, K.H., Elbrond, V.S., Andersen, N.K., Unmack, M.A., 2004. 1142 1058 Claude, J.R., Claude, N., 2004. Hayashi, E., Kobayashi, T., Shiroshita, Y., Kuratani, K., Kinoshita, M., Hara, H., 2011. 1145 1061 Costa, D.L., R.J.A., Tepper, J.S., 1992. 1063 Couderc, J.P., Xiaojuan, X., Zareba, W., Moss, A.J., 2005. Hernandez, S.H., Nelson, L.S., 2010. 1149 Hollo, Z., Homolya, L., Davis, C.W., Sarkadi, B., 1994. 1151 1066 Curran, M.E., Splawski, I., Timothy, K.W., Vincent, G.M., Green, E.D., Keating, M.T., 1995.
Hoymann, H.G., 2007. 1153 1069 Delaunois, A., Dedoncker, P., Hanon, E., Guyaux, M., 2009. Hoymann, H.G., 2012. 1155 Irwin, S., 1968. 1157 1072 Dieterle, F., Sistare, F., Goodsaid, F., Papaluca, M., Ozer, J.S., Webb, C.P., Baer, W., Senagore, A., Schipper, M.J., Vonderscher, J., Sultana, S., Gerhold, D.L., Phillips, J.A., Maurer, G., Carl, K., Laurie, D., Harpur, E., Sonee, M., Ennulat, D., Holder, D., Andrews- John-Baptiste, A., Huang, W., Kindt, E., Wu, A., Vitsky, A., Scott, W., Gross, C., Yang, A.H., 1160 Cleavenger, D., Gu, Y.Z., Thompson, K.L., Goering, P.L., Vidal, J.M., Abadie, E., Schaiff, W.T., Ramaiah, S.K., 2012. 1161 Maciulaitis, R., Jacobson-Kram, D., Defelice, A.F., Hausner, E.A., Blank, M., Thompson, A., Harlow, P., Throckmorton, D., Xiao, S., Xu, N., Taylor, W., Vamvakas, S., Flamion, B., Lima, B.S., Kasper, P., Pasanen, M., Prasad, K., Troth, S., Bounous, D., Robinson- Kaczorowski, G.J., Garcia, M.L., Bode, J., Hess, S.D., Patel, U.A., 2011. 1164 Gravatt, D., Betton, G., Davis, M.A., Akunda, J., McDuffie, J.E., Suter, L., Obert, L., Guffroy, M., Pinches, M., Jayadev, S., Blomme, E.A., Beushausen, S.A., Barlow, V.G., Collins, N., Waring, J., Honor, D., Snook, S., Lee, J., Rossi, P., Walker, E., Mattes, W., Kagstrom, J., Sjogren, E.L., Ericson, A.C., 2007. 1167 1084 Dunlop, J., Bowlby, M., Peri, R., Vasilyev, D., Arias, R., 2008. Kamendi, H.W., Brott, D.A., Chen, Y., Litwin, D.C., Lengel, D.J., Fonck, C., Bui, K.H., 1170 Gorko, M.A., Bialecki, R.A., 2010. 1171 1087 Durmuller, N., Guillaume, P., Lacroix, P., Porsolt, R.D., Moser, P., 2007. Kearney, K., Metea, M., Gleason, T., Edwards, T., Atterson, P., 2010. 1174 1090 Easter, A., Sharp, T.H., Valentin, J.P., Pollard, C.E., 2007. Kramer, K., Kinter, L.B., 2003. 1177 1093 EMA, 2006. Guideline on the non-clinical investigation of the dependence potential of Kramer, J.A., Sagartz, J.E., Morris, D.L., 2007. 1179 medicinal products. 1096 EMA, 2013. Guidance for Industry: Drug Interaction Studies — Study Design, Data Anal- Kraushaar, U., Meyer, T., Hess, D., Gepstein, L., Mummery, C.L., Braam, S.R., Guenther, E., 1182 ysis, Implications for Dosing, and Labeling Recommendations. 1100 Emeigh Hart, S.G., 2005. Laverty, H., Benson, C., Cartwright, E., Cross, M., Garland, C., Hammond, T., Holloway, C., 1186 McMahon, N., Milligan, J., Park, B., Pirmohamed, M., Pollard, C., Radford, J., Roome, 1187 1102 FDA, 2001. ICH S7A Safety Pharmacology Studies for Human Pharmaceuticals. N., Sager, P., Singh, S., Suter, T., Suter, W., Trafford, A., Volders, P., Wallis, R., Weaver, 1188 R., York, M., Valentin, J., 2011. 1189 1105 FDA, 2005. S7B Nonclinical Evaluation of the Potential for Delayed Ventricular Repolar- Lawrence, C.L., Bridgland-Taylor, M.H., Pollard, C.E., Hammond, T.G., Valentin, J.P., 2006. 1191 ization (QT Interval Prolongation) by Human Pharmaceuticals. Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013), J. Hamdam et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx 1194 Lemmer, B., Mattes, A., Bohm, M., Ganten, D., 1993. Pirmohamed, M., James, S., Meakin, S., Green, C., Scott, A.K., Walley, T.J., Farrar, K., Park, 1261 B.K., Breckenridge, A.M., 2004. 1262 1196 Levi, F., Hrushesky, W.J., Borch, R.F., Pleasants, M.E., Kennedy, B.J., Halberg, F., 1982. Pons, M., Schnecko, A., Witte, K., Lemmer, B., Waterhouse, J.M., Cambar, J., 1996. 1264 1199 Lienemann, K., Plotz, T., Pestel, S., 2008. Porsolt, R.D., Lemaire, M., Durmuller, N., Roux, S., 2002. 1267 1202 Lindgren, S., Bass, A.S., Briscoe, R., Bruse, K., Friedrichs, G.S., Kallman, M.J., Markgraf, C., Porsolt, R.D., C.D., Niklaus, Lemaire, Martine, Moser, Paul, Rous, Slyvain, France, Charles 1269 Patmore, L., Pugsley, M.K., 2008. 1205 Litwin, D.C., Lengel, D.J., Kamendi, H.W., Bialecki, R.A., 2011. Pugsley, M.K., Authier, S., Curtis, M.J., 2008. 1272 Rahma, M., Kimura, S., Yoneyama, H., Kosaka, H., Nishiyama, A., Fukui, T., Abe, Y., 2001. 1274 1208 Luft, J., Bode, G., 2002. 1210 Lynch III, J.J., Castagne, V., Moser, P.C., Mittelstadt, S.W., 2011. Redfern, W.S., Carlsson, L., Davis, A.S., Lynch, W.G., MacKenzie, I., Palethorpe, S., Siegl, 1277 P.K., Strang, I., Sullivan, A.T., Wallis, R., Camm, A.J., Hammond, T.G., 2003. 1278 1213 Malik, M., Farbom, P., Batchvarov, V., Hnatkova, K., Camm, A.J., 2002. Redfern, W.S., Strang, I., Storey, S., Heys, C., Barnard, C., Lawton, K., Hammond, T.G., 1282 1216 McGrath, P., Li, C.Q., 2008. Valentin, J.P., 2005. 1283 1218 McMahon, C., Mitchell, A.Z., Klein, J.L., Jenkins, A.C., Sarazan, R.D., 2010. Redfern, W.S., Waldron, G., Winter, M.J., Butler, P., Holbrook, M., Wallis, R., Valentin, 1286 1221 Meyer, O., Jenni, R., Greiter-Wilke, A., Breidenbach, A., Holzgrefe, H.H., 2010. Samson, N., Dumont, S., Specq, M.L., Praud, J.P., 2011. 1289 1224 Miller, D.S., 2002. Shah, R.R., 2006. 1291 1226 Monahan, B.P., Ferguson, C.L., Killeavy, E.S., Lloyd, B.K., Troy, J., Cantilena Jr., L.R., 1990.
Shell, L., Rozum, M., Jortner, B.S., Ehrich, M., 1992. 1293 1229 Moretti, A., Bellin, M., Welling, A., Jung, C.B., Lam, J.T., Bott-Flugel, L., Dorn, T., Goedel, A., Stonard, M.D., 1990. 1296 Hohnke, C., Hofmann, F., Seyfarth, M., Sinnecker, D., Schomig, A., Laugwitz, K.L., 1233 Moscardo, E., McPhie, G., Fasdelli, N., Giarola, A., Tontodonati, M., Dorigatti, R., Meecham, K., Talavera, J., Kirschvink, N., Schuller, S., Garreres, A.L., Gustin, P., Detilleux, J., Clercx, C., 1300 1236 Moser, P., Wolinsky, T., Castagne, V., Duxon, M., 2011. Tontodonati, M., Fasdelli, N., Moscardo, E., Giarola, A., Dorigatti, R., 2007. 1303 1239 Muller, P.Y., Dieterle, F., 2009. Trepakova, E.S., Koerner, J., Pettit, S.D., Valentin, J.P., Committee, H.P.-A., 2009. 1306 1242 Murphy, D.J., 2002. Valentin, J.P., Hammond, T., 2008. 1309 1244 Murphy, D.J., Renninger, J.P., Schramek, D., 2010. Valentin, J.P., Bass, A.S., Atrakchi, A., Olejniczak, K., Kannosuke, F., 2005. 1311 1247 Nattel, S., Carlsson, L., 2006. Valentin, J.P., Bialecki, R., Ewart, L., Hammond, T., Leishmann, D., Lindgren, S., 1314 Martinez, V., Pollard, C., Redfern, W., Wallis, R., 2009. 1315 1249 Ninkovic, J., Bally-Cuif, L., 2006. 1251 Nirogi, R., Shanmuganathan, D., Jayarajan, P., Abraham, R., Kancharla, B., 2012. Wallis, R.M., 2010. 1318 West, R., Gossop, M., 1994. 1320 1254 Peng, S., Lacerda, A.E., Kirsch, G.E., Brown, A.M., Bruening-Wright, A., 2010. Wienkers, L.C., Heath, T.G., 2005. 1322 1257 Pestel, S., Martin, H.J., Maier, G.M., Guth, B., 2006. Winter, M.J., Redfern, W.S., Hayfield, A.J., Owen, S.F., Valentin, J.P., Hutchinson, T.H., 1324 1259 Pestel, S., Krzykalla, V., Weckesser, G., 2007. Please cite this article as: Hamdam, J., et al., Safety pharmacology — Current and emerging concepts, Toxicol. Appl. Pharmacol. (2013),


(microsoft word - cjde brochure - chapitre 2 - anglais- modifi 351.doc)

Educational program for people with diabetes Medication Inv.: 1-6-14729 (Hôtel-Dieu, Hôpital Fleurimont) CENTRE DE JOUR DU DIABÈTE DE L'ESTRIE. Mieux connaître le diabète, Centre hospitalier universitaire de Sherbrooke, 2002, 121 p. PROGRAMME MÉDECINE - PROGRAMME - CLIENTÈLE DIABÈTE. Mieux connaître votre diabète, brochure # 321-1,

Microsoft word - final layton.docx

Something to do with a girl named Marla Singer Journal Title: Free Associations: Psychoanalysis and Culture, Media, Groups, Politics Number 62, September 2011 ISSN: 2047-0622 URL: SOMETHING TO DO WITH A GIRL NAMED MARLA SINGER: CAPITALISM, NARCISSISM, AND THERAPEUTIC DISCOURSE IN DAVID