CHAPTER 13

In Vivo MRI/S for the SafetyEvaluation of Pharmaceuticals

PAUL D. HOCKINGS*a,b AND HELEN POWELLc

aAstraZeneca R&D, PHB In Vivo Biomarkers, 43183 Molndal, Sweden;bMedTech West, Chalmers University of Technology, 41296 Gothenburg,Sweden; cAstraZeneca R&D, Global Safety Assessment, Macclesfield SK104TG, United Kingdom*Email: paul.hockings@astrazeneca.com

13.1 Introduction

Despite the increasing investment in drug discovery and development, only onein nine new medicinal products that enter clinical studies actually reaches theregistration phase, with approximately 30% of compounds failing due totoxicology findings, despite extensive safety testing.1 In addition, a number ofdrugs that had been approved for use in humans have either been withdrawn orhave been issued with black box warnings following more extensive clinicaluse.2 In order to improve this attrition rate, there is a requirement for phar-maceutical companies to explore and employ increasingly sophisticatedmethods to assess the safety profile of compounds. The pharmaceuticalindustry uses medical imaging because it enhances the ability to quantify theimpact of drugs on human health and can be used alongside ordinary orinvasive clinical assessments. Medical imaging can decrease patient numbersand/or the duration of exposure in clinical trials and hence accelerate drugdevelopment. Magnetic resonance imaging (MRI) has been the most widelyused medical imaging technique in the pharmaceutical industry because of itssuperb soft tissue contrast and capability of delivering quantitative 3D

New Developments in NMR No. 2

New Applications of NMR in Drug Discovery and Development

Edited by Leoncio Garrido and Nicolau Beckmann

r The Royal Society of Chemistry 2013

Published by the Royal Society of Chemistry, www.rsc.org

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information on organ anatomy and function.3 It is used in a variety of diseaseareas in both non-clinical and clinical drug efficacy studies; however, there arerelatively few examples of the use of MRI in drug safety studies.4–6 In 2007 one ofthe authors (PDH) conducted an informal survey of a number of non-clinicalimaging groups in the pharmaceutical industry and showed that onlyapproximately 5% of effort (range 0–20%) was devoted to safety imaging studies.This seems a disproportionally small effort considering that MRI is a powerfultool that could potentially be used to reduce attrition in the late pipeline.

The aim of non-clinical safety studies is to evaluate potential risk to humansof new medicinal products. By assessing both on- and off-target effects anddefining the dose–response relationship of any observed adverse effects, safetymargins can be calculated. Such studies therefore need to be conducted inspecies relevant to man in terms of pharmacology, exposure and metabolism,and at doses over and above those to be tested clinically. Current regulatoryguidelines recommended by the International Conference on Harmonisation ofTechnical Requirements for Registration of Pharmaceuticals for Human Use(ICH; http://www.ich.org/products/guidelines/safety/article/safety-guidelines.html)require the evaluation of chronic toxicity, carcinogenicity, safety pharmacologyand reproductive toxicity prior to regulatory approval. Chronic toxicity isassessed in one rodent and one non-rodent species, with studies of at least 6 or 9months duration, respectively, with the examination of tissue morphology atthe microscopic level and organ function by the measurement of clinicalchemistry endpoints as readouts. The carcinogenic potential of phar-maceuticals is evaluated by the conduct of long-term carcinogenicity studiesfollowing the assessment of genotoxic potential both in vitro and in vivo.Reproductive toxicity studies investigate the effect of test compounds on maleand female fertility, and embryonic, fetal and post-natal development. Thesafety pharmacology core battery of studies is used to investigate the effects ofthe test substance on the vital functions, including the cardiovascular,respiratory and central nervous systems with either follow-up or supplementarystudies (e.g. of the renal/urinary system, autonomic nervous system orgastrointestinal system) as required. Other studies assessing, for example,immune or endocrine functions may also be conducted if warranted based onfindings in standard studies. Despite such extensive testing, compounds deemedto be safe to administer to humans can be associated with unexpected toxicityin the clinic, identified either during early clinical studies or, worse still,following launch to the market. In particular, liver and cardiac toxicity havebeen associated with drugs that have either been withdrawn from use or havebeen issued with black box warnings limiting potential use.

Data from first time in man to registration during the period between 1991and 2000 for the ten largest pharmaceutical companies indicate an averagesuccess rate of 11%, with 62% and 45% of all compounds entering Phase IIand III, respectively, failing to progress to the next stage of clinical devel-opment.1 Indeed, during this period 23% of compounds failed at the regis-tration stage following completion of all the clinical trials and submissiondocumentation. Whilst many factors may contribute to the attrition of drug

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candidates during non-clinical and clinical development, including efficacy,pharmacokinetic and commercial reasons, safety issues are estimated toaccount for 31% of all attrition, 65% of which is due to non-clinical toxicityand 35% of which is accounted for by human adverse events. Withimprovements in the estimation of pharmacokinetic parameters and bioavail-ability, toxicity is now the single most common cause of drug attrition.1

Analysis of attrition rates from DuPont-Merck and Bristol-Myers Squibbduring the period between 1993 and 2006 indicates that the two most commoncauses of attrition are cardiovascular and liver toxicity, accounting forapproximately 27% and 15% of attrition of all advanced molecules,respectively.2 The underlying mechanisms of toxicity are often complex; withon-target (mechanism-based) effects, hypersensitivity and immunologicalmechanisms, off-target effects, biotransformation and idiosyncraticmechanisms contributing to the overall rates of attrition.7 Despite extensivemonitoring prior to registration, adverse events have been demonstrated to berelated to 6.5% of patient admissions to hospitals in the UK8 andapproximately one in seven in-patients experience an adverse drug reactionwhilst in hospital, which is a significant cause of morbidity, increasing thelength of stay of patients.9 Such data indicate that in both drug developmentand clinical use, effective intervention strategies are urgently needed to reducethe risk of safety issues.

Whilst considerable effort and investment is being made to improvecompound selection early in the drug discovery and development process by theincorporation of in silico models, better screening tools and in vitro and in vivoefficacy models, similar advances have not been made to the standard toxi-cology testing cascade. For example the pharmaceutical industry has investedheavily in the development of biomarkers to facilitate project progression10 butrelatively little in the development of safety biomarkers to indicate an adverseresponse to a test agent.11 The term safety biomarker encompasses anymeasurement used to diagnose and monitor drug-induced toxicity, includingtraditional soluble markers found in biofluids such as plasma and urine,microscopic analysis of tissue biopsies, clinical tests such as electrocardiograms(ECG) and imaging (most commonly ultrasound). Biomarkers derived frommedical imaging procedures have the advantage that they are comparativelynon-invasive and good for following focal diseases such as cancer or athero-sclerosis; however, they can be expensive to use and difficult to access.

An ideal clinical safety biomarker is a marker that informs about the lowestdose at which subtle, low-grade and reversible toxicities may appear, that willallow toxicities to be monitored in patients participating in studies and can beused to provide early information on benefit/risk-balance to inform projectstop/go decisions. Non-clinical safety biomarkers can be used to a) distinguishbetween compounds in order to select the best possible pharmacological targetsand molecules, taking account of the need to balance efficacy, kinetics,drug–drug interactions, safety, intellectual property and commercialconsiderations, b) enable molecules to progress efficiently from drug discoveryinto clinical development by providing a translational biomarker and c) predict

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susceptibility to an adverse response. New translational biomarkers have highpotential impact and value and have often been the subject of consortia to alignstakeholders and share costs as the resources required to validate/qualify themcan be substantial. Non-clinical safety biomarkers are anchored by correlatingthe marker with histopathology, whereas clinical safety biomarkers generallycannot be. Therefore translation from non-clinical species to man is key forclinical safety biomarkers. While no differences exist in the technology used forefficacy and safety biomarkers, differences do exist in the context of use. Thereare two important aspects to consider when deciding whether a safetybiomarker is fit for purpose. The first is validation, where the measurementperformance characteristics of an assay and the range of conditions underwhich the assay will give reproducible and accurate data are assessed. Thesecond is qualification, which is the fit-for-purpose evidentiary process oflinking a biomarker with biological processes and clinical endpoints.10 Theexpectation is that a safety biomarker will need a higher degree of validationand qualification than an efficacy biomarker to be considered fit for purpose.

Medical imaging is an important source of biomarkers for both clinical andnon-clinical studies although these imaging biomarkers will not replace tradi-tional endpoints in the short term. With increasing knowledge and experiencethis might partly change in the future, especially for

� species or animal models, which are costly, with limited availability and ofhigh ethical/emotional value (e.g. non-human primates)

� assessment of lesion severity at the end of the dosing period and recoveryafter a drug holiday in animals allocated to recovery groups to ensure truereversal of effect

� assessment or evaluation of mechanisms of toxicity during investigative/problem-solving studies

� longitudinal assessment of the development of a lesion in a single animal,replacing the need for necropsies at each individual timepoint, therebyreducing animal numbers.

Rather than provide a comprehensive literature review of the use of magneticresonance imaging (MRI) and spectroscopy (MRS) in safety studies, thischapter will review the application of these techniques to study liver and cardiactoxicity as these result in the greatest number of drug projects failing duringclinical development.

13.2 Hepatotoxicity

Drug-induced liver injury (DILI) is a recurrent cause of delayed progressionand/or attrition of new drug candidates, failed drug licensing, drug withdrawalpost-licensing and of serious illness in man. More drugs have been withdrawnfrom the market due to hepatotoxicity than for any other reason.12,13 Theconsequences of these withdrawals for pharmaceutical companies have beenenormous and there is much interest in preventing further post-approval

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attrition due to hepatotoxicity. One explanation for these withdrawals is thatcurrently available clinical biomarkers for hepatotoxicity do not adequatelypredict which patients may suffer drug-induced hepatic injury. Specifically,DILI mechanisms vary depending on the drug and some types of liver injurysuch as hepatocellular damage and cholestasis are more important to be able tomonitor than others.14 As a result, there is intense interest in finding new non-clinical biomarkers that better predict hepatotoxicity in humans, and betterclinical biomarkers that identify at-risk patients and provide earlier signals ofhepatotoxicity. Currently available biomarkers of hepatocellular injury such asserum alanine aminotransferase (ALT) lack sensitivity and specificity andcannot distinguish between adaption versus progression to liver failure.14

Non-clinically, the emphasis is on finding and qualifying new biomarkersthat help select compounds with no or markedly reduced potential for DILIand on translation of these new biomarkers from the non-clinical to the clinicalarena. DILI occurs via complex mechanisms and can be initiated via multiplemechanisms such as reactive metabolites, mitochondrial injury, lysosomalinjury, hepatobiliary transport inhibition and immune activation.12 Down-stream effects can include steatosis, cholestasis, necrosis and fibrosis.Biomarkers that assess individual mechanisms will improve mechanisticunderstanding and enable in vivo hazard identification and risk assessment. Theprimary value of DILI biomarkers in this regard is to enable

� selection of pharmacological targets that will not result in DILI� selection of compounds having minimal possible potential to cause DILI� monitoring of DILI in non-clinical species, using sensitive and specific

biomarkers that ideally detect early stages of liver dysfunction prior toonset of irreversible liver changes and that translate to man.

13.2.1 Hepatic Steatosis

Hepatic steatosis is a reversible condition where vacuoles of lipid accumulate inhepatocytes. It is the most common liver disease in the Western world and itspresence is associated with obesity and insulin resistance.15 Some drugs such asthe antiarrhythmic agent amiodarone, the antiviral nucleoside analoguefialuridine and the anti-estrogen agent tamoxifen have been reported to causehepatic steatosis; however, there have been relatively few in-depth studies inhumans or experimental animals.16 This may be because hepatic steatosis hasnot historically been a reason for non-approval or withdrawal of drugs fromthe market. However, hepatic steatosis is known to increase the risk forprogression to steatohepatitis and to make the liver more vulnerable to hepa-tocellular injury.17 In some cases drug-induced hepatic steatosis patients canpresent with a rapid evolution of severe hepatic failure, lactic acidosis andultimately death.18

Patients with hepatic steatosis are usually detected by elevated serumaminotransferase levels or ultrasonographic fatty liver; however, there is anabsence of a predictable correlation between abnormalities in liver enzymes and

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histologic lesions.19 Standard non-invasive tests such as the fatty liver index,SteatoTest and NashTest are based on a combination of age, body mass index(BMI) and sex combined with plasma biomarkers reflecting alterations inhepatic function such as ALT, aspartate aminotransferase and gamma-glutamyl transferase, which are not directly involved in the initiation and/orprogression of liver disease.20 Thus, liver biopsy remains the gold standard fordiagnosis of hepatic steatosis. MRS and MRI are non-invasive techniques thathave been shown to have high sensitivity in the detection of steatosis,21 buthave been described as being expensive and too complex to be used in theclinical setting.22 Figure 13.1 shows a typical image and spectra used to evaluatehepatic steatosis in mouse liver. Localized spectroscopy sequences such aspoint-resolved spectroscopy (PRESS) or stimulated-echo acquisition mode(STEAM) are used and the spectra are analyzed by comparing the integrals ofthe water and lipid peaks. These techniques have been used in numerousnon-clinical23,24 and clinical25,26 intervention studies where it was important tofollow the relative changes in individuals over time and it may be in this setting

–10123456

(a)

(b)

liver

waterlipid methylene

Figure 13.1 Assessment of hepatic steatosis in mice. (a) Coronal MRI slice through amouse indicating the position of the liver. Image acquired with a 9.4T/20USR Bruker Biospec scanner using a high-resolution respiratory gated3D FISP sequence with flip angle 41, TR/TE 3.3ms/1.7ms, field of view100�45�45mm and matrix size 428�192�192. (b) Localized 1H MRSfrom a 2�2�2mm3 voxel positioned in the livers of two mice with aPRESS sequence, TR 3 s, TE 6.7ms, SW 4006Hz, 64 averages and 2048data points. Bottom: control mouse with a normal fat : water ratio. Top:mouse on a high fat diet showing accumulation of lipid in the liver.Voxels are positioned well away from large blood vessels and fattyinfiltrations.Data provided by Abdel wahad Bidar, AstraZeneca, Sweden.

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that they have their greatest application. There are also a small number ofstudies where MRI/S has been used to monitor hepatic steatosis as a safetybiomarker. In 2004 Zhang et al. used a three-point Dixon method to quantitatefatty liver in rats induced by an experimental microsomal transfer protein(MTP) inhibitor in order to develop a safety biomarker that could be translatedto human studies.27 Cuchel et al. showed that inhibition of MTP by BMS-201038 resulted in a reduction of low density lipoprotein (LDL) cholesterollevels in patients with homozygous familial hypercholesterolemia, and thattherapy was associated with elevated liver aminotransferase levels and hepaticfat accumulation as detected by MRS.28 Visser et al. used MRS to monitor thepotential accumulation of hepatic triglycerides on treatment with the apoli-poprotein B-100 (ApoB) synthesis inhibitor mipomersen in familial hyper-cholesterolemia patients29 and statin intolerant patients.30 Given the difficultyof obtaining serial liver biopsies in clinical trials, MRI and MRS techniques arerapidly becoming the gold standard methodologies to assess hepatic steatosis.

13.2.2 Hepatobiliary Transporter Inhibition

DILI can arise as a result of multiple initiating factors, but a key mechanism isimpairment of bile formation and flow resulting in cholestasis.14 Impaired bileflow results in increased levels of bile acids and bilirubin in blood andsymptoms such as pruritus, jaundice and eventually liver damage. Manypharmaceutical companies have developed in vitro transporter activity assays toidentify candidate drugs that inhibit hepatobiliary transporters and thereforehave the potential to induce cholestatic DILI in humans.31 However, a currentlimitation of the in vitro transporter inhibition assays is that it is unclear howthese data relate to transient or sustained elevations in plasma bile acids andother markers of impaired liver function, which in turn may or may not beindicative of cholestasis and/or hepatocellular damage. If imaging could beused to assess the functional consequence of transporter inhibition in vivo andthus determine the predictivity of the assays and their value in candidateselection, such approaches could be applied to risk assessment and problemsolving studies in non-clinical species. The translational nature of theserelatively non-invasive methods would also facilitate the assessment of hepa-tobiliary transporter activity in the clinic, in order to provide evidence tosupport future monitoring where indicated.

The MR contrast agent gadoxetate (Gd-EOB-DTPA, gadoxetic acid, Eovistor Primovist, Bayer HealthCare) is a clinically approved hepatobiliary-specificagent that is excreted by both liver and kidney. It is used to detect liver tumorsas it is specifically taken up by hepatocytes and not tumor cells.32 Gadoxetateis injected intravenously, transported from the extracellular space into thehepatocytes by the adenosine triphosphate (ATP) dependent organic aniontransporting polypeptide 1 (OATP1/Oatp1 human/rat) and subsequentlyexcreted into the biliary canaliculi by the multidrug resistance associatedprotein 2 (MRP2/Mrp2 human/rat).33 Candidate drugs that inhibit OATP1 orMRP2 will inhibit the uptake or efflux of gadoxetate, respectively.

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The MRI acquisition can be tuned so that image intensity is proportional togadoxetate concentration. Ulloa et al.34 acquired serial in vivo MR images ofrat liver with a time resolution of one minute in order to determine the uptakeof gadoxetate from plasma into the hepatocyte and excretion into the bile(Figure 13.2). The concentration of gadoxetate in the hepatocyte was calculated

Vehicle

200 mg/kg

500 mg/kg

Baseline 5 minpost contrast

30 minpost contrast

55 minpost contrast

(a)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 10 20 30 40 50 60

Vehicle

200 mg/kg

500 mg/kg

Time (min)

Gad

oxet

ate

[mM

]

(b)

Figure 13.2 Uptake of gadoxetate from plasma into the hepatocyte and excretioninto the bile in rats. (a) Examples of dynamic images for rats treated withvehicle (top), 200mg kg�1 (middle) or 500mg kg�1 (bottom) of a hepa-tobiliary transporter inhibitor at t¼ 0, 5, 30 and 55min. after contrastinjection. Note the enhancement of the small bowel lumen 30min. aftercontrast injection in the vehicle treated animal. No enhancement wasobserved in the bowel of the animal treated with 500mgkg�1. Imagesacquired with a 4.7 T/40 Bruker Biospec and IntraGate FLASH TR/TE60ms/1.4ms, flip angle 301, field of view 60�60mm and matrix size256�256. (b) Mean concentration of gadoxetate in hepatocytes. Errorbars are SEM. 6 rats/group.

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from the liver gadoxetate concentration corrected by the extracellulargadoxetate concentration and the volume fraction of extracellular space in theliver. The rate of change of gadoxetate concentration in the variouscompartments was used to model gadoxetate transport kinetics and thereforethe function of the OATP1 and MRP2 hepatobiliary transporters. Theseresearchers showed that dynamic contrast enhanced magnetic resonanceimaging (DCE-MRI) could detect inhibition of gadoxetate uptake and efflux byan investigational chemokine agonist and that results correlated with clinicalchemistry markers of DILI. This imaging technique has the potential toprovide an in vivo biomarker of hepatobiliary transporter inhibition. It offers arelatively non-invasive alternative to bile duct cannulation studies for non-clinical assessment of effects of test compounds on bile flow in vivo, whichenables reduction in animal numbers compared to the bile duct cannulationmodel, especially in longitudinal studies that otherwise can only be assessedusing a sequential design. In addition this technique can be transferred to theclinical setting as gadoxetate is a clinically approved contrast agent.

13.3 Cardiotoxicity

Cardiotoxicity is another leading cause of drug attrition and is therefore a coresubject in non-clinical and clinical safety testing of new drugs.35,36 Themechanisms of cardiac toxicity are not completely understood but may resultfrom interference with processes such as mitochondrial function, intracellularcalcium homeostasis, selective membrane permeability and oxidant/antioxidantbalance. Some drugs can cause irreversible myocardial damage e.g. doxo-rubicin, while others may cause reversible cardiotoxicity resulting in temporaryleft ventricular dysfunction. Cardiotoxicity as a result of cancer therapy hasbeen widely studied as it is often the dose-limiting factor for patients. There aretwo important classes of cardiotoxic chemotherapeutic agents: anthracyclinesand anti-HER2 (human epidermal growth factor receptor 2) directed therapiesincluding trastuzumab, which induce left ventricular dysfunction and heartfailure.37 It is important to detect left ventricular dysfunction as early aspossible in order to stop further treatment, reduce the chemotherapy doseand/or to commence standard medical treatment for heart failure. Cardiacimaging is recommended in all patients with symptoms of cardiotoxicityregardless of the cumulative dose, for asymptomatic patients on trastuzumabevery 3 months while on therapy and for anthracycline treated patients atcumulative doses of 200, 300 and 400mgm�2, and every 50mgm�2 thereafter(doxorubicin equivalents).37

Cardiac imaging can be both a sensitive and specific methodology to measuresmall changes in cardiac function (see also Chapter 18).38 Non-clinical imagingstudies of potential cardiotoxicity can be used both to better inform risk-benefitevaluations before progressing into clinical development and to validate andqualify a safety imaging biomarker for clinical studies, by examining thereproducibility of the technique and the effect size of a compound challenge sothat clinical studies can be appropriately powered. Imaging biomarkers can be

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coupled with non-imaging biomarkers such as plasma levels of cardiactroponins to improve the sensitivity and specificity for cardiac toxicity. Noconsensus regarding threshold values for these biomarkers has been establishedfor evaluating drug-induced cardiotoxicity in non-clinical or clinical settings.

Cardiac MRI is used because it produces comprehensive structural andfunctional images with excellent soft tissue contrast without the need forionizing radiation. This translates into superior interstudy reproducibilityresulting in better reliability of observed changes and reduced patient numbersin clinical trials.39 In contrast to 2D cardiac ultrasound, it does not rely ongeometrical assumptions to calculate volumes and is therefore reliable even inremodeled hearts with complex geometry.40 Three-dimensional echocardi-ography does not rely on geometrical assumptions either; however, there maybe difficulties in full heart coverage for large hearts, and issues with imagequality.41 In addition, cardiac MRI can detect the presence of myocardialfibrosis and/or inflammation.

There are a number of disadvantages in cardiac MRI compared to alter-native techniques such as echocardiography and radionuclide scans and theseinclude a higher cost and the need to move patients to the imaging suite(compared to ultrasound). Contraindications to MRI include claustrophobia,metal implants such as pacemakers, defibrillators, insulin pumps, aneurysmclips, or any other foreign metallic body, and limitation in large body size(e.g. BMI438 kgm�2). There is a requirement for a regular cardiac rhythm forECG gated image acquisition. A specific problem for non-clinical safety studiesis that the MRI scanners may not be co-localized with non-clinical safetyfacilities.

Hockings et al. showed that cardiac output measured non-invasively by MRIin the dog correlated well with invasive techniques and that cardiac MRI coulddetect changes in cardiac function induced by the plasma volume expanderminoxidil.42 Lightfoot et al. showed that doxorubicin-induced gadolinium lateenhancement in the left ventricular myocardium is associated with a subsequentdrop in left ventricular ejection fraction as well as histopathological evidence ofintracellular vacuolization consistent with cardiotoxicity.43 Similar doxorubicininduced impairment of cardiac function and increases in gadolinium lateenhancement has also been observed by Woodhouse et al. (Figure 13.3 andTable 13.1).44 Doxorubicin was also shown to induce decreases in murinecardiac energetics as measured by spatially localized 31P MRS before leftventricular dysfunction became evident in the mouse.45 The phosphocreatine-to-ATP ratio correlated with peak filling rate and ejection fraction, suggesting arelationship between cardiac energetics and both left ventricular systolic anddiastolic dysfunction. The MANTICORE 101 – Breast clinical trial is designedto determine if conventional heart failure pharmacotherapy can preventtrastuzumab-mediated left ventricular remodeling among patients withHER2þ early breast cancer, as measured by the 12-month change in leftventricular end-diastolic volume using cardiac MRI.46 In addition to thesestudies where imaging biomarkers of cardiac disease are used as endpointsin non-clinical and clinical studies, imaging may also be used to aid the

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qualification of novel plasma biomarkers of cardiac damage. Such solublebiomarkers may be used either to predict susceptibility to cardiac toxicity ordetect cardiac toxicity at an early stage in order to halt or modify the treatmentregime.

13.4 Conclusion

Despite the emergence of new approaches to aid the selection of compoundswith reduced toxicity liability and the harmonization of non-clinical safetystudies to support human clinical trials, toxicity remains a major cause ofattrition during drug development and post-marketing. Alongside furtherdevelopments in in silico and in vitro screening, knock-out and humanizedmodels, imaging has the potential to reduce attrition by providing a relatively

(a) (b)

LeftVentricle

RightVentricle

RightAtrium

LeftAtrium

Poor contractility

Dilated left atrium

Figure 13.3 Doxorubicin-induced cardiac toxicity. Typical long axis images from(a) vehicle and (b) doxorubicin treated rats indicating dilated left atrium.Rats were imaged on a Bruker BioSpec 4.7T system using a retro-spectively gated IntraGate FLASH multislice cine sequence TR/TE120ms/1.4ms, flip angle 301, field of view 45�45mm, matrix 192/256,slice thickness 1mm, repetitions 150.

Table 13.1 Effect of 8 weeks doxorubicin or vehicle treatment on cardiacfunction and late gadolinium enhancement in 2 groups of rats(n¼ 6/group).

LVM[g]

EDV[mL]

ESV[mL]

SV[mL]

EF[%]

FS[%]

LGE[%]

Vehicle Mean 0.66 0.34 0.06 0.28 82 65 5995% C.I. 0.07 0.03 0.01 0.03 3 12 7

Doxorubicin(1.25mgkg�1)

Mean 0.70 0.33 0.17 0.16 50 43 9195% C.I. 0.04 0.04 0.05 0.03 13 13 7t-test 0.33 0.85 0.003 0.0002 0.001 0.04 0.001

LVM left ventricular mass; EDV end diastolic volume; ESV end systolic volume; SV stroke volume;EF ejection fraction; FS fractional shortening; LGE late gadolinium enhancement.

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non-invasive means of identifying and managing risk both non-clinically andclinically.

There are two types of safety biomarkers that need to be considered, namelybiomarkers of harm and biomarkers of lack of harm. Biomarkers of harm canbe used, for example, for internal decision making to close a project early or forpatient monitoring. Biomarkers of lack of harm can be used to progressprojects and to provide evidence to regulators that a compound is safe to doseinto man i.e. if no safety signal is seen, or if the margins are sufficient, then thecompound can be dosed into man with confidence. In both cases work is neededto validate and qualify the biomarker before it can be decision making. Forobvious reasons a great deal more work is necessary in order to be confidentthat the absence of a safety signal will result in lack of harm. Good LaboratoryPractice (GLP) and Good Clinical Practice (GCP) ensure that the dataproduced in safety studies are of high quality, reliable and valid. However, it isoften difficult to implement non-clinical MRI studies to GLP as non-clinicalMRI scanners are not currently equipped with software tools that guaranteeconsistent spectrometer operation or data transfer in compliance with GLP,and the burden of GLP documentation makes compliance for innovativeimaging studies impractical. Regulatory agencies do accept investigatorystudies that are not GLP compliant if the work is critical to a scientificallybased risk assessment and has been conducted to an acceptable standard.

With all the potential benefits that imaging can bring to safety studies, it isessential to have a strategic view on which imaging biomarkers to prioritizeboth for incorporation in standard regulatory studies in the future, but also foruse in investigative studies addressing mechanisms and contributing to riskassessment. Furthermore, since developing new biomarkers is a protractedprocess, being reactive (i.e. only promoting biomarker development for aspecific project need) rather than proactive and deliberate increases the oddsthat, in the near term, the optimal set of biomarkers will not be validated andqualified for use in projects. In order to aid the development, validation andregulatory acceptance of imaging biomarkers, cross-company consortia havebeen established. One example is the Health and Environmental SciencesInstitute (HESI), a non-profit institution that brings together scientists fromacademia, government and industry, which has recently established a projectcommittee on the use of imaging in non-clinical safety assessment. Cardiac,brain and liver imaging have been prioritized as key areas of interest and workis ongoing to develop pre-competitive, cross-site validation studies in theseareas. Importantly, this committee has input from the Food and DrugAdministration (FDA), the body that regulates pharmaceutical approval anduse in the USA, and thus it is hoped that by contributing to the development ofimaging methods for risk assessment and management, such methods will bemore readily accepted by regulatory agencies. The true measure of theimportance of investment in developing imaging biomarkers lies in under-standing the potential cost of further unanticipated toxicity being observed inlate stage projects or worse still once a compound has been marketed, balancedagainst the likelihood of investment in this area mitigating against project

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closure or drug withdrawal, or indeed providing project teams with theconfidence to advance a compound.

In summary, MRI and MRS have the potential to provide relativelynon-invasive, robust and reproducible biomarkers to predict and monitorsafety that ‘‘plug the gaps’’ in our current approaches, and have the additionalbenefit of being applicable to both the non-clinical and the clinical settings.Investment in this area can not only underpin decisions to progress acompound either in the non-clinical or the clinical setting by providing a meansof determining mechanisms of toxicity or monitoring for potential safety issues,but can also support the validation of in vitro assays to aid compound selection.Strategic planning and cross-company consortia are essential to exploit thistechnology and ensure that its potential in the safety assessment of phar-maceuticals is fulfilled.

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