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Pharmacology & Therapeutics xx (2007) xxx–xxx

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JPT-05965; No of Pages 15

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Associate editor: Madhani

Potential of p38-MAPK inhibitors in the treatment of ischaemic heart disease

James E. Clark, Negin Sarafraz, Michael S. Marber ⁎

The Cardiovascular Division, Kings College London, The Rayne Institute, St Thomas' Hospital, London, SE1 7EH, United Kingdom

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OAbstract

Chronic heart failure is debilitating, often fatal, expensive to treat and common. In most patients it is a late consequence of myocardialinfarction (MI). The intracellular signals following infarction that lead to diminished contractility, apoptosis, fibrosis and ultimately heart failureare not fully understood but probably involve p38-mitogen activated protein kinases (p38), a family of serine/threonine kinases which, whenactivated, cause cardiomyocyte contractile dysfunction and death. Pharmacological inhibitors of p38 suppress inflammation and are undergoingclinical trials in rheumatoid arthritis, Chrohn's disease, psoriasis and surgery-induced tissue injury. In this review, we discuss the mechanisms,circumstances and consequences of p38 activation in the heart. The purpose is to evaluate p38 inhibition as a potential therapy for ischaemic heartdisease.© 2007 Elsevier Inc. All rights reserved.

EKeywords: p38; MAPK; Inhibitors; Heart failure; Ischaemia; Infarction

⁎ Corresponding aE-mail address:

0163-7258/$ - see fdoi:10.1016/j.pharm

Please cite this artpharmthera.2007.

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Contents

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1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. p38-Mitogen-activated protein kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. Structure and function of p38-MAPK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04. Mechanisms of p38-MAPK activation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4.1. Mitogen-activated protein kinase kinases. . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.2. Autophosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

5. p38, myocardial ischaemia and ischaemic heart disease . . . . . . . . . . . . . . . . . . . . . . . . 06. Pharmacological inhibitors of p38-MAPK. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 07. Clinical trials of p38-MAPK inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 08. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

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UN1. Introduction

Atherosclerotic vascular disease manifests predominantly asheart disease and stroke, which are the most frequent causesof death in the United Kingdom. Collectively atheroscleroticvascular disease was responsible for 40% of total mortality in theUnited Kingdom in 2004. Despite recent reductions in this high

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uthor. Tel.: +44 20 7188 1008.mike.marber@kcl.ac.uk (M.S. Marber).

ront matter © 2007 Elsevier Inc. All rights reserved.thera.2007.06.013

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mortality, morbidity is increasing as more patients survivemyocardial infarction (MI) and stroke. For example, about1.3 million people in the United Kingdom have survived acuteMI, 2 million have angina and 0.9 million have heart failure. Theextraordinarily high impact of atherosclerosis on the Nation'shealth is reflected in its economic cost. Atherosclerotic diseaseof just the coronary arteries costs the United Kingdom healthcaresystem £3500 million with an estimated further £4400 millionlost to the economy through premature death, illness and infor-mal care (The United Kingdom Heart Attack Study (UKHAS)Collaborative Group, 1998; Office for National Statistics, 2000).

e treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.

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Table 1

t1:1 Study Model End point Inhibitor Outcome

t1:2 (Weinbrenneret al., 1997)

Ex vivo: buffer perfused rabbit heart subjectedto global ischaemia–reperfusion

p38 phosphorylation andcell viability (trypan blue)

SB203580 5 μM during ischaemia Inhibition of p38 activation abolishes protection inpreconditioned hearts and cardiomyocytes

In vitro: isolated rabbit cardiomyocytest1:4 (Wang et al., 1998a) In vitro: neonatal rat cardiac myocytes

cotransfected with p38α/β, activeMKK3/MKK6 or dominant negative isoforms

Cell survival, hypertrophicresponse, apoptosis

Modulation of p38α/β usingoverexpression of dominantnegative isoforms orupstream activators

The hypertrophic response in myocytes is mediated byp38β isoform, whereas over expression of p38α resultsin increase cell death

t1:5 (Nagarkatti & Afi, 1998) In vitro: ischaemia in rat myoblast cellline H9C2

Cell viability (MTT) SB203580 15 μM before/duringischaemia

SB203580 administered prior to ischaemia blockspreconditioning, but is protective during prolongedischaemia

t1:6 (Meldrum et al., 1998) Ex vivo: buffer perfused rat heart exposedto H2O2

LV function, coronary flow,CK release and tissue TNF

SB203580 1 mmol/min prior to insult p38 inhibition decreases myocardial TNF production,cardiomyocyte death and dysfunction

t1:7 (Mackay & Mochly-Rosen, 1999)

In vitro: simulated ischaemia in neonatalrat cardiomyocytes

LDH release SB203580 10 μM during ischaemia p38 inhibition, by SB203580, during ischaemia protectsagainst cardiomyocyte apoptosis

t1:8 (Ma et al., 1999) Ex vivo: buffer perfused rat heart subjectedto global ischaemia–reperfusion

LV Function, apoptosis andCK release

SB203580 10 μM before/duringischaemia

Inhibition of p38 attenuates reperfusion injury byreducing apoptosis and improving cardiac function

t1:9 (Craig et al., 2000) In vitro: neonatal rat cardiac myocytescotransfected with MKK6, TNF-α, TAK-1and IL-6

Apoptosis and IL-1 translationand transcription

SB203580 5 μM Activating both p38 and TNF-α augments myocardialsurvival during stress which is inhibited by SB203580

t1:10 (Hoover et al., 2000) In vitro: cardiac myocytes (transfectedwith MKK6) exposed to sorbitol

α-B crystallin expressionand phosphorylation;MAPKAP-K2 andp38 activation and apoptosis

SB203580 5 μM during insult Inhibition of p38 with SB203580 increased sorbitol-mediated apoptosis

t1:11 (Saurin et al., 2000) In vitro: simulated ischaemia in culturedrat neonatal cardiac myocytes

p38 phosphorylation, CKand LDH release andcell viability (MTT)

SB203580 10 μM during ischaemia Inhibition of p38 activation during prolonged ischaemiareduces injury and contributes to preconditioning-induced cardioprotection p38α is phosphorylated duringischaemia whereas p38β is deactivated

t1:12 (Yue et al., 2000) In vitro: simulated ischaemia–reperfusion inrat neonatal cardiomyocytes

LV function and apoptosis(TUNEL)

SB242719 10 μM Inhibition of p38/JNK leads to cardioprotection, whereasinhibition of ERK pathway exacerbates injuryt1:13 SB203580 10 μM

t1:14 (Mackay & Mochly-Rosen, 2000)

In vitro: simulated ischaemia–reperfusion inrat neonatal cardiomyocytes

p38 phosphorylation, apoptosisand LDH release

SB203580 10 μM during ischaemia Incubation with SB203580 during ischaemia–reperfusion attenuates cell death

t1:15 (Barancik et al., 2000) In vivo: regional ischaemia–reperfusion inpig heart

p38 phosphorylation andATF-2 phosphorylationand Infarct size

SB203580 1 μM during ischaemia Inhibition of p38 during ischaemia decrease infarct sizeand delays cell death

t1:16 (Marais et al., 2001) Ex vivo: buffer perfused rat heart subjectedto global ischaemia–reperfusion

p38 phosphorylation, LVfunction and apoptosis

SB203580 1-10 μM duringischaemia/reperfusion

p38 inhibition by SB203580 during ischaemia andreperfusion is cardioprotective

In vitro: neonatal cardiac myocytest1:18 (Gysembergh

et al., 2001)Ex vivo: buffer perfused rabbit heart subjectedto focal ischaemia–reperfusion

p38 activity and infarct size SB203580 1 μM during ischaemia Inhibition of p38 activity during coronary arteryocclusion is cardioprotective

t1:19 (Schneider et al., 2001) Ex vivo: buffer perfused rat heart subjected toglobal ischaemia–reperfusion

LV function and necrosis SB202190 10 μM before ischaemia p38 inhibition reduces ischaemic injury and does notblock protective effect of preconditioning

t1:20 (Martin et al., 2001) In vitro: simulated ischaemia in neonatal/adultrat cardiocytes overexpressing wild-typep38α MAPK

LDH release SB203580 10 μM during ischaemia SB203580 is cardioprotective through inhibition of p38isoform and not due to inhibition or activation of otherkinases

t1:21 (Rakhit et al., 2001) In vitro: simulated ichaemia–reperfusion injuryin neonatal rat cardiomyocytes

p42/44 and p38 MAPKphosphorylation

SB203580 1 μM during ischaemia SB203580 protected against injury, but p38β isoformdoes not contribute to survival

t1:22 (Sanada et al., 2001) In vivo: regional ischaemic preconditioning indog heart

HSP27 phosphorylation,arterial blood pressure,infarct size and collateral flow

SB203580 1 μM during ischaemia/reperfusion

SB203580 treatment during the preischemic andpostischaemic periods had no significant effect oninfarct size

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t1:24 (Tanno et al., 2003) Ex vivo: buffer perfused mouse heart subjectedto global ischaemia–reperfusion (mkk3−/−

and mkk3+/+)

Infarction/risk volume,p38, TAB1 and HSP27phosphorylation

SB203580 1 μM during ischaemia SB203580 sensitive ischaemic activation of p38 byTAB1-associated autophosphorylation contributes tomyocardial injury

In vitro: global ischaemia in H9c2 myoblastsexpressing wild-type and drug-resistant p38α

t1:26 (Sharov et al., 2003) In vitro: cardiomyocytes isolated from dogswith heart failure simulated by hypoxia,Angiotensin-II or nor-epinephrine

Apoptosis and Fas-L cyclinD1 expression

SB203580 10 μM during stress Inhibition of p38 activity attenuated stress-inducedapoptosis and reversed changes in Fas-L and cyclinD1 expression

t1:27 (Clanachan et al., 2003) Ex vivo: buffer perfused rat heart subjected tohypothermia-rewarming and global ischaemia–reperfusion

LV function SB202190 10 μM during rewarming/reperfusion

SB202190, when present during reperfusion, improvesrecovery of LV function Inhibition of p38 did not protectagainst rewarming-induced injury

t1:28 (Otsu et al., 2003) In vivo: regional Ichaemia–reperfusion inp38α+/+ and p38α+/− mice

p38α activity, infarct size andLV function

p38α+/− mice were used toknockdown p38 activity

Activation of p38α during ichaemia–reperfusion isdetrimental; reduction in p38α expression resultsin protection

t1:29 (Gorog et al., 2004) Ex vivo: buffer perfused mouse heart subjectedto low-flow global ischaemia–reperfusion

LV function, Coronary flowand apoptosis

SB203580 1 μM during ischaemia The p38 activation that accompanies short-termhibernation does not appear to contribute to thecontractile deficit

t1:30 (Koike et al., 2004) In vivo: canine heart transplantation from non-heart-beating donors

Cardiac output (CO) andLV function

FR167653 Dose not reported duringcold storage

Inhibition of p38 activation attenuates ischaemia–reperfusion injury in heart transplantation from non-heart-beating donors

t1:31 (See et al., 2004) In vivo: regional ischaemia in rat hearts LV function and postinfarctionremodelling

RWJ-67657 50 mg/day 7 days postMI for 21 days

RWJ-67657 treatment post-MI had beneficial effects onLV remodelling and dysfunction

t1:32 (Yada et al., 2004) In vivo: regional ichaemia–reperfusion inmouse hearts

Protein kinase activation andkinase activity, Nuclear factorκB activity, inflammatorycytokines and infarct size

FR167653 2 mg/kg i.p.before ischaemia

FR167653-mediated inhibition of p38 activity duringregional ichaemia–reperfusion injury reduces infarct size

t1:33 (Kaiser et al., 2004) In vitro: simulated ischaemia–reperfusion inneonatal cardiomyocytes expressing dominant-negative p38

Cell death, apoptosis, DNAfragmentation and Infarct size

Reduction of endogenous p38 usingoverexpression of dominant negativep38 isoform or upstream activation

p38α functions as a prodeath signalling effector in bothcultured myocytes as well as in the intact heart

In vivo: regional ischaemia–reperfusion indominant-negative MKK6 transgenic mice ordominant-negative p38α transgenic mice

t1:35 (Aleshin et al., 2004) Ex vivo: buffer perfused rat heart subjected toglobal ischaemia–reperfusion

TNFα mRNA expression; LVfunction and CK release

FR167653 1.0 mg/kg i.p. before I/R,and 1.0 mg/L during perfusion

FR167653 inhibited ichaemia–reperfusion-mediatedmyocardial TNFα production and p38 activation andimproved functional recovery

t1:36 (Martindale et al., 2005) In vivo: regional ischaemia–reperfusion inhearts from mice overexpressing cardiac-restricted wild type MKK6

LV dimensions (by echo);αB-crystallin expression;DNA fragmentation

p38 activity was modified by cardiacoverexpression of MKK6

Overexpression of MKK6 resulted in less myocardialdamage following ischaemia–reperfusion and enhancedfunctional recovery

t1:37 (Kabir et al., 2005) Ex vivo: buffer perfused mouse heart subjectedto global ischaemia–reperfusion in presence/absence of antimycin A

p38 and HSP27phosphorylation andinfarct size

SB203580 1 μM at the same time asantimycin A

Cardioprotection initiated by antimycin A is dependantupon p38 activation but independent of the upstreamkinase MKK3; however, during lethal ischaemia,inhibition of p38 activity was protective

t1:38 (Wang et al., 2005) Ex vivo: buffer perfused rat heart subjected toglobal ischaemia–reperfusion

p38, activation and cytokineexpression, activation ofcaspases and LV function

SB203580 20 μM before ichaemia Inhibition of p38 activation during ischaemia resulted inless MAPKAPK2, caspase-1, caspase-3 and caspase-11activation, and TNF, IL-1beta, IL-6 production aftermyocardial ichaemia as well as increasing functionalrecovery of the heart

t1:39 (Okada et al., 2005) In vitro: hypoxia-reoxygenation in neonatal ratcardiomyocyte

MAPKAP phosphorylation;Cyt. C release frommitochondria, caspase-3activation and LDH release

SB203580 10 μM throughout theexperiment

SB203580 abrogated activation of p38 MAPK,translocation of HSP27, and F-actin reorganization,prevented cytochrome C release, caspase-3 activation,and DNA fragmentation

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t1:40 Table 1 (continued )

t1:41 Study Model End point Inhibitor Outcome

t1:42 (Sumida et al., 2005) Ex vivo: buffer perfused rat heart subjected toglobal ischaemia–reperfusion

p38 and JNK activities LVcontractility, CK release,mitochondrial ATP generationand infarct size

SB203580 10 μM during reperfusion p38 inhibition exerts cardioprotection only whencontractile force-induced necrosis is prevented

t1:43 (Liu et al., 2005) In vivo: mouse hearts subjected to regionalischaemia–reperfusion

LV function, cardiacremodelling

SC-409 30 mg/kg/day given after MIfor 12 weeks

Inhibition of p38 MAPK attenuates cardiac remodellingand improves cardiac function in mice with heart failureafter infarction

t1:44 (House et al., 2005) Ex vivo: buffer perfused mouse heart subjectedto global low-flow ischaemia–reperfusion

Infarct size and p38 and HSP27phosphorylation

SB203580 2 μM during ischaemia/reperfusion

Inhibition of p38 during ichaemia–reperfusion injuryprotects against myocardial cell death

t1:45 (Li et al., 2005) In vitro: rat neonatal cardiomyocytesoverexpressing MKK6

LV remodelling, cytokinerelease and LV function

SB239068 20 μM and 1200 ppm indrinking water or culture mediumduring exposure

p38 inhibition prevents induction of inflammatorycytokines in cardiomyocytes and extracellularremodelling in heartIn vivo: hearts from MKK6bE transgenic mice

subjected to ischaemia–reperfusiont1:47 (Gupta et al., 2005) In vitro: adult rat ventricular myocytes

(ARVMs) during sepsisCell fractional shortening, cellviability (MTT), caspase-3activity

SB203580 10 μM before insult SB203580 pretreatment followed by bigET-1administration decreased p38 phosphorylation and down-regulated ET(B) receptor expression in sepsis group

t1:48 (Kim et al., 2006) In vitro:Hypoxia–re–oxygination in neonatalrat cardiomyocyte

Cell viability (trypan blue),apoptosis (TUNEL), necrosis,ROS generation

SB203580 1 μM during ischaemia/

reperfusion

Inhibition of p38α prevented hypoxia re–oxygenationinduced apoptosis of cardiomyocytes

t1:49 (Khan et al., 2006) Ex vivo: buffer perfused rat heart subjected toglobal ischaemia–reperfusion

Coronary flow, LV function,LDH release, infarct size andapoptosis (TUNNEL)

SB203580 10 μM before TNF-α Inhibition of p38 MAPK improved cardiac function afterreperfusion and attenuated ischaemic reperfusion-induced myocardial apoptosis and necrosis

t1:50 (Bellahcene et al., 2006) Ex vivo: buffer perfused mouse hearts frommkk3−/− mice subjected to TNF-α

LV function, p38 and HSP27phosphorylation

SB203580 1 μM before ischaemia Activation of p38 contributes to TNF-α inducedcontractile depression in intact heart and in isolatedcardiac myocytes through MKK3; inhibition of p38abolished contractile depression caused by TNFα

t1:51 (Engel et al., 2006) In vivo: regional ischaemia in rat hearts Left ventricular remodelling,fractional shortening andneovascularisation

SB203580 2 mg/kg at time of surgery SB203580 and FGF-1 induces cardiomyocyte mitosis,reduces scarring, and rescues function after MI

t1:52 (Li et al., 2006) In vivo: angiotensin and L-NAME-mediatedcardiac hypertrophy in rats

LV function, arterialinflammatory cell infiltration,and cardiomyocyte apoptosis

SD-282 60 mg/kg administered for4 days during treatment

SD-282, reduces inflammatory response and apoptosis,resulting in a reduction of myocardial damage, which, inturn, improves cardiac function following angiotensin IIand L-NAME treatment

t1:53 (Vahebi et al., 2007) In vitro: isolated skinned cardiac muscle fibrebundles

Cardiac myofilament function,Phosphorylation of α-Tmand TNI

Reduction of endogenous p38 usingoverexpression of dominant negativep38α isoform or upstream activation

Activation of p38α directly depresses saromeric functionby decreased phosphorylation of α-tropomyosin, whichis reversed by inhibition of p38α activity or by overexpressing dominant negative isoform

t1:54 (Riad et al., 2007) In vivo: diabetic mellitus induced by a singleinjection of streptozotocin

LV function, p38phosphorylation and peripheralICAM-1 and VCAM-1

SB239063 40 mg/kg/day for 43 daysafter induction of diabetes mellitus

Inhibition of p38MAPK reduced cardiac cell adhesionmolecules expression indicating both antiinflammatoryand vasculoprotective effects in the diabetic heart

t1:55 (Clark et al., 2007) In vivo: regional ischaemia in hearts frommkk3−/− and mkk3+/+ mice

LV function, LV remodelling,p38 and HSP27phosphorylation

Knockout of MKK3 Postinfarction LV remodelling continues in the absenceof MKK3 as does p38 activation

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Atherosclerotic plaque rupture/erosion results inMI, which ischaracterised by necrosis and apoptosis of cardiomyocytes.Although the acute condition alone may result in death due toventricular arrhythmias or pump failure, in the patients withsubstantial infarction that survive, a chronic phase of ventricularremodelling occurs. Remodelling, is a maladaptive processcharacterised by cardiomyocyte apoptosis, fibrosis, thinning ofthe ventricular wall at the site of infarction, ventricular chamberenlargement and hypertrophy of surviving cardiomyocytes(Pfeffer & Braunwald, 1990; Swynghedauw, 1999; Udelsonet al., 2003). These events may, eventually, lead to heart failurewhich is frequently lethal despite current best care. Therefore,intervention to minimise pathological cardiac remodelling ishighly desirable to reduce the mortality and the incidence andseverity of congestive heart failure after MI.

Various intracellular signalling pathways are thought to playa critical role in the myocardial response to ischaemia andconsequent pathological remodelling. Multiple mitogen-acti-vated protein kinase (MAPK) are activated during ischaemiaand may contribute to the structural and functional changes.MAPK are highly conserved serine/threonine kinases that areactivated by a dual phosphorylation of a Thr-X-Tyr motif, inresponse to wide a variety of stimuli such as cytokines, osmoticand other environmental stresses and consequently play a role innumerous cell functions including growth and proliferation(English et al., 1999; Pearson et al., 2001). Three of the fivemajor MAPK cascades have been extensively studied in theheart: extracellular signal-regulated kinase (ERK1 and ERK2),c-Jun N-terminal kinases (JNK1 and JNK2) and p38 kinases. Ithas been shown that JNK and p38 contribute to, whereas ERK/ERK2 protect against, apoptotic cell death. Although the mech-anisms by which p38 and JNK induce apoptosis may be cell andstimulus specific, there is overwhelming evidence that theactivation of p38-MAPK (or p38) that occurs during prolongedischaemia accelerates injury since its inhibition by pharmaco-logical or genetic means slows the rate of infarction/death(Saurin et al., 2000; Martin et al., 2001; see Table 1). Althoughthis evidence is based on animal data, it seems likely similarmechanisms operate in the human heart since p38 is identicallyactivated by ischaemia (Han et al., 1995; Cain et al., 1999; Cooket al., 1999; Lee et al., 2000; Lemke et al., 2001) and earlyclinical trials indicate a potential benefit (de Winter et al., 2005).Thus, superficially at least, inhibitors of p38 have therapeuticpotential in ischemic heart disease (Force et al., 2004).

2. p38-Mitogen-activated protein kinase

p38-MAPK are activated by a wide range of extracellularinfluences, including radiation, ultraviolet light, heat shock,osmotic stress, proinflammatory cytokines such as interleukin(IL)-1 and tumour necrosis factor (TNF)-α, and certain mitogens(Sugden & Clerk, 1998) in addition to myocardial ischaemia(Bogoyevitch et al., 1996; Saurin et al., 2000; Luss et al., 2000;Ping & Murphy, 2000). Furthermore, the consequent activationof p38-MAPK is intimately involved in multiple cellular re-sponses, including growth, proliferation, differentiation, anddeath (English et al., 1999; Ono & Han, 2000). Perhaps not

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surprisingly these cellular effects have clear consequence(s),translating into involvement in complex pathophysiologies,such as wound healing (Lim et al., 1998), inflammatory arthritis(Badger et al., 1996), sepsis (Kotlyarov et al., 1999), acute res-piratory distress syndrome (Carter et al., 1999), and malignanthypertension (Behr et al., 2001).

Four p38 isoforms (α,β, δ and γ) exist, which have preservedstructure but variable sensitivity to pharmacological inhibition.All 4 isoforms have a Thr180-Gly181-Tyr182 (TGY) dual phos-phorylation motif which is used by investigators to infer acti-vation. p38α and β have high sequence homology and sharesensitivity to pharmacological inhibition by prydinyl imidazolemolecules (such as SB203580) but have only 60% homologywith p38γ and δ, which are resistant to SB203580 (SB) inhi-bition (Eyers et al., 1999). Of the SB-sensitive isoforms, p38α isthe predominant form in human and rodent myocardium (Lemkeet al., 2001; Rakhit et al., 2001; Sanada et al., 2001; Braz et al.,2003). Studies with knockout mice and cells have shown thatp38α is essential for embryonic development as knockout of theα isoform results in embryonal lethality, but mice lacking p38β,p38γ, and p38δ are viable (Allen et al., 2000; Adams et al., 2000;Tamura et al., 2000; Brancho et al., 2003).

3. Structure and function of p38-MAPK

The catalytic site of p38 lies in a pocket between the N- andC-terminal domains. These domains are connected by a singlehinge and the L16 loop of the C-terminal domain which wrapsback around the N-terminal domain and controls the relationshipbetween the relatively rigid domains (see Fig. 2). In addition, inthe inhibitor bound nonphosphorylated state, there is a mis-alignment between the N- and C-lobes which prevents thecooperation between a lysine residue (Lys53) in the N-terminallobe and aspartic acid residue (Asp168) in the C-terminal lobe,imperative to binding and stabilization of the α phosphate groupand adjacent ribose of ATP, respectively (Wilson et al., 1996;Gum et al., 1998). Therefore, it is widely thought that thenondual phospho- form of p38 is inactive as a result of stericobstruction of the peptide-binding channel and low ATP affinity.

Thr180 and Tyr182 are located on a flexible “activation loop”that guards the active site. Dual phosphorylation of these 2amino acids in response to exotoxin, cytokines, physical stress(such as hyperosmolarity), and chemical oxidant stress, such ashydrogen peroxide (Han et al., 1994; Freshney et al., 1994;Rouse et al., 1994; Raingeaud et al., 1995) is thought to cause theactivation loop to refold and move out of the peptide-bindingchannel. This movement is then thought to exert a “crank-handle” effect on the overall tertiary structure of the kinasereorienting the N- and C-, terminal lobes so that Lys53 andAsp168 move towards one another by 2.5–5 Å. This alters theconformation of the catalytic site enabling the cooperationnecessary for ATP binding and allowing substrate access(Wilson et al., 1996; Diskin et al., 2007). The docking groovesused by substrates and activators consist of 2 regions (see Fig. 2),the CD region and the ED region (Tanoue et al., 2000). The CDregion is part of a shallow groove formed by the acidic residuesAsp313, Asp316, Glu81, and the aromatic residues Phe129 and

e treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.

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Tyr311. The ED region is part of a deeper groove formed byresidues 159–163 at one side and residues Gln120, His126 andPhe129 at the opposite side (Haar et al., 2007). It is believed thatthese 2 binding regions facilitate activator (MAPK kinase 3,MKK3) and substrate (MK2 and MEF2) binding (Chang et al.,2002; Haar et al., 2007).

4. Mechanisms of p38-MAPK activation

4.1. Mitogen-activated protein kinase kinases

Although the intracellular activation cascade for p38 undermost physiological conditions is still unclear, several upstreamMAPK kinases (MKK) have been identified from in vitroanalysis, including MKK3 and MKK6 (Derijard et al., 1995;Han et al., 1996). MKK4 is predominately involved in JNKactivation but is able to activate p38-MAPK, at least, in vitro(Deacon & Blank, 1997). Using MKK-targeted mouse lines, it

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Fig. 1. Mechanisms of p38-MAPK activation. Classical activation by MKK3/MKmechanism ②. TCR-mediated Tyr323 phorphorylation by ZAP70 is depicted as maddition, TAB1 is a p38 substrate. PhosphoTAB1 is less able to activate TAK1 (★).and phosphoTAB-1. Heavy lines represent an interaction; dotted lines represent a m

Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in thpharmthera.2007.06.013

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1has been shown that, in response to most stress stimuli, MKK31and MKK6 are the principal MKK activating p38α and β,1respectively (see Fig. 1). MKK3 and MKK6 are in turn acti-1vated by phosphorylation by a MKK kinase (MKKK). The1MKKK, being responsible for activation of the p38 cascade,1appears to be cell type and stimulus specific, and several have1been implicated (Yamaguchi et al., 1995; Moriguchi et al.,11996; Ichijo et al., 1997; Hutchison et al., 1998; Gallo &1Johnson, 2002; Ge et al., 2002; Cheung et al., 2003).1However, p38 activation is not limited to this traditional1phospho-relay signalling cascade. Since SB203508 (the most1widely used p38 kinase inhibitor) occupies the catalytic site,1without inhibiting upstream MKK, it should only inhibit the1phosphorylation events downstream of p38 without inhibiting1the dual-phosphorylation of p38 itself (Young et al., 1997).1However, certain conditions, such as myocardial ischaemia,1cause a SB-sensitive form of p38 dual phosphorylation. Two1mutually exclusive explanations for these observations are

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K6 is depicted as mechanism ①. TAB1-induced autoactivation is depicted asechanism ③. TAB1 activates TAK1, which in turn activates MKK3/MKK6. InPharmacological inhibition of p38-MAPK diminishes p38 dual phosphorylationodification (phosphorylation); open arrows represent a multi-element pathway.

e treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.

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(i) that p38 is able to autophosphorylate its activation loop or(ii) that SB203580 inhibits a kinase upstream of p38 involved inits activation by trans-phosphorylation during ischaemia.

4.2. Autophosphorylation

Ge and co-workers elegantly reported that auto-phosphory-lation of p38 can occur, facilitated by an interaction with the non-enzymatic adaptor protein transforming growth factor-β-activated protein kinase-1 (TAK1) binding protein-1 (TAB1;Ge et al., 2002). TAB1 is known to perform a similar function byinducing the autophosphorylation of TAK1, which in turnactivates MKK3/MKK6. In vitro co-expression experimentshave shown that the interaction of TAB1 and p38α leads tophosphorylation of the TGY activation motif. TAB1-dependentp38α activation appears to play a role in the injury responseduring myocardial ischaemia (Tanno et al., 2003; Fiedler et al.,2006), myocyte-derived dendritic cell maturation (Matsuyamaet al., 2003), and peripheral T-cell anergymaintenance (Ohkusu-Tsukada et al., 2005). The interpretation of the TAB1-p38interaction was, however, complicated by Cohen's group whodemonstrated that the phosphorylation of TAB1 on Ser423 andTyr431 was p38-MAPK-dependent and hence prevented bySB203580. The authors proposed a feedback control mechanismof TAK1 activity, whereby p38 activity inhibits TAK1, throughthe phosphorylation of TAB1. Inhibition of p38 activity (bySB203580) abolishes this feedback control of TAK1, causingunopposed activation of the parallel JNK pathway andconsequently IKK (Cheung et al., 2003). This is depicted inFig. 1. Although TAB1 andMKK3/MKK6 mechanisms interactthrough the potential modulation of TAK1, other possibilitiesalso exist. For example there is some evidence to suggest thatTAB1 causes p38 redistribution to the cytoplasm and mayrestrict access to downstream targets, such as MAPK-activatedprotein kinase 2 (MAPKAPK2). This is in direct contrast to thepattern seen with MKK3/MKK6 (Lu et al., 2005).

Using MKK3/MKK6 double knockout and MKK4/MKK7double knock out mouse embryonic fibroblasts (MEF), Kanget al. have shown that peroxynitrite-induced phosphorylation ofp38α is associated with an ∼85 kDa disulfide complex in wildtype MEF (Kang et al., 2006). This association was diminishedin MKK3/MKK6 knockout MEF (Kang et al., 2006). Theauthors suggested that phosphorylation of p38 mediated byTAB-1 can be modulated by a yet unknown binding partner(s) ina manner dependent on a disulfide complex (Kang et al., 2006).

In addition to TAB-1-mediated activation, p38 can alsoautophosphorylate through an alternative pathway in responseto T-cell antigen receptor (TCR) activation (Dong et al., 2002;Rincon & Pedraza-Alva, 2003). In this pathway, activation ofTCR leads to recruitment of a Syk family kinase, ZAP-70,which directly phosphorylates p38 on Tyr323 (Rouse et al.,1994). In a recent study it was shown that the phosphorylationof p38 on Try323 can be blocked in the presence of the DNAdamage inducible gene Gadd45a, an autoimmune suppressor.Absence of Gadd45a has been shown to result in chronicphosphorylation of p38, T-cell hyperproliferation and autoim-munity (Salvador et al., 2005a, 2005b). Following ZAP-70

Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in thpharmthera.2007.06.013

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phosphorylation of p38, an autophosphorylation event similarto that induced by interaction with TAB-1 occurs resulting indual phosphorylation, and activation of the kinase.

However, regulation of p38 kinase activity in vitro, at least isnot solely dependent on upstream kinases and binding partners.Diskin and co-workers, using a in vitro mutation approach, madeintrinsically active p38 isoforms based on activating mutationspreviously found in the yeast MAPK kinase p38/Hog1 (Bellet al., 2001). Single andmultiple point mutations of human p38αresulted in high intrinsic activity independent of activation bydual phosphorylation. Structural analysis of these p38 mutantshas identified a hydrophobic core stabilised by 3 aromaticresidues, Tyr69, Phe327 and Trp337, in the vicinity of the L16Loop region. It is believed that the hydrophobic core is an in-herent stabiliser that maintains the low basal activity level ofunphosphorylated p38 (Diskin et al., 2004). Upon activation,however, a segment of the L16 Loop, including Phe327 becomesdisordered allowing ATP and substrate binding. The mutation ofthese amino acids involved in the hydrophobic core results in theconformational changes imposed naturally by dual phosphory-lation, namely destabilising the hydrophobic core and lockingthe kinase in a constitutively active state. In addition, in thisactive state, p38 is able to autophosphorylate in an invitro kinaseassay (Diskin et al., 2004). More recently p38β, p38γ and p38δmutants were similarly constructed (Askari et al., 2007). In thesemutants, a highly conserved aspartic acid located in the acti-vation loop (Asp170 in Hog-1; Asp176 in p38α, p38β and p38δ;and Asp179 in p38) was mutated. The spontaneous kinaseactivity of p38β, p38γ and p38δ appeared to be lower than thedual phosphorylated wild-type isoforms whereas the p38αisoform presented the highest spontaneous activity. Therefore, itis apparent that modifications of the amino acids in the hydro-phobic core along with the mutations in Asp176 are capable ofactivating p38s (Askari et al., 2007), the former likely explainingthe mechanism by which ZAP-70 induces autoactivation(Mittelstadt et al., 2005). In addition, these mutants provide atool to dissect isoform-specific downstream signalling (Askariet al., 2007).

5. p38, myocardial ischaemia and ischaemic heart disease

Ischaemic heart disease remains the leading cause of death,accounting for approximately 1 quarter of all deaths in theUnited Kingdom. Currently, the most effective method ofreducing mortality in such patients is to achieve rapidreperfusion by lysis or mechanical disruption of the occlusivecoronary thrombus and plaque. The mortality from acute MIunder these circumstances is inversely related to the amount ofmyocardial salvage achieved by reperfusion.

There is increasing evidence from preclinical investigationsthat inhibition of p38 during prolonged ischaemia slows the rateof infarction/death and inhibits the production of inflammatorycytokines, such as TNF-α, IL-1 and IL-8, which aggravateischaemic injury (Young et al., 1997; see Table 1 for summary).It was fist demonstrated as early as 1996 that p38α and βare activated in response to ischaemia and reperfusion in theheart (Bogoyevitch et al., 1996). Since then, using gene transfer

e treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.

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techniques, the α isoform that has been implicated in myocyteapoptosis, consistent with the findings that this isoform alonecontributes to cell death following ischaemia (Saurin et al.,2000; Martin et al., 2001). p38s phosphorylate a number ofknown transcription factors to alter their transactivating po-tential influencing gene expression. However, the immediatedownstream targets of p38 that aggravate myocardial injuryare still largely unknown. One downstream substrate of p38αis MAPKAP2, which can, in turn, phosphorylate HSP27, aheat shock protein, which is thought to confer a number of pro-tective effects (Kim et al., 2005). In addition, phosphorylation ofMAPKAP2 can also result in phosphorylation of factors thattransactivate cytokine genes, such as TNF-α, a cytokine im-plicated in chronic heart failure. Interestingly, TNF-α alsoactivates p38 and thus p38 has been considered as the keystonein an autoamplifying cytokine cascade by most investigators andan attractive target for antiinflammatory drug development (Leeet al., 2000; Kuma et al., 2005).

Furthermore, a proapoptotic role for p38α and/or p38βduring myocardial ischaemia is suggested by protection of car-diac myocytes from ischaemic damage using a selective p38α/p38β isoform inhibitor, SB203580 (Wang et al., 1998a). Usingadenoviral-mediated expression of p38α and p38β in rat neo-natal cardiomyocytes our group have previously shown that after2.5 hr simulated ischaemia p38α was activated, whereas p38βactivation was significantly inhibited (Saurin et al., 2000).Inhibition of p38α activation during prolonged ischaemia, butnotβ, resulted in an increase in cell viability (Saurin et al., 2000).This strongly supported Wang et al. who suggested that p38αactivation in cardiac myocytes is sufficient to cause apoptosiswhereas activation of the β isoforms leads to protection andhypertrophy (Wang et al., 1998a). However, there is some evi-dence to suggest that p38 isoforms may have potential protectivefunction and suggest a possible adverse effect of prolonged p38inhibition in the heart. Glembotski's laboratory have demon-strated chronic activation of p38 through overexpression ofMKK6 in the heart can result in improved functional recoveryfrom ischaemia and MI (Martindale et al., 2005). The protectiverole of p38β has also been investigated in a recent study by Kimand co-workers who have shown that activation of p38β bycarbon monoxide promotes the nuclear translocation of heatshock factor-1 (HSF-1), which regulates the expression ofcytoprotective HSP70 in cells and tissues (Kim et al., 2005).HSF-1 can also serve as a negative regulator of proinflammatorygenes, including IL-1β, and TNF-α (Xie et al., 2002). The roleof p38s (the α isoform predominantly) in myocardial ischaemicinjury has been studied extensively since the findings of (Wanget al. 1998a). These studies, which in the main suggest p38activation during ischaemia worsens injury and depresses LVfunction, are too numerous to review and appear in Table 1.

There is little information in the literature regarding the rolesof either the γ and δ isoform of p38 in the myocardium duringischaemia. Conserved cardiac expression of p38γ amongst sev-eral different species suggests that this isoform may play animportant role in the heart and therefore is unlikely to be func-tionally redundant (Court NW et al., 2002). p38γ is localised inthe cytoplasm of the cardiac myocyte and is reported to have a

Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in thpharmthera.2007.06.013

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3punctate distribution (Court NW et al., 2002), and p38δ mRNA3is broadly expressed in a wide variety of mouse and human3tissues including the heart (Wang et al., 1997; Beardmore et al.,32005). The C-terminal tail of p38γ allows its interaction with3PDZ domains of its substrate protein(s) and association with3α1-syntrophin and SAP90/PSD95 in skeletal muscle and3neuronal synapses, respectively (Zhang et al., 2003; Hasegawa3& Cahill, 2004; Sabio et al., 2004). p38γ-catalysed phosphory-3lation of hDlg (the mammalian homologue of the Drosophila3tumour suppressor Dlg) triggers its dissociation from the3cytoskeleton, indicating that this may regulate the integrity of3intercellular-junctional complexes, cell shape, volume and cell3polarity in response to many kinds of external stimuli. In support3of these findings, Parker et al. identified a novel p38δ substrate as3stathmin, a cytoplasmic protein that was previously reported to be3a substrate of several intercellular signalling kinases which have3been linked to regulation of microtubule (MT) dynamics in a3phosphorylation-dependent manner (Belmont & Mitchison,31996). This may suggest that a common theme in p38 pathway3activation may be the re-organisation of the cytoskeletal frame-3work to enhance cell survival in times of stress such as ischaemia3(Parker et al., 1998). Moreover, both p38γ and p38δ phos-3phorylate the MT-associated protein Tau in neurons in vivo.3Hyperphosphorylated Tau is the major component of the paired3helical filaments, which constitute one of the main neuropatho-3logical hallmarks of many neurodegenerative disorders (Sabio3et al., 2005). So, although there is only circumstantial evidence to3support a role for γ and δ isoforms in the heart, this is likely an3emerging area of research. In the absence of isoform-selective3pharmacological inhibitors, this is, to some extent, aided by the3availability of a number of p38 isoform-targeted mouse lines3(Beardmore et al., 2005; Sabio et al., 2005) and spontaneously3active mutants (Askari et al., 2007).

36. Pharmacological inhibitors of p38-MAPK

3Early efforts in drug discovery of small molecule inhibitors of3kinases were met with scepticism that selectivity could ever be3accomplished, due to the high degree of structural similarity in3the adenosine binding pocket among the entire kinome. Thus, it3was somewhat of a surprise when SB203580, the first reported3p38 inhibitor, emerged showing selectivity over the closely3related JNK and ERKMAPK families (Lantos et al., 1984). The3pyridinyl imidazole antiinflammatory agents were soon shown3to be highly selective p38 inhibitors and the bi-cyclic pyridinyl4imidazole SKF-86002 was the first compound reported to inhibit4LPS-stimulated cytokine production (Lee et al., 1988, 1994). It4was not long before investigators explored dual 5-lipooxygen-4ase/cyclooxygenase (LO/COX) and cytokine inhibition as4potential mechanisms for the potent anti-inflammatory activity4of these compounds (Lee et al., 1993), and subsequently,4SB203580 was used as a pharmacological inhibitor to study the4cascade of kinases (via p38) involved in cytokine production4(Gallagher et al., 1997). The structures of representative classes4of p38 inhibitors are shown in Fig. 3.4The crystal structures of pyridinyl imidazole-p38α complexes4have recently become available and suggest that SB203580 binds

e treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.

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to the active site of both phosphorylated (active) and unpho-sphorylated (inactive) p38 in an ATP-competitive manner (seeFigs. 2 and 3). These inhibitors bind to an aryl-specificity pocketbehind the site, which is normally occupied by the adenine ring ofATP. The interaction occurs between the 4-pyridinyl group(analogous to the N-1 adenine of ATP) and the N-H of Met109. Inaddition, studies have also have implicated Thr106 as a key residueconferring selectivity (Wilson et al., 1996; Gum et al., 1998). The2 adjacent residues, His107 and Leu108, alongwith Thr106 lie at theback of the ATP pocket and are identical in p38α and p38β, butare different in p38γ and p38δ (Met106, Pro107 and Phe108, re-spectively) which are insensitive to SB203580 inhibition. Using amutagenesis approach it has been shown that if these 3 residues inp38α and p38β are changed to Met-Pro-Phe (as found in p38γand p38δ) the mutant kinase is no longer inhibited by SB203580.By contrast, introduction of the Thr-His-Leu sequence of p38αinto p38γ or p38δ confers sensitivity to SB203580. Takentogether, these studies have identified Thr106 as the key residueforming the aryl-specificity pocket (Saccani et al., 2002).

In addition to novel prydinyl imidazole compounds, a newgroup of selective p38 inhibitors are the arly-pyridinyle-hetero-cylces. In these compounds, the imidazole core is replaced byother heteroaryl scaffolding, and consequently these compoundsgenerally exhibit in vitro potency similar to prydinyl imidazoles(Boehm et al., 2001). Other structurally diverse p38 inhibitorsinclude a subset of novel non-aryl-pyridinyls such as triaza-napthalenones, N,N′-diary ureas, benzzophenones, pyrazole

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Fig. 2. Crystal structure of p38 with SB203580 occupying the ATP binding site.The Thr106 residue (4), which is important for binding of pyridinyl imidazoleinhibitors, and the 2 residues within the activation loop that are phosphorylated(Thr180 (2) and Try182 (1)) are highlighted. Tyr323 (3), which has been implicatedin TCR-mediated activation of p38 is also shown. SB203580 is shown in green.The activation loop is shown in orange. ED and CD activator/substrate bindingregions are highlighted. The C-terminal extension that forms the L16 loopbridging the domains is also indicated (created in JenaLib Jmol; PDB entry1a9u; Wang et al., 1998b).

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ketones, indole amides, diamides, quinazolinones, and pyridy-laino-quinazolines (Cirillo et al., 2002). Unlike the imidazole-based p38α inhibitors, the urea-containing inhibitors act in anoncompetitive manner (Kulkarni et al., 2007). Crystallographicstudies of urea-containing p38α inhibitors, such as BIRB-796,have revealed that these compounds bind to at a site remote fromATP pocket, and induce a significant movement of Phe169, suchthat this residue fills the ATP pocket, preventing ATP binding(Pargellis et al., 2002). Thus, inhibitors of p38 can be dividedinto 2 groups dependent upon their mode of binding to p38; theseare (i) active site or “gatekeeping” inhibitors (such as SB203580)and (ii) those which bind remotely and interfere with ATP bind-ing indirectly (such as BIRB-796).

Enthusiasm for “blanket” pharmacological inhibition of p38is tempered by the fact that this kinase is involved in innu-merable biological processes and therefore not surprising thatunder many circumstances its activation leads to myocardialprotection rather than injury (Weinbrenner et al., 1997; Craiget al., 2000; Hoover et al., 2000; Communal et al., 2000; Forceet al., 2004; Zheng & Zuo, 2004; Martindale et al., 2005). Thisparticularly seems to be the case when p38 activation occurs as aconsequence of an intervention that precedes lethal myocardialischaemia, such as ischaemic or pharmacological precondition-ing (Nagarkatti & Afi, 1998; Marais et al., 2001; Sanada et al.,2001). However, in these studies the same inhibitor, at the sameconcentration, reduces injury if present solely during lethalischaemic injury (Nagarkatti & Afi, 1998; Marais et al., 2001;Sanada et al., 2001; Tanno et al., 2003). The cause of this ap-parent paradoxical observation may relate to an attenuation ofp38 activation during lethal ischaemia by its prior transientactivation. Thus there is ample evidence from cardiac as well asother research fields that p38 activation can have beneficialconsequences whilst it is also incontrovertible that restrictingp38 inhibition to the activation that accompanies lethalmyocardial ischaemia reduces infarction (Nagarkatti & Afi,1998; Marais et al., 2001; Sanada et al., 2001; Marais et al.,2005; Table 1).

However, if we are to consider currently available p38inhibitors with the aim of treating chronic conditions, it is likelygreater levels of selectivity will be required to avoid the inhi-bition of beneficial forms of activation. As more kinases areinvestigated, a better understanding of selectivity over thekinome has followed. With expanded kinase panels, it has nowemerged that “classic” p38α inhibitors like SB203580, whichwere once described as being selective over certain kinases, nowhave been shown to have similar, and sometimes lower IC50s.These actions are a result of similarities in the inhibitory bindingsite or in particular the hydrophobic pocket equivalent to thatformed by Thr106 (Fabian et al., 2005). So if broad inhibition ofp38 is not the answer, what is? At present, the majority ofpharmacological inhibitors of p38 are selective for the α and βisoforms of the kinase. It is clear from the published data that theduring prolonged ischaemia the α isoform plays an importantrole in the progression of dysfunction. Perhaps a more rationalapproach to inhibit p38 in a site- and condition-specific mannermight be to target the activation of the kinase pathway upstreamof p38 itself, such as TAB1 or MKK3/MKK6. Using a model of

e treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.

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Fig. 3. Structure of representative classes of p38 MAPK inhibitors. p38 inhibitors can be divided into 2 groups dependant upon their mode of binding to p38; active siteinhibitors, such as SB203580 and RJW-67657, bind competitively to the ATP site of the enzyme whereas others bind remotely and interfere with ATP bindingindirectly (such as BIRB-796).

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coronary artery ligation in a mkk3-targetted mouse line, we haverecently demonstrated that removing MKK3 does not alterpathological remodelling and progression to ventricular dys-

Please cite this article as: Clark, J. E. et al. Potential of p38-MAPK inhibitors in thpharmthera.2007.06.013

4function after MI (Clark et al., 2007). Maybe this is not sur-5prising considering the multitude of pathways involved in5ischaemia and inflammation but it does, perhaps, highlight the

e treatment of ischaemic heart disease. Pharmacol Ther (2007), doi:10.1016/j.

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potential importance of other pathways which warrant furtherinvestigation.

7. Clinical trials of p38-MAPK inhibition

Despite the apparent dichotomy in preclinical research of theconsequences of p38 inhibition, 2 types of molecular inhibitorsof p38, aryl-prydinyl heterocycles (namely SB242235 and RWJ-67657) and non-aryl-prydinyl heterocycles (VX-745, BIRB-796and RO3201195), have so far advanced to clinical trials. Resultsof phase I and early phase II trials were also promising. In onestudy, orally administered SB242235 (1–500 mg), which hasbeen shown to have potent antiinflammatory effects in a ratmodel of arthritis (Badger et al., 2000), was well tolerated andsuppressed production of TNF-α, IL-1β, IL-6, and IL-8 in adose-dependent manner within 3 hr (Adams et al., 2001). Arandomised clinical study to examine the efficacy of RWJ-67657to combat the effects of endotoxin on normal healthy volunteerwas recently carried out. Development of flu-like symptoms,which were associated with raised serum levels of TNF-α, IL-6and IL-8, were reduced in a dose dependent manner by RWJ-67657 (Fijen et al., 2001).

However, clinical studies have not been limited to healthycontrols; VX-745 (Vertex) has been given to patients with activerheumatoid arthritis (Haddad, 2001; Weisman, 2002), and al-though the drug is well tolerated, it was associated with adverseeffects, such as elevation in liver transaminases. Preclinical safetyevaluations of this drug in animals have revealed that at thatconcentrations VX-745 can cross the blood–brain barrier andexert adverse neurological side effects (Weisman, 2002). For thesereasons, further investigation onVX-745 has been suspended. Therise in the level of liver transaminases has also been observed in aseries of double-blinded, randomised, placebo-controlled studiesof BIRB-796 in healthy volunteers. This compound also sup-pressed neutrophil activation ex vivo, but no inhibition of LPS-induced TNF-α production was observed (Wood et al., 2002).

Currently, the most clinically advanced p38 inhibitors are theScios compound (now Johnson & Johnson) SCIO-323 for treat-ment of stroke and the Vertex compound VX-702, which hasbeen tested in patients with acute coronary syndromes in whompercutaneous coronary intervention (PCI) is planned. Therewere no adverse side effects and the serum level of C-reactiveprotein (CRP), which is considered a risk factor in MI patients,was suppressed (de Winter et al., 2005). However, althoughmany p38 inhibitors have advanced to phase I, II or III clinicaltrial, many of these studies have been stopped prematurely dueto adverse side effects. One reason for this might be the cross-reactivity against other kinases or other cellular signalling mole-cules. An alternative explanation is that p38-dependant sig-nalling is vital to normal cell function necessitating a greaterunderstanding of mechanisms of activation in the hope that thiswill reveal circumstance-specific targets.

8. Summary and conclusions

The p38 kinase pathway has been studied intensely since itsdiscovery in the early 1990s. It has been implicated in various

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biological processes, such as cell growth, apoptosis and inflam-mation. In the heart p38 is activated by various pathologicalconditions, such as ischaemia and pressure overload. Studiesusing genetic and pharmacological inhibitors of p38 suggeststhat this kinase plays a key role in left ventricular matrix re-modelling (Petrich & Wang, 2004), cell survival followingischaemia–reperfusion (Ma et al., 1999; Shao et al., 2006) andpossibly, in post MI remodelling (Ren et al., 2005). Investigatorshave shown that multiple isoforms of p38 are activated inresponse to specific stimuli and take part in distinct signallingpathways, which result in activation of specific downstreamsubstrates. From the existing evidence, it appears that p38α andp38β are differentially regulated during myocardial stresses andthat the consequences of activation of each isoformmay differ bycell type. This highlights the likelihood that different memberswithin a single kinase family can play distinct roles in the heartduring ischaemia. Despite continued interest in the p38 pathwayfew studies to date have addressed the role of p38 isoforms otherthan p38α during ischaemia. Furthermore, since there are noisoform-specific pharmacological inhibitors of p38 activity, thecontribution of each isoform remains unclear.

Understanding the physiological roles of each p38 isoformand identifying their mechanism(s) of activation and potentialsubstrates are important avenues that may lead to pharmaco-logical inhibitors with greater circumstance selectivity therebyavoiding the potential pitfalls of chronic systemic inhibition.

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