European Journal of Pharmacology 601 (2008) 88–93

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European Journal of Pharmacology

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Neuropharmacology and Analgesia

The acute and chronic phases of chronic relapsing experimental autoimmuneencephalomyelitis (CR EAE) are ameliorated by the peroxynitrite decompositioncatalyst, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinatoiron(III) chloride, (FeTPPS)

Christopher Bolton ⁎, Gwen S. Scott, Terence Smith, Roderick J. FlowerCentre for Biochemical Pharmacology, John Vane Science Centre, The William Harvey Research Institute, St. Bartholomew's, The London and Queen Mary's School of Medicine and Dentistry,Charterhouse Square, London, EC1M 6BQ, UK. NeuMatRx, Truro, Cornwall TR3 6NT, UK

⁎ Corresponding author. Tel.: +44 20 7882 6081; fax:E-mail address: c.bolton@qmul.ac.uk (C. Bolton).

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.10.029

a b s t r a c t

a r t i c l e i n f o

Article history:

There is increasing evidence Received 6 May 2008Received in revised form 28 September 2008Accepted 13 October 2008Available online 22 October 2008

Keywords:Experimental autoimmune encephalomyelitisMetalloporphyrinPeroxynitriteFeTPPSNeuroinflammation

that the oxidative radical, peroxynitrite (ONOO−), is involved in the pathogenesisof inflammatory diseases including multiple sclerosis and the animal counterpart, experimental autoimmuneencephalomyelitis (EAE). Compounds that impede the actions of ONOO− have proved useful in the control ofEAE. In particular, catalytic isomerisation of ONOO− to inactive nitrate, through the use of metalloporphyrins,curtails the cellular response to inflammatory stimuli and halts the progression of neuroinflammation duringEAE. The present study examined the pharmacological effects of the metalloporphyrin and ONOO−

decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinatoiron(III)chloride (FeTPPS) on theacute and relapse phases of chronic relapsing (CR) EAE. Administration of FeTPPS to CR EAE-inoculated Biozzimice commenced either therapeutically and immediately prior to the emergence of acute or relapsesymptoms, or prophylactically, from the onset of remission of acute neurological signs. Drug therapy reducedacute and relapse symptoms but, and in contrast to the former phase, was of limited benefit in preventinghistological changes during the latter stage of disease. In contrast, prophylactic FeTPPS was effective inlimiting CNS pathology and neurological deficits. The findings confirm the inhibitory effects of FeTPPS onacute stage EAE. Moreover, the study extends previous observations by verifying compound efficacy onrelapsing disease. Use of metalloporphyrins, such as FeTPPS, again highlights the important role played byONOO− in the development of inflammatory diseases such as EAE.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

The potent oxidative molecule peroxynitrite (ONOO−) acts as animportant inflammatory mediator in a variety of pathological condi-tions including ischemia, meningitis and lung dysfunction (Szabo,1996; Kastenbauer et al., 1999; Szabo et al., 2002; Kim et al., 2005;Salom et al., 2007). In particular, there is substantial evidence thatONOO− has a role in the pathogenesis of central nervous system (CNS)disorders, including multiple sclerosis and the animal counterpart,experimental allergic encephalomyelitis (EAE) (Cross et al., 1997; Vander Veen et al., 1997; Cross et al., 1998; Hooper et al., 1998; Torreilleset al., 1999; Hooper et al., 2000, 2001; Phares et al., 2007). Moreover,many studies have demonstrated the presence of nitrotyrosineresidues, a marker of ONOO− production, in CNS tissues frommultiplesclerosis patients and animals with EAE, (Cross et al., 1997; Van der

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Veen et al., 1997; Cross et al., 1998; Oleszak et al., 1998; Hooper et al.,2000; Liu et al., 2001; Calabrese et al., 2002) indicating that themolecule is actively involved in the disease process.

Several studies have confirmed ONOO− generation by cells andtissues within the multiple sclerosis- and EAE-diseased CNS andconsidered the contribution made by the molecule to the pathogen-esis of the diseases. (Cross et al., 1997; Van der Veen et al., 1997; Crosset al., 1998; Ischiropoulos et al., 1992; Pryor and Squadrito, 1995). Forexample, ONOO− production activates a variety of potentiallydeleterious biochemical pathways leading to oxidation, lipid perox-idation, nitration of protein tyrosine residues, DNA strand breakageand activation of poly (ADP-ribose) polymerase (Szabo, 1996; Groves,1999). Moreover, several in vitro studies have strongly implied ONOO−

induces damage to neurons, oligodendrocytes and the neuroendothe-lium (Beckman et al., 1990; Mitrovic et al., 1994; Scott et al., 2003,2004; Knepler et al., 2001; Jack et al., 2007) which represent keytargets for attack in the pathogenesis of EAE and multiple sclerosis.

Pharmacological intervention to specifically or indirectly limitthe disruptive effects of ONOO− has proved beneficial in the controlof EAE and promotion of recovery from the disease. For example,

Fig. 1. Effect of therapeutic administration of FeTPPS on neurological scores during theacute phase of CR EAE. Male Biozzi mice, inoculated for CR EAE, were dosed i.p., twicedaily, with either vehicle or FeTPPS at 10 mg/kg body weight or 20 mg/kg body weightfor 5 days from acute disease-associated body weight loss, 15–18 days post-inoculation,prior to the onset of neurological deficits. Animals were sacrificed, for the removal ofspinal columns, 21–24 days post-inoculation. Results are from two combined studieswhere n=number of mice/treatment. Data was analysed using the Kruskal–Wallis test,incorporating Bonferroni correction, with post-hoc Mann–Whitney U-test. ⁎⁎Pb0.01,⁎⁎⁎Pb0.001 compared to vehicle.

Table 1Neurological status of CR-EAE-sensitised mice treated with vehicle or FeTPPS during thecourse of disease

Treatment(mg/kg body weight)

Neurological status

No signs(score 0)

Non-paralysed(scores 1–3)

Paralysed(scores 4–6)

AVehicle 0/30 30/30 26/30c

(10) 4/25a 21/25a 0/25b

(20) 20/26b 6/26b 0/26b

BVehicle 0/15 15/15 15/15(20) 1/11 10/11 0/11b

CVehicle 0/9 9/9 8/9c

(20) 8/10b 2/10b 0/10b

Acute diseased mice received vehicle or FeTPPS therapeutically and i.p., twice daily, at10 mg/kg body weight or 20 mg/kg body weight (A) from days 15–18 post-inoculation.Acute disease-recovered mice were treated either prophylactically from the remissionof symptoms (B), 24–26 days post-inoculation, or therapeutically from the onset ofrelapse, 32–35 days post-inoculation, with FeTPPS, i.p., twice daily, at 20 mg/kg bodyweight (C). Animals in treatments A and B were sacrificed, for the removal of spinalcolumns, 21–24 days post-inoculation and 30–32 days post-inoculation and group C,44 days post-inoculation.Non-paralysed and paralysed animals had neurological scores between 1 to 3 and 4 to 6respectively. Data was analysed using Fisher's exact test with Yates continuitycorrection.

a Pb0.05 compared to vehicle.b Pb0.001 compared to vehicle.c All vehicle-dosed mice showed non-paralytic disease but 4 animals in A and 1

animal in C failed to develop paralytic symptoms. The onset, loss and duration ofneurological signs in treatments A, B and C were not significantly different from thevehicle group (data not shown).

89C. Bolton et al. / European Journal of Pharmacology 601 (2008) 88–93

administration of the ONOO− scavenger uric acid to EAE-sensitisedmice maintains normal blood–brain barrier function, restores neuro-vascular integrity and inhibits CNS inflammation (Hooper et al., 2000;Kean et al., 2000). Interestingly, multiple sclerosis patients show atendency towards reduced serum uric acid levels that correlate withdisease activity and neurovascular dysfuction (Drulovic et al., 2001;Spitsin et al., 2001; Sotgiu et al., 2002; Toncev et al., 2002).Consequently, raising the circulating levels of uric acid, by adminis-tration of the nucleoside inosine, has been proposed as a noveltreatment formultiple sclerosis (Koprowski et al., 2001;Mousavizadehet al., 2003). Alternative ONOO− scavengers, such as mercaptoethyl-guanidine (MEG) and guanidinoethyldisulphide (GED) are also capableof altering the neurological profile of EAE (Scott et al., 2001a,b). Themetalloporphyrin 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulphona-tophynyl)porphyrinatoiron (III), (FeTMPS), a potent catalyst of ONOO−

that isomerises the molecule to produce inactive nitrate, curtails theneurological and histological progression of acute EAE (Cross et al.,2000).

The present study has further investigated the role of ONOO− in amodel of EAE with a chronic relapsing (CR) EAE profile of disease. Theinvestigation has utilised the metalloporphyrin and ONOO− decom-position catalyst 5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrina-toiron(III)chloride (FeTPPS), in several dosing regimes, to influence thepathogenesis of the acute and chronic phases of CR EAE. Resultsshowed that therapy with FeTPPS during the acute and relapse phaseof the disease curtailed the development of neurological signs andreduced histological changes in CNS tissues. Prophylactic doses of thedrug, prior to relapse, enhanced the suppressive influence of the drug.Use of the CR EAEmodel further emphasises the important role playedby ONOO− in the pathogenesis of the disease.

2. Materials and methods

2.1. Materials

Incomplete Freund's adjuvant, Mycobacterium tuberculosis H37Raand Mycobacterium butyricum were obtained from Fisher Scientific(Loughborough, UK). Phosphate buffered saline (PBS), formalin,paraffin wax, haematoxylin and eosin, luxol fast blue and cresyl violetwere purchased from Sigma-Aldrich Company Ltd. (Poole, UK). FeTPPSwas obtained from Merck Biosciences Ltd., (Nottingham, UK).

2.2. Animals and induction of CR EAE

Male Biozzi mice (Harlan Olec, UK), 8 to 10 weeks old, wereinoculated for CR EAE as previously described (Bolton et al., 1997).Briefly, mice were injected, in each flank, with 0.15 ml of an emulsioncontaining lyophilised homologous spinal cord, PBS and incompleteFreund's adjuvant supplemented with M. tuberculosis H37Ra and M.butyricum. Mice were re-sensitised with fresh inoculum 7 days afterthe initial inoculation. The experiments were undertaken in accor-dance with the UK Animals Procedures Act of 1986.

2.3. Neurological assessment of EAE

Animals were weighed daily and assessed for neurologicalsymptoms from day 10 post-inoculation. Symptoms of EAE werescored as follows: 0=normal, 1=piloerection and partial flaccid tail,2=complete flaccid tail, 3=hind limb hypotonia, 4=partial hind limbparalysis; 5=complete hind limb paralysis, 6=hind limb and forelimbparalysis, 7=moribund/dead.

2.4. Administration of FeTPPS

FeTPPS was dissolved in sterile PBS vehicle and administered, viathe intraperitoneal (i.p.) route, twice daily, at a dose of either 10mg/kgbody weight or 20 mg/kg body weight. CR EAE-inoculated micereceived FeTPPS, for 5 consecutive days, from the onset of eitheracute- or relapse-associated body weight loss, as defind by a 1 g reduc-tion in body weight over 24 h, between days 15 and 18 post-inoculationand days 32 to 35 post-inoculation, respectively. Also, FeTPPS was givenprophylactically, i.p., twice daily, from the remission of acute neurologicalsymptoms that began 24 days post-inoculation to 26 days post-inoculation. Control CR EAE-sensitised mice received vehicle, i.p., twicedaily.

Fig. 2. Effect of therapeutic dosing with FeTPPS on body weight change during the acutephase of CR EAE. Male Biozzi mice, inoculated for CR EAE, were dosed i.p., twice daily,with either vehicle or FeTPPS at 10 mg/kg body weight or 20 mg/kg body weight for5 days from acute disease-associated body weight loss, 15–18 days post-inoculation,prior to the onset of neurological deficits. Results are from 2 combined studies wheren=number of mice/treatment. Data was analysed using the Kruskal–Wallis test,incorporating Bonferroni correction,with post-hoc Mann–Whitney U test. ⁎⁎Pb0.01,⁎⁎⁎Pb0.001 compared to vehicle.

Fig. 3. Effect of FeTPPS, administered prophylactically from the remission of acutesymptoms, on the relapse phase of CR EAE. Male Biozzi mice, inoculated for CR EAE,were dosed with either vehicle or FeTPPS at 20 mg/kg body weight, i.p., twice daily,from days 24–26 post-inoculation. Animals were sacrificed, for the removal of spinalcolumns, 30–32 days post-inoculation. n=number of mice/treatment. Data wasanalysed using the Kruskal Wallis test, incorporating Bonferroni correction, withpost-hoc Mann–Whitney U test. ⁎Pb0.05, ⁎⁎Pb0.01, ⁎⁎⁎Pb0.001 compared to vehicle.

90 C. Bolton et al. / European Journal of Pharmacology 601 (2008) 88–93

2.5. Histological assessment of EAE

A minimum of 6 spinal cords were collected from mice oncompletion of the dosing schedule for each treatment. Each animalwas sacrificed, by carbon dioxide inhalation, bled, via cardiacpuncture, and the spinal column removed by blunt dissection. Thespinal cord was collected by flushing PBS, via a 5 ml syringe and 17gauge needle, through the distal end of the column. Standardhistological techniques, based on the methods described by Smith etal. (2000) and Groom et al. (2003) were used. Briefly, spinal tissue wasfixed in formalin and embedded in paraffin wax. Sections, 10μm thick,were cut at one standard depth, from each spinal cord, and stainedeither with haematoxylin and eosin for the detection of lesions orluxol fast blue, eosin and cresyl violet for the assessment ofdemyelination. Lesion number in a section of each entire spinal cordwas assessed ‘blind’ by light microscopy. The intensity of staining anddemyelination in an individual section of complete spinal cord weresimilarly assessed and scored using a scale from − to ++++.

2.6. Statistical analysis

Results from one experiment, or two combined studies, wereexpressed as the mean±S.E.M. of n observations where nN6 and thecritical significance levelwas 0.05. Neurological status andmean lesion

Table 2Histological analysis of spinal cords from vehicle or FeTPPS-dosed CR EAE-diseased Biozzi m

Treatment(mg/kg body weight)

Staining intensity of lesions

n − −/+ + ++

AVehicle 10 0 0 0 3(10) 6 0 0 3 3(20) 6 0 4 2 0

BVehicle 6 0 0 0 1(20) 6 1 1 4 0

CVehicle 6 0 0 0 1(20) 6 0 0 2 4

FeTPPS or vehicle was administered, i.p., twice daily, either therapeutically during acute disimmunisation, prior to the relapse phase (B), or therapeutically from the onset of relapse (C

a Pb0.01 compared to vehicle. Results were analysed using Unpaired Student's t-test. Animpost-inoculation and 30–32 days post-inoculation and group C, 44 days post-inoculation.

number were analysed using Fisher’s exact test with Yates continuitycorrection and Unpaired Student's t-test, respectively. Mean neurolo-gical scores and mean body weight changes were subjected to theKruskal–Wallis test, incorporating Bonferroni correction for repeatedmeasures, with post-hoc Mann–Whitney U test.

3. Results

3.1. FeTPPS therapy of the acute phase of CR EAE

The administration of vehicle to CR EAE-inoculated mice did notinfluence the neurological profile of disease compared to the undosedcontrol group (Fig. 1) CR EAE-sensitised mice receiving FeTPPS, at10 mg/kg body weight for 5 days, from the onset of acute disease-associated body weight loss, 15 to 18 days post-inoculation, hadsignificantly reduced neurological scores 2 to 5 days after thecommencement of treatment (Pb0.01–b0.001). The values werefurther decreased, during the treatment period, in animals dosedwith FeTPPS at 20 mg/kg body weight (Pb0.001).

A uniform incidence of disease was observed in the vehicle group(Table 1A). Significantly fewer animals dosed with FeTPPS, at either10mg/kg bodyweight or 20mg/kg bodyweight, showed neurological,

ice

Mean lesion number Demyelinationscore+++ ++++ ±S.E.M.

4 3 63±4 –

0 0 36±6a –

0 0 12±3a –

4 1 73±7 20 0 16±5a 1

3 2 84±11 20 0 71±8 2

ease (A), beginning days 15–18 post-immunisation, prophylactically, 24–26 days post-), commencing 32–35 days post-immunisation.als in treatments A and B were sacrificed, for the removal of spinal columns, 21–24 days

91C. Bolton et al. / European Journal of Pharmacology 601 (2008) 88–93

non-paralytic, signs of disease (Pb0.05 and Pb0.001 respectively).Furthermore, none of the animals receiving low- or high-dose therapyexperienced acute paralytic symptoms.

FeTPPS-induced protection from acute disease was accompaniedby reduced body weight changes in drug-treated mice, compared tocontrols, (Fig. 2). The drug-associated reduction in body weight losswas also observed in all successive dosing regimes (data not shown).Mice dosedwith compound at 10mg/kg bodyweight had significantlyless body weight loss during the treatment phase (Pb0.01–b0.001).The changes in bodyweight were further reduced in animals receivinghigh-dose drug therapy (Pb0.001).

The staining intensity of the cellular infiltration in the spinal cordsfrom FeTPPS-dosed mice was reduced (Table 2A). Furthermore, themean number of lesions in the group treated with drug at 10 mg/kgbody weight was significantly less than controls (Pb0.01) and wasfurther reduced by increasing the dose (Pb0.01). Demyelination wasnot assessed in tissues removed frommice at the acute stage of CR EAEas myelin disruption is not typical of the initial neurological phase ofthe disease.

3.2. Prophylactic FeTPPS administration from the remission of acutedisease

Prophylactic dosing with vehicle or FeTPPS, at 20 mg/kg bodyweight, to mice in remission from acute disease, commenced 24 dayspost-inoculation to 26 days post-inoculation. Vehicle-treated animalsshowed characteristic relapse symptoms beginning approximately35 days post-inoculation (Fig. 3). In contrast, the relapse phase of CREAE was significantly suppressed in mice dosed with drug (Pb0.01–b0.001). Furthermore, administration of FeTPPS completely preventedthe development of paralytic symptoms (Table 1B). The dosing regimealso lessened lesion intensity and demyelination and significantlyreduced lesion number (Pb0.01) (Table 2B).

3.3. FeTPPS therapy of the relapse phase of CR EAE

Mice selected for the undosed and vehicle groups had a similarprofile of disease over the treatment period (Fig. 4). The administrationof FeTPPS, at 20 mg/kg body weight for 5 days, from the beginning ofrelapse phase-associated body weight loss, 32 days post-inoculation,significantly reduced neurological scores, 2 to 6 days following thecommencement of therapy (Pb0.05–b0.001). In addition, significantlymore mice receiving the compound remained either disease-free or

Fig. 4. Effect of therapeutic administration of FeTPPS on neurological scores during therelapse phase of CR EAE. Male Biozzi mice, inoculated for CR EAE, were dosed witheither vehicle or FeTPPS at 20 mg/kg body weight for 5 days, i.p., twice daily, fromdisease-associated body weight loss, 32–35 days post-inoculation, prior to the onset ofneurological deficits. Animals were sacrificed, for the removal of spinal columns,44 days post-inoculation. n=number of mice/treatment. ↓=commencement of dosing.Results were subjected to the Kruskal–Wallis test, incorporating Bonferroni correction,with post-hoc Mann–Whitney U test. ⁎Pb0.05, ⁎⁎Pb0.01, ⁎⁎⁎Pb0.001 compared tovehicle.

without non-paralytic symptoms (Pb0.001) (Table 1C). Moreover,drug-treated animals did not showparalytic signs of CR EAE (Pb0.001).Interestingly, drug therapy did reduce the intensity, but not thenumber, of lesions or the extent of demyelination in spinal tissues(Table 2C).

4. Discussion

The present study has used the Biozzi mouse model of CR EAE todemonstrate the significantly beneficial prophylactic and therapeuticeffects of the ONOO− decomposition catalyst FeTPPS on the neurolo-gical deficits characteristic of the acute and relapse phases of thedisease. Furthermore, the dosing regimes influenced the formation ofinflammatory lesions and demyelination in the spinal cords of drug-treated mice.

FeTPPS is amember of themetalloporphyrin family that inactivatesONOO− through a complex series of oxidation and reduction reactionsthat conclude with the decomposition of the radical via nitriteformation (Shimanovich and Groves, 2001). The metalloporphyrinshave been used extensively, in vitro and in vivo, to control ONOO−-associated damage in a variety of tissues. For example, in vitro useblocks thymocyte proliferation, prevents inflammatory mediatorrelease from mast cells, reduces activated microglial toxicity, restoresmyocyte function and preserves astrocyte viability (Misko et al., 1998;Choi et al., 2001; Xie et al., 2002; Kang et al., 2003; Kim et al., 2005; Yuet al., 2005). In vivo, the drugs counteract aberrant myocardialcontractility, prevent cerebral and intestinal ischemia, suppressdopaminergic neurotoxicity, inhibit endotoxaemia, reduce renaldamage and possess antinociceptive properties (Imam et al., 2001;Chirino et al., 2004; Lancel et al., 2004; Thiyagarajan et al., 2004;Cuzzocrea et al., 2006; Dhar et al., 2006; Stefanutti et al., 2007; Yeo etal., 2008).

Previous studies in EAE showed the metalloporphyrin FeTMPSmodified monophasic, adoptively-transferred EAE in the SJL mouse(Cross et al., 2000). Prophylactic doses of the compound suppressedacute neurological impairment in cell transfer recipients, compared todiseased animals receiving the inactive, iron-depleted compoundTMPS. In addition, CNS inflammation, demyelination and nitrotyr-osine immunoreactivity were reduced but inducible nitric oxidesynthase expression remained unchanged, indicating drug efficacythrough the reduction of ONOO− production rather than NO depletion.The significance of ONOO− in the pathogenesis of EAE models is againimplied through the use of MEG and GED which react with andthereby scavenge the molecule. Scott et al. (2001a) found low-doseMEG or GED was ineffective at influencing EAE but higher doses diddelay onset and reduce the incidence of disease.

Our results confirm the pharmacological actions of an additionalmetalloporphyrin, FeTPPS, in a CRmodel of EAE. The studyalso extendsthe previous observations of Cross et al. (2000) and Scott et al. (2001a)by demonstrating therapeutic efficacy during acute disease. Moreover,the neurological profile, but not the histological features, of the relapsephase of CR EAE were modified by therapy with FeTPPS. Furthermore,prophylactic administration of FeTPPS, from the remission of acutesymptoms, proved effective at suppressing the subsequent symptomsand histological changes observed during relapse. The work in CR EAEre-emphasises the role played by ONOO− in the immunologically-driven events that occur prior to the manifestation of disease. Inaddition, the data strongly indicates a function for the molecule inprecipitating and perpetuating the symptoms of disease.

The induction and development of EAE induces profoundbiochemical changes that are effected by reactive molecules such asONOO−, formed by the rapid combination of nitric oxide withsuperoxide. For example, disturbances to mitochondrial function,tyrosine nitration, poly (ADP) ribose activation and lipid peroxidationfeature in the pathogenesis of EAE and are all subject to mediation byONOO− (Cross et al., 1997; Scott et al., 2001b; Qi et al., 2007; Zargari

92 C. Bolton et al. / European Journal of Pharmacology 601 (2008) 88–93

et al., 2007). ONOO− also has cellular targets and the blood–brainbarrier has been identified as a prime site for the deleterious actions ofthe molecule in EAE (Hooper et al., 2000; Kean et al., 2000). Loss ofneurovascular integrity, as a result of ONOO−-mediated cytotoxicity,would facilitate themovement of extracellular fluid and inflammatorycells into CNS tissues with resultant neuronal dysfunction. Interest-ingly, recent in vitro work by us indicates the blood–brain barrier isrelatively resistant to the cytotoxic actions of ONOO− and suggests theinvolvement of other mediators in neurovascular damage during EAE(submitted for publication).

Collectively, the current and previous EAE-related studies provideuseful information on the relative efficacy of a class of drugs thattarget a defined molecule involved in the pathology of EAE. However,the compounds do not exclusively inhibit the actions of ONOO−. Forexample, the metalloporphyrins scavenge a wide-range of reactiveoxygen species and the MEGs act as anti-inflammatory agents bydown-regulating inducible nitric oxide synthase and cyclooxygenase-2 expression (Brahn et al., 1998; Patel and Day, 1999; Moochhala et al.,2005; Badn et al., 2007; Castello et al., 2008). Therefore, themodulatory effects of the metalloporphyrins and the MEGs in modelsof EAE may also be attributed to their additional actions on mediatorsoperating during neuroinflammatory disease.

The present work with FeTPPS reveals different therapeuticresponses to an identical, high-dose treatment regime administeredduring either the acute or relapse phase of CR EAE. In particular, ourstudies showed the drug to be similarly efficacious at suppressing thesymptoms of acute and relapse disease, but less efficient in controllingCNS inflammation in the latter phase of CR EAE. The dissimilar actionsof FeTPPS on the two neurological stages of CR EAE suggest differentmechanisms in operation which govern the development of disease.CR EAE is characterised by pathological features including demyelina-tion, axonal degeneration and spasticity (Croxford et al., 2008) thatemerge sequentially with the chronicity of the condition and,presumably, are due to the successive activation of aberrant inflam-matory and immunological systems. Interestingly, a similar progres-sive profile of disease symptoms occurs during the evolution ofmultiple sclerosis (Compston and Coles, 2002). Therefore, thesuccessful management of chronic neurological diseases, such asmultiple sclerosis, may require specific pharmacological interventiondesigned to target the mechanisms responsible for the separate butassociated elements of disease.

Acknowledgement

The work was supported by a grant from The William HarveyResearch Foundation.

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