WO2008090331A1

WO2008090331A1 - Serotonin reuptake inhibitors for treating arthritis

Info

Publication number: WO2008090331A1
Application number: PCT/GB2008/000217
Authority: WO
Inventors: Brian M. Foxwell; Sandra Sacre
Original assignee: Imperial Innovations Limited
Priority date: 2007-01-22
Filing date: 2008-01-22
Publication date: 2008-07-31
Also published as: GB0701171D0

Abstract

The present invention relates to serotonin reuptake inhibitors (SRIs) including selective serotonin reuptake inhibitors (SSRIs) and serotonin norepinephrine reuptake inhibitors (SNRIs) for treating inflammatory arthritis and related conditions. The present invention also relates to a kit comprising a pharmaceutical composition comprising a serotonin reuptake inhibitor and instructions indicating that the composition is for use in treating inflammatory arthritis and related conditions. In addition, the present invention relates to a method of treating inflammatory arthritis and related conditions comprising administration of a serotonin reuptake inhibitor. The method is preferably carried out on a subject in need of treatment. The inflammatory arthritis is preferably rheumatoid arthritis.

Description

Serotonin reuptake inhibitors for treating arthritis

Rheumatoid arthritis is a chronic inflammatory disease occurring in 0.5%-l% of the population. This disease affects the joints and is characterized by thickening of the synovial membrane. This disease leads to progressive debilitation in joint function which results in pain, disability, loss of man power and shorter life expectancy. In addition there are a number of similar related conditions; psoriatic arthritis, juvenile arthritis ankylosing spondelytis, Crohn's disease and psoriasis.

Current treatments for rheumatoid arthritis have a number of disadvantages, including expense and/or severe side effects. At present, steroids such as dexamethasone and methyl-prednisolone, are widely used in the treatment of rheumatoid arthritis. While treatment with steroids can be effective, there are a number of serious side effects. These side effects include hypertension, growth deficiencies in younger patients, osteoporosis, cataracts, psychosis, elevated blood sugar, glaucoma, etc. In addition, some patients are resistant to long-term use of steroids or become so with time.

New and alternative treatments currently used for rheumatoid arthritis are based on biologicals such as antibodies and soluble receptors. The most widely used of these is based on blocking TNF function with neutralizing antibodies or soluble receptors and has defined TNF as a key pathway in the inflammatory processes in RA. This type of anti-TNF therapy has been successful in the treatment of a number of diseases, with a substantial proportion of patients (approximately 60%) showing significant clinical benefit Besides treating the inflammation, anti-TNF therapy also prevents joint destruction and thus is considered to have disease modifying anti-arthπtic drug (DMARD) activity However, it is extremely expensive and this places a heavy financial burden either on the patient or the healthcare system or both and this seriously limits availability. Many patients in the developed world and the majoπty in the developing world are not able to afford this treatment. In addition, possible side effects of anti-TNF therapy include anaphylaxis and cytopenia Moreover systemic neutralisation of TNF leads to increased susceptibility to infection and the long-term effects are still unknown Currently it is not possible to take anti-TNF drugs orally, which is a disadvantage Moreover of those patients that do respond approximately 50% will become refractory to treatment in 2 years due to the development of immunity to the biological treatment

Another class of drugs are the disease modifying anti-rheumatic drugs (DMARDS). An example of these is methotrexate, an anti-metabolite drug, which is widely used for the treatment of rheumatoid arthritis, psoπatic arthritis and psoπasis. Methotrexate has been successful in the treatment of these diseases, but can cause substantial side effects, such as severe skin reaction, infections such as pneumonia, severe damage to liver, kidneys, lungs and gastrointestinal tract. Again a significant number of patients do not respond or become refractory with time.

A number of DMARD pharmaceutical agents containing gold are also used in the treatment of rheumatoid arthritis Examples of such agents include gold sodium thiomalate and auranofin Potential side effects from being treated with antiinflammatory gold agents are oral ulcers, altered taste, seπous skin rashes, renal problems, inflammation of the intestines (enterocolitis), liver injury and lung disease. Furthermore, resistance to gold has been known to develop in patients

A further class of drugs are the non-steroidal anti-inflammatory drugs (NSAED' s). These are used to alleviate symptoms but not modify disease progression and includes the Cox 2 inhibitors "VIOXX"^®, (a registered trademark of Merck & Co., Inc) and "CELEBREX" ^®, (a registered trademark of G.D. Searle & Co). However there is major concern about the safety of these drugs with evidence of increased cardiovascular risk.

As a result of lack of efficacy, development of resistance, unacceptable side-effects and expense of existing treatments and route of administration, it is hugely desirable to find alternative treatments for rheumatoid arthritis. Thus there is a huge unmet medical need for an orally-available, well tolerated, inexpensive drug that could selectively block the production of TNF associated with pathological inflammation found in RA and related conditions.

SRIs (including SSRIs and SNRIs) are well known for use in the treatment of depression and other related conditions such as OCD. SRIs (including SSRIs and SNRIs) would be expected to promote inflammation as serotonin is associated with inflammation. In addition, other studies have indicated that sertraline, a SSRI, exacerbates inflammation in the carrageenan-induced model of paw inflammation (Abdel-Salam et al., Pharmacol Res 48, 157-165, 2003).

The first aspect of the present invention provides a serotonin reuptake inhibitor (SRI) for treating inflammatory arthritis and related conditions.

The inflammatory arthritis is anti-TNF responsive arthritis, as are the related conditions psoriatic arthritis, juvenile arthritis, ankylosing spondelytis, Crohn's disease and psoriasis. These drugs are envisaged to be useful in diseases where treatment with anti-TNF has shown to be beneficial e.g. Crohn's disease and psoriasis, but not useful in diseases where anti-TNF has been shown to be ineffective or to exacerbate disease, e.g. systemic lupus erythematosus, multiple sclerosis and ulcerative colitis. These drugs would not be expected to be beneficial for the treatment of sepsis where TNF is produced by lipopolysaccharide (LPS) stimulation. These drugs are unable to inhibit LPS induced TNF. This indicates that these drugs do not possess general anti-inflammatory properties. In a preferred embodiment the inflammatory arthritis is rheumatoid arthritis and the related conditions are psoriatic arthritis, juvenile arthritis, ankylosing spondelytis, Crohn's disease and psoriasis.

A serotonin reuptake inhibitor of the invention is any compound that inhibits reuptake of serotonin. This includes selective serotonin reuptake inhibitor (SSRI) compounds and serotonin norepinephrine reuptake inhibitor (SNRI) compounds.

As is known in the art, a SRI is a drug that increases the amount of serotonin (5- hydroxytryptamine or 5-HT) by inhibiting its reuptake into cells. The drug is an inhibitor of the serotonin transporter (5-HTT), a monoamine transporter protein.

A SRI of the invention inhibits TNF production.

Information on SRIs can be found in Wilson et al., J. Med. Chem. 43, 3103-3110, 2000 and Tatsumi et al., European Journal of Pharmacology 340, 249-258, 1997.

The serotonin reuptake inhibitor of the invention can be amoxapine, amitriptyline ("ELAVIL", "ENDEP", "TRYPTANOL", "TREPILINE"), clomipramine

("ANAFRANIL"), desipramine ("NORPRAMIN", "PERTOFRANE"), dothiepin hydrochloride ("PROTfflADEN", "THADEN"), doxepin ("ADAPIN", "SINEQUAN"), imipramine ("TOFRANIL"), lofepramine ("GAMANIL", "LOMONT"), nortriptyline ("PAMELOR"), protriptyline ("VIVACTIL") or trimipramine ("SURMONTIL").

The selective serotonin reuptake inhibitor of the invention can be citalopram ("CELEXA", "CEPRAMIL", "EMOCAL", "SEPRAM", "SEROPRAM"), escitalopram oxalate ("LEXAPRO", "CIPRALEX", "ESERTIA"), fluoxetine ("PROZAC", "FONTEX", "SEROMEX", "SERONIL", "SARAFEM", "FLUCTIN" (EUR)), fluvoxamine maleate ("LUVOX", "FAVERIN"), paroxetine ("PAXIL", "SEROXAT", "AROPAX", "DEROXAT", "PAROXAT"), sertraline ("ZOLOFT", "LUSTRAL", "SERLAIN"), dapoxetine or St Johns Wort

The serotonin-norepinephnne reuptake inhibitor of the invention can be venlafaxine ("EFFEXOR XR" and "EFEXOR"), nefazodone ("SERZONE"), milnacipran ("DALCIPRAN" and "IXEL"), desipramine ("N0RPRAMINE" and "PERTOFRANEIS") or duloxetine ("CYMBALTA").

The use may be of combinations of two or more SRIs, which may be for simultaneous, separate or sequential use.

The SRI may be used in combination with a further anti-inflammatory agent. Administration of the SRI and other anti-inflammatory agent can be simultaneous, separate and/or sequential The SRI, in combination with another pharmaceutical agent, can act additively or synergistically.

The other anti -inflammatory agent may be termed a non-steroidal anti-inflammatory agent (NSAID), a disease modifying anti-rheumatic drug (DMARD), a statin (including HMG-CoA reductase inhibitors such as simvastatin), a biological agent (biologicals), a steroid, an immunosuppressive agent, a salicylate and/or a microbicidal agent. Non-steroidal anti-inflammatory agents include anti-metabolite agents (including methotrexate) and anti-inflammatory gold agents (including gold sodium thiomalate, aurothiomalate or gold salts, such as auranofin). Biologicals include anti-TNF agents (including adalimumab, etanercept, infliximab, anti-EL-1 leagents, anti-IL-6 reagents, anti-B cell reagents (retoximab), anti-T cell reagents (anti-CD4 antibodies), anti-IL-15 reagents, anti-CLTA4 reagents, anti-RAGE reagents), antibodies, soluble receptors, receptor binding proteins, cytokine binding proteins, mutant proteins with altered or attenuated functions, RNAi, polynucleotide aptemers, antisense oligonucleotides or omega 3 fatty acids Steroids include cortisone, prednisolone or dexamethasone. Immunosuppressive agents include cylcospoπn, FK506, rapamycin, mycophenolic acid Salicylates include aspirin, sodium salicylate, choline salicylate and magnesium salicylate. Microbicidal agents include quinine and chloroquine. For example, the SRI may be administered in combination with one or more of an NSAID, DMARD, or immunosuppressant.

The further anti-inflammatory agent is administered by any appropriate route, for example oral (including buccal or sublingual), suppositories, topical (including buccal, sublingual or transdermal), or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Where the further anti-inflammatory agent is administered orally, it may be administered as part of the same composition as the SRI.

The SRIs of the present invention are those SRIs which are non-toxic to living cells.

The present invention also provides the use of a serotonin reuptake inhibitor (SRI) in the manufacture of a medicament for treating inflammatory arthritis and related conditions.

The second aspect of the invention is a kit comprising a pharmaceutical composition comprising an SRI and instructions indicating that the composition is for use in treating inflammatory arthritis and related conditions.

Compositions in accordance with the invention may be supplied as part of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable earner. This pharmaceutical composition may be in any suitable form. It may be provided in unit dosage form and will generally be provided in a sealed container. The kit of the invention may comprise a plurality of said unit dosage forms.

The oral pharmaceutical compositions may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions).

Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, steaπc acid or salts thereof Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc

For the preparation of solutions and syrups, excipients which may be used include for example water, polyols and sugars For the preparation of suspensions oils (e g vegetable oils) may be used to provide oil-in-water or water in oil suspensions

The pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts, buffers, coating agents or antioxidants They may also contain further therapeutically active agents

Route of administration may include, parenterally (including subcutaneous, intramuscular, intravenous, by means of, for example a dπp patch), some further suitable routes of administration include (but are not limited to) oral (including buccal and sublingual), rectal, nasal, topical, infusion, vaginal, intradermal, intrapeπtoneally, intracranially, intrathecal and epidural administration or administration via oral or nasal inhalation, by means of, for example a nebuliser or inhaler, or by an implant

For administration via the oral or nasal inhalation routes the may be delivered using a mechanical form including, but not restricted to, an inhaler or nebuliser device

Further, where the oral or nasal inhalation routes are used, administration is by a SPAG (small particulate aerosol generator) may be used

Dosages of the substances of the present invention can vary between wide limits, depending upon the condition to be treated, the health of the individual to be treated, etc and a physician may determine appropπate dosages to be used The dosage may be repeated as often as appropπate The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy The amount of active ingredient which can be combined with a earner matenal to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration The amount of active ingredient which can be combined with a earner material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect

The compositions and uses described in this application are envisaged to have human, animal and veterinary applications They are preferably applicable to mammals, in particular humans but are also applicable for use in production animals, in particular sheep, cows, pigs, chickens and goats, as well as companion animals, in particular cats and dogs and sporting animals, such as horses

The third aspect of the invention is a method of treating inflammatory arthritis and related conditions comprising administration of a serotonin reuptake inhibitor In the third aspect of the invention, the method is preferably earned out on a subject in need of treatment

In the present invention, the term "treatment" includes prophylactic treatment (i e prevention) In most circumstances, prevention of inflammatory arthntis or a related condition is unlikely to be earned out Usually, it is only when the presence of inflammatory arthntis or a related condition is diagnosed in a subject that prevention means are applied However, prophylactic treatment may be appropnate if there is i) a known family history of significant inflammatory arthntis or a related condition or if tests (e g genetic tests) identify that an individual has a predisposition to inflammatory arthntis or a related condition

The preferred embodiments, as descnbed for the first aspect of the invention, are the same for other aspects of the invention mutatis mutandis

The present invention is descnbed with references to the drawings, in which

Figure 1 illustrates inhibition of spontaneous cytokine release from RA synovial membrane cultures by citalopram and fluoxetine hydrochlonde ("PROZAC") Graph (A) illustrates a dose dependent inhibition of TNF levels and IL- lβ levels by fluoxetine hydrochloπde ("PROZAC") as measured by ELISA Graph (B) illustrates a dose dependent inhibition of TNF in cells incubated with citalopram

Figure 2 illustrates inhibition of disease progression in the CIA model of rheumatoid arthritis by citalopram Graph (A) illustrates clinical score, Graph (B) illustrates histology scoπng. All statistics were calculated using a Mann- Whitney one tailed test, **=P<0.01, *=P<0.05, (n=6).

Figure 3 illustrates inhibition of disease progression in the CIA model of rheumatoid arthritis by fluoxetine hydrochloπde ("PROZAC"). (A) illustrates clinical score (B) illustrates paw swelling. All statistics were calculated using a Mann-Whitney one tailed test **=P<0.01, *=P<0.05.

Figure 4 Graph (A) illustrates inhibition by fluoxetine hydrochlonde of TNF production from human macrophages activated by LPS (10ng/ml), Flagelhn (lOng/ml), R848 (lμg/ml) and malp-2 (lOng/ml) Graph (B) illustrates TNF production in murine RAW264.7 macrophages stimulated with 20μg/ml poly IC in the presence of media alone or fluoxetine hydrochloπde Graph (C) illustrates inhibition by fluoxetine hydrochloπde of TNF production in CpG stimulated muπne RAW264 7 macrophages.

Figure 5 illustrates that inhibition of TNF production by R-848 is common to the serotonin reuptake inhibitors. Graph (A) illustrates TNF production from macrophages stimulated with lμg/ml R848 or 10ng/ml LPS in the media alone or in the presence of 30μg/ml citalopram, lOμg/ml fluoxetine hydrochloπde ("PROZAC") or 5μg/ml sertraline ("ZOLOFT") Graph (B) illustrates TNF levels from macrophages stimulated with 10ng/ml Pam3, 10ng/ml LPS, 10ng/ml Flagelhn, lμg/ml R848 and 10ng/ml malp-2 in media alone or in the presence of 5μg/ml sertraline ("ZOLOFT") Figure 6 illustrates that TLR3, 7, 8 and 9 are expressed in the rheumatoid synovial membrane cultures but only TLR3 and 8 induce TNF production Graph (A) RT-PCR illustrates the presence of TLR3, TLR7, TLR8 and TLR9 mRNA in 3 separate donors Graph (B) illustrates FACS analysis of cell surface and intracellular expression of TLR3, TLR8 and TLR9. Representative histogram plots of 3 independent donors are shown Graph (C) illustrates TNF production in rheumatoid membrane synovial cells after stimulation with 20μg/ml Poly IC (TLR3), lμg/ml imiquimod (TLR7), lμg/ml R-848 (TLR7/8) and 2μM CpG (TLR9).

Figure 7 illustrates the effect of serotonin on TNF and EL-I levels in RA membrane cell cultures Graph (A) illustrates addition of serotonin to RA membrane cell cultures for 24 hours (n=5-6). (B) Addition of chloroquine to RA membrane cell cultures for 24 hours (n=4). *=P<0.05

The present invention is descπbed with reference to the following non-limiting examples:

Examples

General Materials and Methods

Reagents

Cell culture reagents used were Penicillin-Streptomycin, RPMI 1640 and DMEM obtained from Cambrex (Belgium), Indomethacin from Sigma (USA) and FBS from PAA (Austπa). The TLR hgands used were chloroform extracted Escherichia coli (E.coh) LPS, resiquimod (R-848), muπne CpG (ODN1826), human CpG (ODN 2006) and imiquimod from Invivogen (USA), Macrophage-activating hpopeptide (Malp-2), Flagellin (puπfied) and serotonin (5-HT) hydrochloπde were from Alexis (UK) Fluoxetine hydrochloπde ("PROZAC") and chloroquine diphosphate salt, Sertraline ("ZOLOFT") and citalopram were purchased from Sigma (USA) Pam₃cys- ser(lys)₄ 3HCl (Pam3) was from Alexis (UK) The antibodies used for FACS were FITC conjugated α-TLR3, α-TLR8 and α-TLR9 from Imgenex (USA) and a IgGl- FITC control from BD Pharmingen. Reverse transcriptase-polymerase chain reaction (RT-PCR)

RNA was isolated using the Qiagen RNA Blood isolation kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Total RNA was reverse transcribed with Superscript II RNase H^" reverse transcriptase (Gibco Life Technologies, Carlsbad,

CA) and oligo (dT) primer. For human TLR3 amplification, the primers 5'-

GCAAACACAAGCATTCGGAATCT-3' and 5'-

TTGAAGGCTTGGGACCAAGGCA-S¹ were used with an annealing temperature of

62°C. For human TLR7 amplification, the primers 5'- TCTACCTGGGCCAAAACTGTT-3' and 5'- GGCAC ATGCTGAAGAG AGTTA-3' were used with an annealing temperature of 6O⁰C. For human TLR8 amplification, the primers 5'- CCG ACTTGG A AGTTCTAG ATC-3¹ and 5¹-

AATGCTTCATTTGGGATGTGCT-3' were used with an annealing temperature of

60⁰C. For human TLR9 amplification, the primers 5'- GTGCCCCACTTCTCCATG- 3¹ and 5'- GGCACAGTC ATGATGTTGTTG-3¹ were used with an annealing temperature of 55⁰C. Amplification was performed in a Dyad PCR machine (MJ

Instruments). Subsequent PCR amplification consisted of 35 cycles.

ELISA (enzyme-linked immunosorbent assay) Macrophages or RA synovial membrane cell cultures were stimulated with lOng/ml chloroform-extracted LPS, 20μg/ml poly IC, 2μM CpG (murine or human), 10ng/ml Pam3, 10ng/ml Flagellin, 10ng/ml Malp-2 or lμg/ml R-848 in complete media for 6h. Supernatants from RA cultures were harvested after 24 hours. AU reagents other than LPS were free from LPS contamination as assessed using the limulus amebocyte lysate (LAL) assay from Cambrex (USA).

Sandwich ELISAs were employed to measure TNF, (Pharmingen, UK), IP-10 (R&D, USA) and IL- lβ (Bioscource, USA). Absorbance was read on a spectrophotometric ELISA plate reader (Labsystems Multiscan Biochromic) and analyzed using Ascent software V2.6 (Thermo Labsystems, Cambridge, United Kingdom). All results are expressed as the mean cytokine concentration ± SD obtained from triplicate cultures per condition. Cell viability was not significantly affected over this time period when examined by the 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay (Sigma).

Statistical methods Mean, standard deviation (SD) and statistical significance were calculated using GraphPad version 3 (GraphPad Software Inc., USA). For statistical analysis, a one tailed student's t-test of paired data was used with a 95% confidence interval and the Mann-Whitney test.

Cell culture

RA synovial membrane cells were isolated from patients undergoing joint replacement surgery as previously descπbed (Foxwell et al., Proc. Natl. Acad. Sci. USA 95,8211-8215, 1998). All patients gave wπtten informed consent and the study was approved by the local ethics committee. Immediately after isolation, cells were used for mRNA analysis, stained by FACS or cultured at IxIO⁵ cells/well in 96-well tissue culture plates (Falcon, UK) in RPMI 1640 containing 10% (v/v) FBS and 100 U/ml penicillin/streptomycin. Peπpheral blood monocytes were isolated and cultured as previously descπbed (Foxwell et al., Proc. Natl. Acad. Sci. USA 95,821 1-8215, 1998). Macrophages were deπved from monocytes after differentiation for 4 days with 100 ng/ml M-CSF (PeproTech, UK). The mouse macrophage cell line RAW 264.7 was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.

Example 1 Rheumatoid arthritis synovial membrane cells were cultured for 24 hours in the presence of media alone or media containing 5μg/ml orlOμg/ml fluoxetine hydrochloπde ("PROZAC"). Supernatants were harvested and levels of cytokines (A) TNF or IL- 1 were measured by ELISA Addition of fluoxetine hydrochloπde ("PROZAC") at the time of culture led to a dose dependent decrease in the levels of TNF and IL-I (Figure 1, Graph A). Rheumatoid arthritis synovial membrane cells were also cultured in the presence of media alone or media containing lOμg/ml, 20μg/ml and 30μg/ml citalopram and levels of TNF were measured. TNF production was dose dependently inhibited in cells treated with citalopram (see Figure IB).

To check if the ability of citalopram and fluoxetine hydrochloride to inhibit these cytokines was due to increased serotonin in the cultures, the cells were incubated with 10 or lOOμM serotonin for 24 hours. After 24 hours supernatants were collected and TNF and IL-I levels determined (n=5). The serotonin had no effect on the amount of TNF (Figure 7, Graph A) produced from these cultures, but increased the amount of IL-I being produced at the higher dose of serotonin (Figure 7, graph A).

These results illustrate that citalopram inhibits the spontaneous release of TNF and fluoxetine hydrochloride inhibits the spontaneous release of TNF and EL- lβ from rheumatoid synovial membrane cultures.

Example 2

The murine CIA model has been shown to be clinically similar to human rheumatoid arthritis with a comparable synovitis, bone erosion and panus formation.

Male DBA mice (8-10 weeks of age) were immunized at the base of the tail intradermally with 200μg type II collagen emulsified in CFA (Difco Laboratories, West Molesey, U.K.). The onset of arthritis was considered to be the day that erythema and/or swelling were first observed, and arthritic mice were given a daily clinical score per limb from 0 to 3, with 0 = normal, 1 = slight erythema and swelling, 2 = pronounced edematous swelling and 3 = joint deformity with ankylosis, resulting in a maximum score of 12 per animal. Citalopram or fluoxetine hydrochloride treatment started on the day of onset and was administered daily for 7 days.

The experiment was started at the onset of arthritis (day 1). Mice were given an interperitoneal injection of vehicle (PBS) or 25mg/kg of citalopram or fluoxetine hydrochloπde once a day for 7 days Mice were assessed for (Figures 2A and 3A) clinical score on a daily basis.

Results after treatment with citalopram Mice treated with citalopram at the day of onset of arthritis (day 1) exhibited an attenuated disease progression over a 7 day peπod. Disease pathogenesis was assessed by measuring clinical score The mice treated with citalopram showed a significant improvement to the control group from day 2-3 onwards (Figure 2, Graph A) Histology samples taken from the hind paws of mice treated with citalopram showed a decreased histology score over the control group (Figure 2B).

These results illustrate that citalopram inhibits disease progression in the CIA model of rheumatoid arthritis.

Results after treatment with fluoxetine hydrochloride

Mice treated with fluoxetine hydrochloπde at the day of onset of arthritis (day 1) exhibited an attenuated disease progression over a 7 day peπod. Disease pathogenesis was assessed by measuπng clinical score. The mice treated with fluoxetine hydrochloπde showed a significant improvement to the control group (Figure 3, Graph A, B). This effect of fluoxetine hydrochloπde was dose dependent as 10mg/kg showed a significant improvement throughout the study from the vehicle control, whilst the lOmg/kg group showed a small but not significant improvement (Figure 3, Graph A). This was also reflected in the measurement of hind paw swelling that also showed a significant improvement throughout the study for both the 10mg/kg and 20mg/kg treated groups (Figure 3, Graph B).

These results illustrate that fluoxetine hydrochloπde inhibits disease progression in the CIA model of rheumatoid arthπtis

Example 3

Pπmary human macrophages and munne RAW 264 7 cells were incubated with fluoxetine hydrochlonde for 30 minutes and then stimulated for 6 hours with TLR ligands (R-848, LPS, flagellin, malp-2, poly IC or CpG). Human macrophages showed a decreased production of TNF in response to R-848 but not LPS, flagellin or Malp-2 in the presence of fluoxetine hydrochloride (Figure 4, Graph A).

Murine RAW 264.7 cells were used to examine poly IC and CpG induced TNF, as unlike human macrophages they respond to poly IC a TLR3 ligand and CpG, a TLR9 ligand to produce TNF. Fluoxetine hydrochloride was able to inhibit TNF production in these cells on stimulation with poly IC (Figure 4, Graph B) and CPG (Figure 4, Graph C).

This shows that signaling from toll-like receptors (TLRs) 3, 8 and 9 are selectively inhibited by fluoxetine hydrochloride and that it does not have general antiinflammatory properties as it was unable to inhibit TNF stimulated by activation of the other TLRs.

Example 4

Selective inhibition of TLRs by fluoxetine hydrochloride is also observed for other anti-depressants (SRIs). Citalopram, fluoxetine hydrochloride ("PROZAC") and sertraline ("ZOLOFT") all decreased TNF production in human macrophages stimulated by R848, but did not inhibit LPS induced TNF in the same cells (Figure 5, Graph A). Figure 5B illustrates that sertraline ("ZOLOFT"), like fluoxetine hydrochloride selectively inhibits TLR8 induced TNF in human macrophages (Figure 5, Graph B).

Example 5

TLR3, 7, 8 and 9 are expressed in human RA synovial tissue. mRNA from synovial tissue was extracted, DNAase treated and then analysed by RT-PCR. Expression of message for TLR3, 7, 8 and 9 was detected by RT-PCR from the mixed population of cells in human rheumatoid synovial tissue in three unrelated donors 1-3 (Figure 6, Graph A). TLR3, 8 and 9 were further investigated using FACS. Cells were washed, then blocked with 10% human serum in PBS containing 0.01% azide for 30 minutes at 4°C or for intracellular staining cells were fixed in 2% paraformaldehyde and permeabilised with 0.1% saponin (Sigma, USA) before blocking. Cells were then incubated with FITC conjugated α-TLR3, α-TLR8, α-TLR9 or isotype controls for lhr at 4⁰C and then washed before analysis on a Becton-Dickinson LSR flow cytometer. A directly conjugated antibody was not available to examine TLR7 expression.

TLR3, 8 and 9 protein expression was detected at low levels on the cell surface but was more readily detected upon intracellular staining (Figure 6, Graph B).

To check if these TLRs were functional in synovial membrane cultures, cells were stimulated with the appropriate TLR ligands at the time of seeding the cells (Figure 6, Graph C). These cells spontaneously release cytokines into the media. TNF production was increased on stimulation of cells with Poly IC for TLR3 (334% ±60 P=0.0002, n=12) and R-848 for TLR7/8 (515% ±76 P<0.0001, n=l l), but imiquimod, a TLR 7 ligand and CpG a TLR9 ligand (see Figure 6, Graph C) were unable to stimulate further release of cytokines over the spontaneous levels. This demonstrates that TLR 3 and 8 are both functional in rheumatoid synovial membrane cultures.

In summary, these results shows that TLRs 3, 7, 8 and 9 are expressed in rheumatoid membrane synovial cells but only TLRs 3 and 8 functionally induce increased TNF production in rheumatoid membrane synovial cells.

Example 6

The main mechanism of action of the antidepressants (SRIs) used in this study is to increase the amount of serotonin by inhibiting serotonin reuptake receptors. As we have shown that two serotonin reuptake inhibitors suppress TNF production from human rheumatoid synovial membrane cultures, serotonin was tested in these cultures. At the time of seeding the cells 10 or lOOμM serotonin was added to the culture media Supernatants were collected after 24 hours and the levels of TNfF and IL-I were compared with the spontaneously produced levels in the culture Serotonin had no effect on TNF levels (n=6) (Figure 7, Graph A) but significantly increased the levels of IL-I (n=5) (Figure 7, Graph A).

Fluoxetine hydrochloπde ("PROZAC"), citalopram and sertraline have been demonstrated as being able to inhibit selective TLRs TLR3, 7, 8 and 9 are classically viewed as having an endosomal localization. Chloroquine has been reported to inhibit the function of TLR3, 7, 8 and 9, by increasing the pH inside vesicles, which is essential for functional signaling from TLR3, 7, 8 and 9. To confirm that the inhibition of cytokine release from RA cultures may be through the ability of these SRIs to inhibit signaling through endosomal TLRs, we also measured cytokine release in cells incubated with chloroquine There was a dose dependant effect of chloroquine with the best inhibition being observed at lOOμM, both TNF and EL-I release were inhibited (Figure 7, Graph B).

Thus, chloroquine inhibits spontaneous TNF and EL-I release but serotonin does not

Claims

1 A serotonin reuptake inhibitor (SRI) for treating inflammatory arthritis or a related condition

2 The serotonin reuptake inhibitor according to claim 1, wherein the serotonin reuptake inhibitor is a selective serotonin reuptake inhibitor or a serotonin norepinephπne reuptake inhibitor

3 The serotonin reuptake inhibitor according to claim 1, wherein the serotonin reuptake inhibitor is fluoxetine, citalopram or sertraline

4 The serotonin reuptake inhibitor according to any one of claims 1 to 3, wherein the inflammatory arthritis is rheumatoid arthritis

5 The serotonin reuptake inhibitor according to any one of claims 1 to 4, wherein the serotonin reuptake inhibitor is administered in combination with a further anti-inflammatory agent

6 The serotonin reuptake inhibitor according to claim 5, wherein the further anti-inflammatory agent is a non-steroidal anti-inflammatory agent (NSADD), a statin, a disease modifying anti-rheumatic drug (DMARD), a biological agent, a steroid, RNAi, aptemers, an immunosuppressive agent, a salicylate and/or a microbicidal agent

7 Use of a serotonin reuptake inhibitor (SRI) in the manufacture of a medicament for treating inflammatory arthritis or a ielated condition

8 A kit composing a pharmaceutical composition compπsing a serotonin reuptake inhibitor (SRI) and instructions indicating that the composition is for use in treating inflammatory arthritis or a related condition

9. The kit of claim 8, wherein the serotonin reuptake inhibitor is a selective serotonin reuptake inhibitor or a serotonin norepinephπne reuptake inhibitor

10 The kit of claim 8, wherein the serotonin reuptake inhibitor is fluoxetine, citalopram or sertraline

11 This kit according to any one of claims 8 to 10, wherein the inflammatory arthritis is rheumatoid arthritis.

12 The kit according to any one of claims 8 to 11 wherein the serotonin reuptake inhibitor is administered in combination with a further anti-inflammatory agent.

13. The kit according to claim 12, wherein the further anti-inflammatory agent is a non-steroidal anti-inflammatory agent (NSAED), a statin, a disease modifying anti- rheumatic drug (DMARD), a biological agent, a steroid, an immunosuppressive agent, a salicylate and/or a microbicidal agent

14. A method of treating inflammatory arthritis or a related condition in a subject, comprising administration of a serotonin reuptake inhibitor to the subject.

15. The method of claim 14, wherein the serotonin reuptake inhibitor is a selective serotonin ieuptake inhibitor or a serotonin norepinephπne reuptake inhibitor

16 The method of claim 14, wherein the serotonin reuptake inhibitor is fluoxetine, citalopram or sertraline

17 The method of any one of claims 14 to 16 wherein the subject is a mammal.

18 The method of claim 17, wherein the mammal is a human.

19 The method of any one of claims 14 to 18, wherein the inflammatory arthritis is rheumatoid arthritis. 20 The method according to any one of claims 14 to 19, wherein the serotonin reuptake inhibitor is administered in combination with a further anti-inflammatory agent

21 The method according to claim 20, wherein the further anti-inflammatory agent is a non-steroidal anti -inflammatory agent (NSAID), a statin, a disease modifying anti-rheumatic drug (DMARD), a biological agent, a steroid, an immunosuppressive agent, a salicylate and/or a microbicidal agent