WO2007096596A2 - Use of il-8 for the treatment of crohn' s disease - Google Patents

Abstract

The invention relates to use of a pro-inflammatory agent such as IL-8 or muramyl dipeptide (MDP) for the manufacture of a medicament for Crohn's disease. Preferably the pro-inflammatory agent is a neutrophil chemoattractant. Preferably the pro- inflammatory agent is EL-8. The invention also relates to a method of treating Crohn's disease in a subject, said method comprising administering to said subject an effective amount of a pro-inflammatory agent such as IL-8, and to IL-8 for use in the treatment of Crohn's disease. The invention also relates to products comprising IL-8 for topical treatment. The invention also relates to methods for determining the responsiveness of a subject to MDP comprising determining the CARD15 genotype of the subject.

Description

IL-8 Therapy

Field of the Invention

The present invention is in the field of Crohn's disease (CD; inflammatory bowel disease (EBD); ulcerative colitis). Ia particular the invention relates to proinflammatory and/or neutrophil chemoattractant therapies and compositions for treating Crohn's disease.

Background to the Invention

Crohn's disease (CD) is a chronic inflammatory disorder, the incidence of which rose dramatically in the latter part of the 20^th century. It primarily affects the bowel, but can also involve the musculoskeletal system, skin and eyes. Key symptoms include refractory inflammatory ulceration and damage to the intestine. The aetiology remains an enigma although many diverse causes have been proposed.

The diagnosis is currently established on the basis bf clinical, endoscopic, radiological, and histological findings. Characteristic pathological appearances include the formation of "skip lesions" (discrete regions of inflamed bowel separated by uninvolved mucosa), aphthous ulceration and fistulation; these relate to the presence of -an underlying granulomatous transmural inflammation. The search for a microbial cause has been extensive due to the similarity between features of CD and those produced by infection with organisms such as Mycobacteria. The other principal theory is that inflammation arises as a primary "autoimmune" phenomenon, for which both humoral and cellular mechanisms have been implicated.

The view in the art is that Crohn's arises from abnormal modulation of inflammation, based on inadequate suppression so that inflammation becomes chronic. Chemoattraction of neutrophils is a known phenomenon. EL-8 is known as a chemoattractant. However, using EL-8 is technically difficult. Firstly, IL-8 is a protein which means it is susceptible to proteases. Furthermore, intact proteins can be difficult to use or to absorb in patients. These and other characteristics make IL-8 extremely hard to work with.

A large number of chemoattractants are known in the art. Polysaccharide chemoattractants are most popular for use in the bowel since they offer the advantage of being present in the gut in healthy individuals. In addition, the chemoattractant lipopolysaccharide (LPS) is a known chemoattractant derived from bacterial coats. This is well understood and easy to work with, and straightforward to syπthesise in vitro. Other known chemoattractants include enterotoxin, C5a and FMetLeuPhe.

PCT/USOl/24857 discloses GM-CSF therapy for Crohn's disease. GM-CSF treatment for Crohn's disease is an unpleasant and painful treatment. GM-CSF has toxic side effects and induces a febrile response. Furthermore, GM-CSF is a non-specific treatment which affects both neutrophils and macrophages. At best, GM-CSF treatment will raise neutrophil levels in the blood simply by a mass action type effect, stimulating their production by, and emigration from, bone marrow. GM-CSF has no effect on recruitment of neutrophils circulating in the blood to a site of insult or injury.

GM-CSF has been used to try to treat active disease, but not as a prophylactic.

Korzenik et al. (2005 NEJM vol 352 pp2193-201) disclose a trial of Sargramostim (GM-CSF) in active Crohn's disease. Patients received 6ug/kg subcutaneously for 56 days. Although there was no statistically significant outcome at the primary endpoint, the secondary endpoint showed a significant effect in the sargramostim group. This group also showed significant improvements in quality of life, together with decreased disease severity.

Further prior art treatments have included combination of lactococcus with IL-10. However, once again this is an anti-inflammatory treatment. Based on the understanding of Crohn's disease as a chronic inflammatory disorder, the prior art has expectedly focused on suppression of inflammation and on IL-8 antagonists in particular.

The present invention seeks to overcome problem(s) associated with the prior art.

Summary of the Invention

The present invention is based on the surprising discovery that there is an IL-8 deficiency in Crohn's disease. Specifically, the deficiency is in stimulating adequate IL-8 production. This unexpected finding enables a deeper understanding of Crohn's disease.

As is surprisingly disclosed by the present inventors, a deficiency in Crohn's disease is understood to be the migration of neutrophils into the affected area. In particular, the defect is in the recruitment of neutrophils. Prior art therapies such as the use of GM-

CSF have focused on increasing the numbers of neutrophils. For example, GM-CSF treatment increases neutrophil output from bone marrow. It is possible that GM-CSF treatment also enhances activation ef neutrophils. However^ this treatment can never increase recruitment of neutrophils. It is surprisingly disclosed herein that recruitment of the neutrophils is the rate limiting step leading to some of the defects in the acute or local inflammatory response in Crohn's disease. Prior art methods such as GM-CSF treatment merely increase the number of circulating neutrophils. However, there is no defect in circulating neutrophil .number in Crohn's disease.

Thus, in a broad aspect the present invention relates to the use of a neutrophil chemoattractant in the treatment or prevention of Crohn's disease.

Based on these findings, the inventors decided to apply IL-8 therapies in Crohn's disease for beneficial effect. Without knowledge of the IL-8 deficiency in Crohn's disease, which is disclosed herein the first time, then the numerous technical difficulties involved in working with IL-8 coupled with its proteinacious nature would dissuade a person skilled in the art from attempting to use it in the context of Crohn's. Furthermore, if a skilled worker decided to try to base a therapy for Crohn's on a neutrophil chemoattractant, then there are numerous established and well understood chemoattractants known in the art, each of which is more tractable and experimentally useable than IL-8.

Furthermore, the view in the prior art is that Crohn's is caused by an exaggerated inflammatory response. The use of a pro-inflammatory agent is therefore counterintuitive and goes against the established view in the art.

In a broad aspect the invention relates to the use of a pro-inflammatory agent in the treatment or prevention of Crohn's disease.

Thus, contrary to the established understanding in the art, the present invention provides IL-8 based therapies for Crohn's disease.

The advantages of the present invention include the use of IL-8 as a prophylactic.

IL-8 may advantageously be used topically at surgery to induce better wound healing and increased anastomotic healing.

In the prior art, efforts are being focused on IL-8 antagonists. Clearly, given the teachings of the present application, this is precisely opposite to the beneficial effects disclosed herein.

In support of the above, the inventors have disclosed that LPS is of limited use in the context of Crohn's. LPS receptors are downregulated in non-inflamed bowel, limiting its utility as a prophylactic agent. LPS is endogenously in high concentrations in both normal and Crohn's bowel, so further increasing its concentration is unlikely to be of benefit. Furthermore, it is believed that MDP is of use and that the most useful application is likely to be in, but not restricted to, the large bowel. MDP is likely to be of most use in patients having at least one wild-type CARD 15 allele.

It should be noted that the EL-8 therapies of the present invention are not working through a vascular effect but are working through a chemoattractant effect Le. a recruitment effect. According to the present invention IL-8 treatment is applicable for all Crohn's patients. Preferably IL-8 treatment is used to address severe colonic Crohn's disease. Advantageously, the present invention propels neutrophils into the bowel wall and therefore directly addresses the defect in acute inflammation.

Preferably the neutrophil chemoattractant treatments disclosed herein are topical. Topical treatments find particular application in small bowel disease and in large bowel disease.

In a broad embodiment, the invention relates to the use of IL-8 agonists in the treatment of acute inflammation, preferably in the treatment of acute (local) inflammation caused by Crohn's disease. It is disclosed herein for the first time that there is an IL-8 deficiency in Crohn's disease.

Without wishing to be bound by theory, it is suggested herein that macrophage make IL-8, and that this attracts neutrophils. It is further suggested that macrophage cause vascular dilatation (through release of mediators such as nitric oxide), which also serves to bring in more neutrophils. When these mechanisms break down, and efficient clearance of luminal contents from the bowel wall fails, it results in increased macrophage activation and increased IL-6 levels and therefore produces chronic inflammation. Thus, according to the present invention, techniques producing increased local blood flow may be used either as a prophylactic or as a therapeutic as described herein in order to ameliorate these effects. Furthermore, enhancement of relevant (local) IL-8 levels, either by supply of IL-8 or induction of IL-8 production, may be used in order to ameliorate these effects, or as a prophylactic. In a preferred embodiment, a combination of IL-8 and blood flow stimulation is employed. Prior art techniques such as GM-CSF treatment relate only to bone marrow production of immune cells, and can generate high cell numbers in the systemic circulation but have no specific effect ie. levels of monocytes, macrophages and neutrophils are all elevated. Furthermore, this approach is mostly used for active disease and is toxic, inducing a febrile response upon injection. By contrast, the present invention, particularly in the preferred embodiment of IL-8 administration, advantageously induces neutrophil recruitment to tissue, and provides an effect specific to neutrophils, and exerts minimal systemic side effects.

Thus, in one aspect the invention provides use of a pro-inflammatory agent for the manufacture of a medicament for Crohn's disease.

Preferably said pro-inflammatory agent is IL-8 or muramyl dipeptide (MDP), preferably said pro-inflammatory agent is IL-8.

Preferably said pro-inflammatory agent is a neutrophil chemoattractant.

In another aspect, ttie invention provides a method of treating Crohn's disease in a subject, said method comprising administering to said subject an effective amount of a pro-inflammatory agent.

In another aspect, the invention provides IL-8 for use in the treatment of Crohn's disease.

In another aspect, the invention provides IL-8 for use in the treatment of impaired acute inflammation.

In another aspect, the invention provides use of IL-8 in enhancement of acute inflammation. In another aspect, the invention provides use of IL-8 in the enhancement of acute inflammatory response in Crohn's disease.

In another aspect, the invention provides use of IL-8 as a topical therapy to aid healing following surgery.

In another aspect, the invention provides use of a neutrophil chemoattractant in the treatment of Crohn's disease.

Ia another aspect, the invention provides use of muramyl dipeptide (MDP) as a proinflammatory agent.

In another aspect, the invention provides use of muramyl dipeptide (MDP) to increase IL-8 production.

In another aspect, the invention provides muramyl dipeptide (MDP) for use in the treatment of Crohn's disease.

In another aspect, the invention provides muramyl dipeptide (MDP) for use in the treatment of impaired acute inflammation.

In another aspect, the invention provides a method for determining whether a subject is likely to respond to MDP as a pro-inflammatory agent, said method comprising determining the CARD 15 genotype of said subject, wherein detection of at least one CARD 15 wild type allele indicates that the subject is likely to be responsive to MDP as a pro-inflammatory agent. The invention also relates to a method of treating a subject with MDP comprising determining the CARDl 5 genotype of said subject, and administering MDP only to a subject having at least one CARD 15 wild type allele.

In another aspect, the invention provides a composition comprising a proinflammatory agent and a PDEV inhibitor. In another aspect, the invention provides a composition as described above for use as a medicament.

In another aspect, the invention provides a composition as described above for use in the treatment of Crohn' s disease.

In another aspect, the invention provides use of a composition as described above for the manufacture of a medicament for Crohn's disease.

In another aspect, the invention provides a method of treating Crohn's disease in a subject, said method comprising administering to said subject an effective amount of a PDE5 inhibitor and an effective amount of a pro-inflammatory agent such as IL-8.

Preferably the PDE5 inhibitor is sildenafil citrate, vardenafil or tadalafil.

Preferably the PDE5 inhibitor is sildenafil citrate (Viagra™).

hi another aspect, the invention provides a kit comprising at least one dose of a proinflammatory agent such as EL-8 and at least one dose of a PDEV inhibitor, together with instructions for their administration to a subject having Crohn's disease.

The invention also relates to products comprising IL-8 for topical treatment. Thus, in another aspect, the- invention provides a patch, dressing or swab comprising IL-8. In another aspect, the invention provides a cream, foam, enema or suppository comprising IL-8. The patch, dressing or swab, cream, foam or suppository is for the topical treatment of Crohn's disorders, ie. for the topical enhancement of the acute inflammatory response, preferably the acute local inflammatory response. These products are preferably to be directly applied to the area in need of treatment, such as the bowel. Preferably they are applied topically according to need. They may be applied prophylactically in advance of insult or injury to the tissue being treated.

Preferably the pro-inflammatory agent is IL-8. A key concept in the present invention is the change in approach compared to the prior art. The prior art is directed at suppressing inflammatory responses. This was based on the understanding of Crohn's as a chronic inflammatory disorder. However, the approach in the present invention is markedly different in that it centres on the enhancement or elevation of an inflammatory response in Crohn's, that is to say enhancement or elevation of the acute inflammatory response which is disclosed as the primary defect in Crohn's by the present inventors.

Detailed Description of the Invention

Definitions

In the context of Crohn's disease, 'treatment of acute inflammation' refers to enhancement or normalisation of acute inflammation or acute local inflammation, ie. treatment of the impaired acute inflammation which underlies Crohn's. Specifically,

'treatment of acute inflammation' in the context of Crohn's does not mean suppression of acute inflammation since, as disclosed herein, this acute response is impaired in

Crohn's and needs to be restored in order to ameliorate or avoid the long term or chronic inflammation typically seen in Crohn's disease.

In discussion of PDEV inhibitors, these may be classified as "short acting" or "long acting". Long acting inhibitors generally have a longer half life. Long .acting inhibitors are preferred. Whether an inhibitor is short or long acting may be easily determined following the established view in the art as set out in Seftei (2004 Clin Cardiol Apr;27(4 Suppl 1):I14-I19).

A 'CARD 15 mutant' allele refers to a non-wild-type allele. There are several non- wild-type CARD 15 alleles known. Each or any of those is considered a 'CARDlS mutant' as described herein. Thus, an individual will be regarded as a 'CARD15 mutant' if that individual possesses no wild-type CARD 15 alleles. Thus, a heterozygous individual possessing one wild-type allele will not be considered a 'CARD 15 mutant' even though they possess one mutant (polymorphic or non-wild- type) allele. 'CARD 15 mutants' as described herein are individuals having no CARDl 5 wild-type alleles. Such individuals may be homozygous for a particular CARD 15 polymorphic allele, or may be heterozygous in the sense that they possess two non-matching non-wild-type alleles; what is important is the absence of at least one wild-type CARD15 allele.

A 'pro-inflammatory agent' has its normal meaning in the art. The pro-inflammatory agent may be any chemical entity having the effect of promoting acute inflammation. Preferably the pro-inflammatory agent is one which promotes acute local inflarnmation. The pro-inflammatory agent may be a cytokine, preferably a chemokine. Preferably the pro-inflammatory agent is a neutrophil chemoattractant Preferably the pro-inflammatory agent is IL-8 or MDP, most preferably IL-8.

Crohn's Disease

Acute inflammation such as acute local inflammation is required to clear foreign material from the bowel wall. One view is that the defect in Crohn's is an immunodeficiency in which a constitutionally weak response predisposes to accumulation of any intestinal contents that breach the mucosal barrier. Consequent failure of the clearance of bacteria and other foreign material results in granuloma formation and chronic inflammation as a secondary phenomenon. Whilst polymorphisms in CARD15 do not underlie this phenotype, they incapacitate the NOD2 pathway which can compensate for this impairment of innate inflammation. Current treatment of the secondary chrome inflammation seen in active CD with immunosuppressants might exaggerate the underlying lesion and promote chronicity.

It is disclosed herein that Crohn's does not present a physical/structural problem with blood vessels, but is rather caused by a low/impaired inflammatory response. Furthermore, Crohn's is characterised herein by a generalised lack of inflammation, not just restricted to the bowel. As well as finding application in Crohn's disease, the present invention also finds application in any idiopathic granulomatous disorder, of which sarcoidosis is one example. Sarcoidosis is characterised by a granulomatous inflammatory response similar to that observed in Crohn's disease. Although sarcoidosis is predominantly concerned with the lungs, it is known to affect any organ and has a similar pathology to Crohn's disease. Indeed, the cytokines underlying aspects of the immune response are common to those in Crohn's disease. Furthermore, many of the symptoms overlap between sarcoidosis and Crohn's disease. Thus, in one aspect of the invention, the invention provides for the treatment of sarcoidosis using phospodiesterase 5 inhibitor as described herein. In another aspect, the invention provides for the treatment of sarcoidosis using a pro-inflammatory agent, preferably IL-8. Advantageously PDE and pro-inflammatory treatments may be combined.

The prior art has focused on changes in blood vessel organisation in Crohn's. By contrast, the present inventors disclose that the defects underlying Crohn's disease are not related to structural changes in the vasculature, but are in fact related to regulational deficiencies in blood flow. One of the key findings presented herein is that it is dysregulation of the responses concerning blood flow which underlie Crohn's disease, which is contrary to the thinking in. the art of peripheral vascular disease. There is no evidence of peripheral vascular disease in Crohn's disease. Furthermore, there are no problems with the structure of the blood vessels in Crohn's disease and no inflammation of the vessels. However, the defect has been pinpointed to the changes in blood flow which should be elicited by local injury and acute (local) inflammation.

It is this response which is compromised in Crohn's disease. The present invention addresses this and builds on this novel finding.

It is a key distinction from the prior art that the present invention is based on regulational methods and therapies whereas the prior art has been focused on characterising structural defects in Crohn's disease. Furthermore, prior art therapies have been focused on only the inflamed region. However, as is disclosed herein, the present invention relates to a generalised treatment which would be of use to the subject as a whole. Furthermore, by characterising the underlying defect, local or topical therapies resulting in increased blood flow may also be applied according to the -present invention. It is a further advantage of the present invention that the behaviour of uninvolved vessels can advantageously be restored to behaviour closer to that of a normal subject than that of a Crohn's affected subject by the methods disclosed herein. 5 However, it should be noted that the behaviour of uninvolved vessels may in fact be an effect downstream of inflammation, whereas the emphasis of the present invention is on the pro-inflammatory response itself.

IL-8

10.

It is disclosed herein that Crohn's macrophages have a defect in producing IL-8 after stimulation with specific agonists. In the context of an acute inflammatory reaction, this leads to reduced IL-8 production and subsequently lower numbers of recruited neutrophils. Failure to recruit adequate numbers of neutrophils leads to defective

15 clearance of bacteria and the chronic inflammation characteristic of Crohn' s.

According to the present invention, a key aim of therapy is to replace the IL-8 that is not produced normally and/or sufficiently in Crohn's patients. According to the present invention, this can be achieved by supplying IL-8 exogenously, or by 0 supplying an alternative neutrophil artractant exogenously, or by stimulating endogenous production by macrophages through a different signaling pathway.

It must be noted that GM-CSF does not achieve any of these aims. It does not increase the numbers of neutrophils at site of insult to the tissues. Nor would it increase IL-8 5 production at these sites. Its action to increase systemic neutrophil numbers would not exert any major clinical effects. Although both G-CSF and GM-CSF can cause neutrophil chemotaxis in vitro, there is no evidence that they have this effect when administered to patients in vivo, and the data available show that they are likely to suppress it which is the opposite of the teaching of the present invention. Indeed, GM- 0 CSF diminishes migration across endothelium in vitro and diminishes neutrophil chemotaxis to IL-8 in vitro. Moreover, G-CSF may increase migration across endothelium in vitro, but reduces migration into tissues in vivo. Furthermore, the prior art does not demonstrate that techniques taught herein such as use of exogenous IL-8 could correct the problem. It was not known in the art that defect(s) in cytokine production correlate with Crohn's, nor whether any such defect would be causal or epiphenomenal. By contrast, we show that there is an intrinsic failure of IL-8 production by Crohn's macrophages. We show that replacement of this specific factor (IL-8) corrects the defect. Based on these findings, the invention relates to use of IL-8 as a therapeutic agent in Crohn's disease.

In one aspect the invention relates to the use of IL-8 as a topical therapy for enhancing acute and/or acute local immune response.

Preferably IL-8 is used as a slow release formulation.

Preferably IL-8 is used as an enteric coated formulation.

Preferably digestion of the IL-8 protein by alimentary proteases is avoided. To this end, protease inhibitors may be co-administered with IL-8, and/or proteins may be used as exipients or fillers in administration of the IL-8 to competitively inhibit protease action.

A preferred mode of administration for the IL-8 medicaments, of the invention is by enema.

Preferably when using slow release formulations, for example orally ingested formulations, these are designed to release the EL-8 in the terminal ileum.

According to the present invention IL-8 can be used to treat any type of Crohn's disease.

Preferably IL-8 is used to treat severe colonic Crohn's disease.

The advantages of the present invention include the use of IL-8 as a prophylactic. IL-8 may advantageously be used topically at surgery to induce better wound healing and increased anastomatic healing.

In a preferred embodiment, a combination of IL-8 therapy and blood flow stimulation therapy (eg. vasodilation treatment) is employed.

It is demonstrated herein that following acute trauma to either the skin or bowel, Crohn's patients fail to recruit neutrophils. This impairs their ability to clear bacteria and other organic bowel contents that gain access to the bowel wall, underlying the chronic granulomatous inflammation characteristic of active disease. We disclose evidence derived from injecting these patients subcutaneously with killed Escherichia coli that this defect translates into a significant impairment in the local inflammatory response. This in turn engenders a systemic pro-inflammatory state.

In both the skin and bowel, we teach that the diminished cellular recruitment correlates with attenuated production of IL-8, an established potent stimulus to neutrophil recruitment. We demonstrate that deficient IL-8 production is directly responsible for the failure of cell migration, and show that replacement of IL-8 in skin windows (either using exogenous recombinant protein or boosting endogenous production with the immune adjuvant muramyl dipeptide) augments neutrophil numbers to normal levels.

The invention also relates to topical delivery of IL-8 to Crohn's lesions in the gastrointestinal tract. This can be accomplished directly or by supplying an immune stimulant that leads to increased IL-8 production. According to the present invention,

IL-8 in the bowel selectively penetrates the mucosa in regions of ulceration and induces an acute inflammatory response, such as an acute local inflammatory response.

This advantageously accelerates clearance of bacteria and other organic materials that have accumulated, hastening remission. CAKD15

Detailed genome-wide linkage analyses in Crohn's affected families have identified a number of susceptibility loci (IBD1-IBD8) for CD. The strongest association is attributable to the N0D2/ CARDl 5 gene located within the IBDl locus. Carriage of a single disease-associated polymorphism confers a 2-4-fold increased risk of CD; this rises to 20-40-fold in the presence of two variant alleles. In the prior art, the mechanism of this predisposition has remained unclear.

NOD2ICAKD15 encodes a cytoplasmic protein, expressed predominantly in mononuclear phagocytes. It is involved in recognition of muramyl dipeptide (MDP), a component of peptidoglycan present in cell walls of gram-positive and gram-negative bacteria and found in high concentrations in the bowel lumen. Binding of MDP activates the transcription factor NF-κB, leading to induction of genes for pro- inflammatory cytokines. Consequently, the polymorphisms associated with CD that abolish the response to MDP should attenuate inflammation. This contrasts both with the pathology of Crohn's lesions, which are obviously inflamed and contain proinflammatory cytokines, and the therapeutic efficacy of immunosuppressive drugs. The situation is further confused by observations from transgenic mice possessing Nod2 mutations that produce a truncated protein. Whilst this was believed to be identical to one of the human polymorphisms, the effect appeared opposite: MDP induced elevated NF-κB activation and more efficient processing and secretion of IL- lβ. This favours a pro-inflammatory mechanism.

Despite the robust association, ^"NOO2/CARD15 is not strongly mechanistically xelated to the causation of CD. Polymorphisms exhibit very limited penetrance and occur in only 40% of patients (predominantly those with ileal disease) as well as in 15% of

■ healthy individuals. The possibility exists that polymorphisms in NOD2/C4&D75 are not in themselves causal, but modify the immune response in CD lesions elicited by some other mechanism. The experimental sections of this document examine acute inflammatory responses in CD, particularly acute local inflammatory responses in CD, and to relate them to NOD2/CAKD15 genotype.

CARD 15 was previously thought to be involved in down regulation of inflammation. However, the present inventors used muramyl dipeptide to explore the function of CARDl 5. Muramyl dipeptide (MDP) is a pro-inflammatory material. However, when applied to CARD 15 mutants, MDP loses its pro-inflammatory effect. It is shown herein that these observations are caused by an impairment of the pro-inflammatory response in CARD 15 mutants. Thus, in contrast to the prior art view that CARD 15 is a down regulator of inflammation, it is shown herein for the first time that in fact CARD 15 is involved in the pro-inflammatory response. Thus, CARD 15 mutants show an impaired pro-inflammatory response.

Thus, in one embodiment, the invention relates to a method for determining whether a subject would be responsive to MDP as a pro-inflammatory, said method comprising determining the CARD 15 genotype of said subject. Detection of homozygous or heterozygous CARD 15 wild type genotype indicates that the subject is likely to be responsive to MDP as aφro-mflammatory material. Detection of homozygous mutant CARD 15 genotype, indicates that the subject is unlikely to react in a: pro-inflammatory manner to the application of MDP.

It is disclosed herein for the first time that muramyl dipeptide is pro-inflammatory. Using muramyl dipeptide (MDP) gets neutrophils in some patients but not others - as noted above this is due to the CARD.15 genotype of those patients. It should be noted that IL-8 treatment is not effected by CARD15. This has been demonstrated using double knockout subjects. Furthermore, it should be noted that approximately 15% of the population are heterozygous for CARD 15, and that approximately 1% of the population are homozygous negative CARD 15 mutants. However, in the context of Crohn's disease, approximately 50% of Crohn's patients are CARD 15 heterozygous, and approximately 30% are homozygous CARD15 negative. Thus, prior art studies of MDP have tended to conclude that it is anti-inflammatory since a lack of response was usually observed, and anti-inflammatory cytokines such as IL-IO were upregulated. The present inventors show that normally pro-inflammatory cytokines are upregulated in advance of any anti-inflammatory cytokines and that it is the loss of this proinflammatory phase in Crohn's disease that is of importance. However, as disclosed herein for the first time, the underlying reason for lack of response to MDP is found in the CARD 15 genotype, and not in the presumed anti-inflammatory effect of MDP. Thus, it is demonstrated herein for the first time that MDP is in- fact pre-inflammatory. It is further demonstrated that CARD 15 homozygous negative mutants will not respond to MDP.

Thus, the present invention relates to use of muramyl dipeptide (MDP) as a proinflammatory agent. Preferably, MDP is used in the context of at least one wild type CARD 15 allele, preferably homozygous wild type CARD 15 alleles. Preferably, MDP is not used hi the context of homozygous negative CARD 15 mutants.

It is important to note that simple CARD 15 heterozygotes can respond to MDP, although this action may be somewhat attenuated, so higher doses of MDP may advantageously be administered to CARD 15 heterozygotes in order to compensate.

It is an advantage of the invention that the IL-8 therapies described herein are not affected by the CARD 15 genotype of the patients.

Vasodilators

Agents capable of stimulating increased local blood flow in the context of acute- inflammation find application in the present invention. In particular, agents capable of stimulating or increasing blood flow to the intestinal mucosa find application in the present invention.

hi one embodiment, systemically acting vasodilators such as beta-blockers, nitrates, calcium antagonists, ACE inhibitors, angiotensin receptor blockers, alpha blockers, hydralazine and/or vasoactive diuretics may also be used in the present invention. Clearly, this use depends on systemic vasodilatation- not diverting blood away from the site of the defect such as the bowel due to a greater vascular volume in the peripheries. Agents having this diverting effect are not suitable for use in the context of the present invention.

Particularly preferred are locally regulated or site-specific vasodilators, of which the class of phosphodiesterase-5 inhibitors is the most preferred. A preferred example of this class is sildenafil (sildenafil citrate or Viagra™). Among the advantages of this class of inhibitor are the fact that they act at sites where some inflammation is present.

It must be noted that most vasodilators (e.g. nicotinic acid, nitrates such as glycerol nitrate etc.) do not provide a locally regulated response, whereas it is a key feature of the present invention that only vasodilators providing a 'local' or stimulus-induced vasodilatory effect are employed. Thus, it is not a general vasodilation which is required, it is the enhancement or prolonging of the enhanced flow response to injury/insult which is key to addressing the defect in Crohn's.

We show that injection of healthy volunteers with killed Escherichia coli subcutaneously into the tissues induces a large increase in blood flow around the inoculation site. At least 50% of this increase can be inhibited by infusion of 1-NMMA (but not noradrenaline), indicating involvement of nitric oxide.

We show an abnormally low blood flow response to bacterial injection in Crohn's disease, a condition in which a failure of neutrophil recruitment may play a critical role in the pathogenesis. Tissue perfusion is closely related to neutrophil extravasation from capillaries to sites of tissue insult. Although the underlying abnormality in. neutrophil accumulation at acute inflammatory sites may primarily relate to deficient cytokine production, reduced perfusion appears to contribute to (or compound) the problem. Therefore, if the flow at sites of lesions can be enhanced or normalized according to the present invention, the inflammatory response and neutrophil emigration are improved and disease remission accelerated. We have shown that phosphodiesterase-5 inhibitors such as sildenafil work to correct the abnormal blood flow in Crohn's disease, advantageously improving healing of Crohn's lesions. Thus, we show that therapeutic agents of this class are useful in treatment of active inflammatory lesions according to the present invention. Furthermore, the invention relates to use of therapeutic agents of this class in patients with quiescent disease as prophylactics to prevent recurrence. This prophylaxis may be particularly advantageous in patients who have recently undergone intestinal resections, of whom almost all develop endoscopic evidence of anastomotic recurrence within 1 year.

Phosphodiesterase-5 Inhibitors

The data presented herein have extensively exemplified the use of the preferred example of this class of compounds, sildenafil (l-[[3-(6,7-dihydro-l-methyl-7-oxo-3- propyl- lH-pyrazolo[4,3-^pyrirnidin-5-yl)-4-ethoxyphenyl]sulfonyl]-4- methylpiperazine citrate; Pfizer' s Viagra™).

Preferably a longer acting phosphodiesterase-5 inhibitor such as vardenafil, preferably vardenafϊl hydrochloride (piperazine: l-[[3-(l,4-dihydro-5-methyl-4-oxo-7- propylimidazo[5,l-/l[l,2,4]triazin-2-yl)-4-ethoxyphenyl]sulfonyl]-4-ethyl- monohydrochloride; Bayer's Levitra™ / Nuviva™) or tadalafil (C₂₂H₁₉N₃O₄; Lilly ICOS 's Cialis™) is used. These have preferred pharmacokinetic properties which can advantageously reduce dose amounts and/or dosing frequency. This is beneficial to the patient in terms of fewer interventions/administrations, and provide a smoother effect profile than a compound which is rapidly cleared and therefore requires more frequent redosing to maintain efficacy. Of these compounds, tadalafil is most preferred due to having the longest acting properties.

Furthermore, any agent that increases local perfusion should have the desired therapeutic effect according to the present invention. Delivery could either be local (using a non-absorbable agent that only penetrates through ulcerated mucus) or systemic; the former would be predicted to have a more favourable side effect profile. Preferred PDE5 inhibitors axe the longest lasting or those having the longest half life. In other words, the longest acting inhibitors are preferred. This is because Crohn's disease is a chronic disorder. The most convenient form of treatment is a once per day administration. Thus, PDE5 inhibitors with shorter half lives are likely to require more frequent administration, or higher doses, or both.

In terms of effectiveness of the PDE5 inhibitor, both short and long lasting PDE5 inhibitors may be used in the present invention. However, it is advantageous to use longer lasting inhibitors for the reasons noted above. Long acting PDE5 inhibitors are preferred because they provide throughout the day coverage with the minimum number of administrations. Short acting agents are also effective if taken at multiple points during the course of the day, taking into account their half-life as discussed herein.

Preferably the PDE5 inhibitor is administered as a sustained release formulation.

Pharmaceutical Compositions

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the vasodilating agent(s) and/or neutrophil chemoattractant(s) of the present invention and a pharmaceutically acceptable carrier, diluent or excipient (including, combinations thereof).

The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solύbilising agent(s):

Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be administered by a number of routes.

Where the agent is to be administered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions maybe administered in the form of tablets or lozenges which can be formulated in a conventional manner.

If the agent/modulator is a protein, then said protein may be prepared in situ in the subject being treated. In this respect, nucleotide sequences encoding said protein may be delivered by use of non-viral techniques (e.g. by use of liposomes) and/or viral techniques (e.g. by use of τetroviral vectors) such that the said protein is expressed from said nucleotide sequence.

In a preferred embodiment, the neutrophil chemoattractant/pro-inflammatory pharmaceutical of the present invention is administered topically. Preferably me pharmaceutical is in a form that is suitable for topical delivery.

Administration

The term "administered" includes delivery by viral or non-viral techniques. Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors. Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs), genetically modified bacteria bearing nucleic acids expressing the agent(s) and combinations thereof.

The components of the present invention may be administered alene but will generally be administered as a pharmaceutical composition — e.g. when the components are is in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the components can be administered (e.g. orally or topically) in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications. If the pharmaceutical is a tablet, then the tablet may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate,, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, kydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The routes for administration (delivery) include, but are not limited to, one or more of: oral (e.g. as a tablet, capsule, or as an ingestable solution), topical, mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, . intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal,, buccal, vaginal, epidural, sublingual. Preferred are oral and/or rectal delivery.

It is to be understood that not all of the components of the pharmaceutical need be administered by the same route. Likewise, if the composition comprises more than one active component, then those components may be administered by different routes.

In a preferred aspect, the pharmaceutical composition comprising proinflammatory/neutrophil chemoattractant such as EL-8 is delivered topically, preferably delivered topically to the intestine, preferably delivered topically to the intestinal mucosa.

Preferably the vasodilator/PDEV inhibitor is delivered orally.

If a component of the present invention is administered parenterally, then examples of such administration include one or more of: intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the component; and/or by using infusion techniques.

For parenteral administration, the component is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Alternatively, the components) of the present invention can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The components) of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. They may also be administered by the ocular route. For ophthalmic use, the compounds can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the components) of the present invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Pharmaceutical Combinations

The agent(s) of the present invention may be administered with one or more other pharmaceutically active substances. By way of example, the present invention covers the simultaneous, or sequential treatments with an agent according to the present invention and one or more steroids, analgesics, antivirals or other pharmaceutically active substance(s).

It will be understood that these regimes include the administration of the substances sequentially, simultaneously or together.

Specifically, the vasodilator/PDEV inhibitor may be delivered sequentially, simultaneously or together with the pro-inflammatory agent/neutrophil chemoattractant.

Dose Levels

Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. Depending upon the need, the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, -such as from 0.1 to 10- mg/kg, more preferably from 0.1 to 1 mg/kg body weight.

Do&ing/Administration of PDEV inhibitors

For tadalafil, preferred dosing is from 5-20mg per day, preferably lOmgper day for an average adult male.

For vardenafil, preferred dosing is from 2.5-20mg per day, preferably lOmg per day for an average adult male.

For sildenafil, preferred dosing is from 25-1 OOmg per day, preferably 50mg per day for an average adult male.

Frequency of Administration

For tadalafil, preferred dosing frequency is according to its standard regimen eg. once per day for an average adult male.

For vardenafil, preferred dosing frequency is up to three times per day for an average adult male.

For sildenafil, preferred dosing frequency is up to three times per day, or even up to four times per day, for an average adult male.

When considering dosing frequency, the guidance regarding daily dose should be borne in mind. Preferably the chosen daily dose is divided equally between the separate administration events. For example, for a sildenafil dose of 50mg per day, preferably this may be given as three doses of 16.7mg, or even as four doses of 12.5mg. Provision / Administration of pro-inflammatory agent

Preferably the pro-inflammatory agent is a neutrophil chemoattractant, preferably IL-8.

Provision of the agent such as IL-8 could be local delivery by means of an oral preparation, enema or other means of local delivery such as genetically modified bacteria (for example as taught in Farrar J Appl Microbiol 2005;98(5):l 191-7) or gene therapy with viral vectors. Delivery to the intestine is particularly .advantageous as this cytokine would only penetrate the bowel^" wall through damaged mucosa, thereby going directly to the site where it is needed.

Preferred dose levels for this agent are 1-lOOug/ml of agent in a topical treatment of approximately 5mls, preferably lOug/ml in a topical treatment of approximately 5mls.

The preferred doses of topical IL-8 specified are those which we show corrected the defect in skin windows (see examples). Due to the nature of its administration, low toxicity is advantageously generated, enabling higher doses to be used. Optimal therapeutic conditions are best determined by trial and observation, such as a clinical trial.

In another embodiment, provision of the agent such as IL-8 could be achieved by augmenting the activity of endogenous pathways leading to IL-8 production, or by a pro-inflammatory stimulus acting locally. It will be noted that GM-CSF is excluded by the requirement to be local-acting, since GM-CSF works systemically. It is a key feature of the present invention that the pro-inflammatory agent/stimuhis is local acting.

In another embodiment, any macrophage agonist that boosts IL-8 production could be used. The defect in Crohn's is in stimulating adequate IL-8 production rather than an absolute deficiency. Thus, rather than adding exogenous IL-8, its local production could instead be stimulated for therapeutic benefit. Intestinal lesions are amenable to treatment with peptides and proteins, since in Crohn's disease they occur principally in the portions of the bowel where digest-ion, is already completed, so that once delivered the active agent is less lflcey to be degraded or lost by digestion.

IL-8 may be provided by stimulating its production from cell(s) in the subject, for example using the immune adjuvant muramyl dipeptide (MDP).

IL-8 may be provided by stimulating (or enhancing) its production locally from macrophage.

Further Applications

The invention may also be advantageously applied to the treatment of sarcoidosis and any other granulomatous disorder, preferably sarcoidosis.

The invention particularly be advantageously applied to the treatment of large bowel disease.

The present invention will now ^"be described by way of examples, which are intended to be illustrative rather than limiting in nature, and in which reference is made to the following figures:

Brief Description of the Drawings

Figure 1 shows bar charts of impaired neutrophil accumulation and EL-8 production in traumatised rectum or ileum in Crohn's disease. Numbers of cells staining for (a) MPO or (b) IL-8 in biopsy sections from healthy controls (HC) and CD and UC subjects in complete remission (numbers detailed). Numbers (mean + SEM) of cells averaged from 5 high power fields shown. Figure 2 shows bar charts and a photograph of neutrophil, cytokine and early inflammatory mediator accumulation in skin windows.(a) Neutrophil emigration into skin windows was impaired in CD₅ irrespective of CARD15 genotype (mean + SEM). Topical application of MDP corrected this only in CARD15 w/w subjects, (b) Neutrophil emigration in CD normalised by topical recombinant EL-8. (c) IL-8 and IL- lβ principal cytokines detected in skin windows at 24 hr. IL-8 (d) and IL- lβ (e) were abnormally low in all CD patients compared with healthy control& (HC), and patients with UC or RA. MDP amplified the response in all cases except CD CARD 15 m/m. Levels of C3a, histamine, PGE₂ and LTB₄ (f) were similar in HCs and CD, and albumin levels (g) were similar in all groups studied.

Figure 3 shows scatterplots, bar charts and a table of changes in gene expression in, and cytokine secretion by, peripheral blood monocyte-derived macrophages exposed to MDP. Cells from 6 healthy (ai), 3 CD CAKD15 m/m (an), 3 CD CARD15 w/w ileal disease (aiii) and 3 CD CAKDl 5 w/w colonic disease (aiv) subjects assayed on Affymetrix U133A microarrays comparing gene expression by cells with and without MDP. Transcripts for IL-8 (Φ) and IL- lβ (-0-) are identified except in CAKDl 5 m/m cells, which failed to respond (aii). Transcripts with the greatest increases in expression are listed in the table (b). These transcripts are translated as shown by induction of secretion of IL-8 (c), IL-I β (d), TNFα (e) and IL- 10(f) by macrophages in response to MDP except in CD m/m cells.

Figure 4 shows diagrams of secretion of IL-8 by HC, CD and UC macrophages in culture in response to (a) wound fluid, (b) C5a (c) TNFα or (d) LPS for 6 hours. Response in CD was independent of CAKD15 genotype. (Mean value shown, *** ρ<0.001) '

Figure 5 shows images and graphs of the effects of subcutaneous injection of is. coli on local and systemic parameters of inflammation, (a) Injection of bacteria led to an area of erythema and swelling over the subsequent 48 hours, (b) Blood flow, measured by laser Doppler, was markedly increased by 24hours in healthy subjects (i) but much less so in CD (ii). (c) Time course of changes in blood flow shows less blood flow in CD₃ particularly the colonic variety, and delayed resolution in UC. (d) Bacteria induced blood flow at 24 hr in 3 HC attenuated by intra-arterial 1-NMMA but not norepinephrine (i) and enhanced by 50mg oral sildenafil citrate (ϋ). (e) Local erythema (i) peripheral neutrophilia (ii), serum IL-6 (iii) and CRP levels (iv) in the 4 subject groups.

Figure 6 shows a schematic representation of proposed mechanisms involved in the pathogenesis of Crohn's disease. Damage to the mucosa allows the penetration of gut contents including bacteria (brown material) into the bowel wall. The outcome depends upon the subsequent inflammatory response. A vigorous response leads to the secretion of high levels of IL-8 attracting large numbers of neutrophils which phagocytose and digest the bacteria and foreign material. A weak inflammatory response predisposes to Crohn's lesions because the foreign material is taken up by macrophages to form granulomata and a focus of chronic inflammation. A naturally weak response can be boosted in the small bowel by MDP signaling through the N0D2 pathway of macrophages to induce them to produce IL-8. In the large bowel a similar role might be performed by TLR4 or CD 14. The predisposition to CD is greatly increased by a combination of a low innate inflammatory response coupled to failure of one of the compensatory booster mechanisms.

Figure 7 shows diagrams of patients studied (macrophages with inflammatory stimuli).

Examples

Overview:

Human studies presented herein reveal grossly defective acute inflammation in Crohn's disease. A number of theories have been advanced concerning the aetiology of Crohn's disease (CD), but none mechanistically proven. We tested the hypothesis that CD is a form of immunodeficiency caused by impaired innate immunity. The inflammatory response was investigated in CD patients and control subjects by quantifying neutrophil recruitment and cytokine production following acute trauma to the bowel or skin. IL-8 secretion by cultured monocyte-derived macrophages was measured after exposure to inflammatory mediators. Heat-killed E. coli were injected subcutaneously into patients and controls, and the magnitude of the ensuing local inflammatory and vascular changes and systemic cytokine and acute phase responses determined.

Trauma to the rectum, ileum or skin led to abnormally low levels of neutrophil accumulation (reductions of 78.6%, n=8, p=0.0003; 57.2%, n=3, p=0.05; 49.6%, n=13,

P=O-SxIO^"6) and pro-inflammatory IL-8 (63.3%, n=7, p=0.003; 63.3%, n=3, p=0.05;

45.4%, n=8, p=0.07xl0^"3 respectively) and IL-lβ (49.6%, n=8, p=0.0005) production in CD. EL-8 secretion by cultured CD macrophages was also reduced following exposure to acute wound fluid (37.8%, n=50, P=LSxIO^"6), C5a (47.5%, n=41, p=0.0005) or TNF-α (52.2%, n=27, p=0.07xl0^"3). These results are indicative of a systemic immunodeficiency state. Subcutaneous inoculation of CD patients with killed

E. coli elicited a markedly attenuated local inflammatory reaction, quantified by changes in blood flow (46.9%, n=6, p=0.01 and 76.8%, n=6, p=0.0003 in ileal and colonic CD respectively). The latter was NO-mediated in controls, and partially normalized in patients by sildenafil citrate (Viagra™). In all these experiments, responses were not related to NOD2/CARD15 genotype.

Methods

General note on patients

Patients were not on immunosuppressive medication (and had not been so in the preceding 2 months) and all had quiescent disease (Harvey-Bradshaw score of <3 (determined according to Harvey and Bradshaw Lancet 1980 Mar 8;1(8167):514); serum inflammatory markers within normal limits), unless otherwise stated.

Subjects studied (bowel biopsies)

Patients studied (skin windows)

Patient details (bacterial injection)

This does not preclude the possibility that some subjects^" might have had occult gastrointestinal lesions, and we did not specifically endoscope all patients to exclude this. Subjects were approximately matched for age, sex and smoking history. Serum albumins, the best marker of nutritional status, were all within the normal range in CD patients (mean ± SEM = 45.41 ± 2.86 g/L). Where clinically indicated, serum vitamin B]₂ and red cell folate levels were measured, and were not abnormal in any individual. No patient had active fistulating disease at the time of study. These studies were approved by the Joint UCL/UCLH Committee on the Ethics of Human, project numbers 00/0004, 02/0324 and 04/Q0502/29. Written, informed consent was obtained from all volunteers. No patient was studied more than once in each of the different experimental modalities.

Genotyping for NOD2/CARD15 polymorphisms Genomic DNA was isolated from blood samples using the GCT™ Genomic DNA Purification Kit (DNA Research Innovations Ltd, Kent, UK). All subjects were genotyped for 3 single nucleotide polymorphisms (SNPs) within the ΗOO2ICAKD15 gene: R702W, G908R and L3020fsinsC, and reference SNP IDs rs2066844, rs2066845, rs2066847 respectively (Hugot et al. Nature 2001 May 31;411(6837):599- 603). Wild type genotypes are referred to as wfw, the presence of any one SNP in the genotype (simple heterozygosity) as w/m, and any two (compound heterozygosity or homozygosity) as m/m.

In slightly more detail, genotyping was carried out using the Taqman® SNP Genotyping Assays (Applied Biosystems, Foster City) and ABI 7900HT Sequence

Detection System (Applied Biosystems, Foster City). Primers and probes for SNP 12 were designed through Custom Taqman® SNP Genotyping Assay (Applied

Biosystems, Foster City) and primers and probes were taken from previously published papers and the National Cancer Institute SNP500 website αittp://snp500cancer.nci.nih.govy (Steer et al. Rheumatology (Oxford) 2003

Feb;42(2):304-7; Packer et al. Nucleic Acids Res 2004 Jan l;32(Database issue):D528-D532).

The primerand probe sequences were as follows:

SNP8 forward primer: 5'-GCTGGCTGAGTGCCAGACATG-S'

SNP8 reverse primer: 5'-AGTGGAAGTGCTTGCGGAGG-S'

SNP8 wild type probe: 5'CCTGCTCCGGCGCCAGGC-S'

SNP 8 variant probe: 5'-CCTGCTCTGGCGCCAGGCC-S'

SNP12 forward primer: 5'-CTGTTGACTCTTTGGCCTTTTCAG-S'

SNP12 reverse primer: 5'- CC ACCTC AAGCTCTGGTGATC-3' SNP12 wild type probe: 5' CTCTGTTGCCCCAGAAT-3' SNP12 variant probe: 5'-CTGTTGCGCCAGAAT-S'

SNP13 forward primer: 5'-GTCCAATAACTGCATCACCTACCT-S' SNP13 reverse primer: 5'-ACTTCCAGGATGGTGTCATTCC-S' SNP13 wild type probe: 5'-CTGCAGGCCCTTGA-3' SNP13 variant probe: 5'CTGCAGGCCCCTTGA-3'.

Total KNA was isolated from the lysate using extraction with chloroform, precipitated with isopropanol, washed with 75% ethanol, air-dried, and re-dissolved in DEPC- treated water (Ambion). The quality and quantity of these samples was determined using an Agilent Bioanalyzer. Approximately 5 μg of total RNA from each independent sample was processed to produce biotinylated cRNA targets, which were hybridised to Human Genome U 133 A arrays following standard Affymetrix procedures fhttp ://www.affymetrix.conϊ).

Immunohistochemistry for bowel biopsies

Immunohistochemistry on biopsy sections was performed using the streptavidin-biotin immunoperoxidase method. Sections were de-waxed, re-hydrated, and incubated with 3% H₂O₂ in methanol for 20 min to block endogenous peroxidase activity. Antigen retrieval was performed by pressure-cooking in 0.01 M citrate buffer (pH 6.0) for 2 min at high pressure. The sections were incubated first with serum-free Protein Block (Dako) for 10 min then washed three times in TBST (TBS, 0.5% Tween). Primary antibody was then added for 1 h at room temperature. Separate sections from each sample were treated with either monoclonal anti-human IL-8 (R&D Systems, 1 : 100) or anti-myeloperoxidase (Dako, 1:1,000) antibodies made up in Tris-buffered saline (TBS). Following incubation with primary antibody, sections were washed 3 times with TBS-Tween (TBST), before incubation with appropriate biotinylated secondary antibodies (Dako; 1:300) for 30 min. Sections were again washed in TBST, then incubated with streptavidin-peroxidase conjugate (Dako; 1:500) for an additional 30 min. Sections were developed with 3,3'-diammobenzidine-tetrahydrochloride (Sigma) and 0.1% H₂O₂ solution for 2 min, counterstained with haematoxylin, and mounted with DPX. For negative controls, duplicate sections were used in which the primary antibodies were omitted.

Skin windows Skin windows were made by dermal abrasion of an area of 3 cm x 1 cm on the volar surface of the forearm with medium grade sand paper until capillary bleeding just commenced. The abrasions were overlaid with filter paper saturated with either normal saline alone or containing MDP (100 ng/ml, Sigma) or recombinant human IL-8 (10 μg/ml, PeproTech) in normal saline. The filter paper was then eovered with a layer of Nescofilm sealing film, and then an adhesive dressing. Dressings and filter papers were removed after either 30 min, for measurements of early mediators, or 24 h for measurements of myeloperoxidase and cytokines.

Filter papers were incubated in 400 μl normal saline on a rotating wheel for 30 min at 4⁰C and then centrifuged (13,000 rpm, 5 min, 4⁰C) to release proteins. Cytokines were determined in this supernatant using commercially available protein arrays containing antibodies against 42 different cytokines (RayBiotech). Absolute levels of cytokine and other secreted protein production were quantified by ELISA using commercially available kits following the manufacturers' instructions, including IL-8, IL-lβ (R&D Systems), albumin (Alpha Diagnostic International), histamine (IBL Hamburg), C3a- desArg (ProGen), prostaglandin E₂ and leukotriene B₄ (R&D Systems). Cellular contents were then extracted by incubating the filter papers in a solution of 0.5 M

NaCl and 1.5% Triton X-100 containing Complete Mini protease inhibitor cocktail tablets (Roche). The samples were sonicated (10 x 1 s bursts), centrifuged (13,000 rpm, 5 min, 4°C) and the supernatant measured for myeloperoxidase by oxidation of 4- aminoantipyrine (Sigma), using horseradish peroxidase (Sigma) as a standard (Trevani et al. J Immunol 1999 Apr 15;162(8):4849-57).

Symptomatic effects of injection ϊa all subjects, injection elicited a local inflammatory reaction marked by erythema, oedema, and local pain and tenderness. The erythema spread progressively as documented in Fig.4Ea but after 24 h became gradually more diffuse and in the majority of subjects had disappeared by 72 h. Local pain was maximal approximately 12 h after inoculation and was of moderate severity. Mild systemic symptoms occurred approximately 12 h following injection in about 40% of subjects and almost completely resolved by 24 h. The profile of adverse reactions was not significantly different between any subject groups.. One subject with ulcerative colitis developed local vesiculation and ulceration at the injection sites.

Measurement of acute phase proteins and serum cytokines

Serum concentrations of CRP and SAA were determined by automated microparticle capture enzyme immunoassay as previously described. (Eda et al. Scand J Clin Lab Invest Suppl 1999;230:32-5; Ledue et al. Ann Clin Biochem 1998 Nov;35 ( Pt 6):745- 53; Noursadeghi et al. Clin Exp Immunol 2005 Apr;140(l):97-100) Serum IL-lβ, IL- 6, IL-8, IL-10, IL- 12, TNFα and interferon-γ were quantified using the Bio-Plex Cytokine Assay (Bio-Rad, CA), following the manufacturer's instructions.

Bowel biopsies

Biopsies were taken from the posterior wall of the rectum. 10 cm from the anus in 9 non-inflammatory bowel disease controls, 6 CD and 3 ulcerative colitis (UC) patients. In all these subjects, the endoscopic appearance of the mucosa was entirely normal and histology performed on these initial biopsies showed no evidence of microscopic Crohn's lesions. A second biopsy was taken 6 h later from the site of the initial biopsy 6 h later. Forceps^~were positioned directly above the previous biopsy lesion so that the- jaws encompassed-the mucosal defect. Paired serial biopsies were also taken from both the rectum and neo-terminal ileum in a further 2 patients with familial adenomatous polyposis and 2 CD patients, all of whom had undergone colectomies and ileorectal anastomoses. 1 CD patient had a ileoanal anastomosis in whom biopsies were taken only from the ileum. Only 1 CD and all 3 UC patients were taking 5 -ASA, the remainder were on no medication. Samples were fixed in formalin, embedded in paraffin wax, sectioned and immunostained for myeloperoxidase (Dako, Glostrup, Denmark; 1:1,000) and IL-8 (R&D systems, MN, USA; 1:100) using the streptavidin- biotin immunoperoxidase method. For negative controls, duplicate sections were used in which primary antibodies were omitted. Neutrophils and IL-8-producing cells were counted in a blinded fashion and averaged over 5 randomly selected high power fields (hpf) per section.

Skin windows Skin windows were made by dermal abrasion of a 3 cm x 1 cm area- on the volar surface of the forearm with medium grade sand paper until capillaries were visualized but before bleeding commenced. Abrasions were overlaid with filter paper saturated with either normal saline alone or containing 100 ng/ml MDP (Sigma,. MO, USA) or 10 μg/ml recombinant human IL-8 (PeproTech, NJ₅ USA). The filter paper was covered with a layer of Nescofihn sealing film, and then an adhesive dressing. Dressings and filter papers were removed after either 30 min (for measurements of early mediators) or 24 h for measurements of myeloperoxidase and cytokines. Only one of the 13 CD patients was on any medication (aspirin, hyoscine and loperamide) and 1 UC patient was on mesalazine.

Filter papers were incubated in 400 μl normal saline on a rotating wheel for 30 min at 4⁰C to elute proteins and then centrifuged (15,000 g, 5 min, 4°C) to pellet filter papers in cells. Cytokines were determined in the supernatant using commercially available protein arrays containing antibodies against 42 different cytokines (RayBiotech, GA, USA). Absolute levels of cytokines and other secreted protein production were quantified by ELISA using commercially available kits including IL-8, IL- lβ (R&D Systems), albumin (Alpha Diagnostic International, TX, USA), histamine (IBL Hamburg, Hamburg, Germany), C3a-desAr-g- (Progen, Heidelberg, Germany), prostaglandin E₂ and leukotriene B₄ (R&D Systems).

Cellular contents were extracted by incubating the centrifuged filter papers in a solution of 0.5 M NaCl and 1.5% Triton X-100 containing Complete Mini protease inhibitor cocktail tablets (Roche, Basel, Switzerland). The samples were sonicated (10 x 1 s bursts), centrifuged (13,000 rpm, 5 min, 4⁰C) and the supernatants measured for myeloperoxidase by oxidation of 4-aminoantipyrine (Sigma), using horseradish peroxidase (Sigma) as a standard. (Shimizu et al. Biochem Biophys Res Commun 1979 Nov l4;91(l): 108-13) Macrophage cultures

Peripheral venous blood was collected into 5 U/ml heparin and mixed with an equal volume of balanced salt solution (0.14 M NaCl, 0.01 % anhydrous D-glucose, 5 μM CaCl₂, 98 μM MgCl₂, 0.54 mM KCl₅.14.5 mM Tris-HCtpH 7.6). Mononuclear cells were isolated by centrifugation (15 min, 800 g, 20 ⁰C) over Ficoll-Paque PLUS (Amersham Biosciences, Buckinghamshire, UK), .and washed repeatedly with ice-cold phosphate-buffered saline (PBS; Oxoid, ON, Canada) to remove platelets. Cells were resuspended in RPMI-1640 medium (Invitrogen, Paisley, UK)/10% normal human serum at a density of approximately 5xlO⁶ cells/ml, and cultured for 5 days (37⁰C, 5% CO₂) in standard tissue culture dishes (VWR, Poole, UK). Medium was changed on days 1, 3 and 5.

Some day 5 cells were incubated with or without 100 ng/ml MDP for 15 h in RPMI- 1640/10% normal human serum. Cells were then lysed in TRIzol (Invitrogen), and cRNA prepared. Global gene expression profiles were determined using Affymetrix Human Genome U133A microarrays:

Microarray analysis In the event that insufficient RNA was isolated, equal amounts of sample from subjects were combined to produce adequate starting material. These were then treated as a new individual sample. Expression patterns from samples pooled in this way were completely consistent with results obtained from individuals. Each array demonstrated control parameters within recommended limits (Raw Q :_30, background ≤.00, GAPDH 3':5' ratio<4). Global normalisation was performed on each chip and the data scaled against a reference chip. After exclusion of highest and lowest 2% of the data points from each chip, the mean and standard deviation of the Iog₂(signal) from each chip was calculated and a mean-corrected log₂ value produced for each data-point. The scale factor for the chips were calculated as the ratio of mean corrected log₂ values of the reference [R] and each target chip [T] as:

[Σ(Log2Signal_R-MeanLog2Signal_R)]/[∑( Log2Signal_τ-MeanLog2Signal_τ)] The data was analysed using the commercially available Spotfire® visualisation and analysis program (www.spotfire.com). Probes were filtered for those that showed at least 2-fold change in expression, and then sorted using hierarchical clustering. A Euclidean distance matrix was generated for all probes, which were- then hierarchically joined as shown in the dendrograms. The full-length genes were annotated using Inpharmatica's annotation system that relies on the Biopendium™.(63) All tnicroarray data are available as MAGE-ML files from <website>, and will be submitted in MIAME compliant format to the Array Express database held at the EBI. Post- genomic verification was provided by measuring cytokine secretion using ELISAs for IL-8, IL-lβ, TNP-α, IL-IO and DL-12 (R&D Systems).

Post-genomic verification of changes in cytokine gene expression was provided by quantifying their secretion into culture supernatants using appropriate ELISA kits (R&D Systems).

Further peripheral blood monocyte-derived macrophages from healthy controls, CD and UC patients were cultured for 5 days in serum-free conditions in X-Vivo-15 medium (Cambrex, MD, USA) before re-plating air equal densities in 96-well culture dishes. These were allowed to adhere overnight, then stimulated for 6 h with either wound fluid collected from new surgical incisions (1.5 mg/ml protein; wound fluid always derived from-control surgical patients without CD; (Wound fluid^" was collected from surgical swabs, used to remove blood and fluid from a clean surgical wound 2 min after incision, which were then soaked in ice cold normal saline. This fluid was placed immediately on ice, centrifuged at 1000 g for 10 min at 4°C, snap frozen and stored at -7O⁰C. It was diluted 5-fold in medium and added to cells.), 500 ng/ml C5a (Sigma), 5 ng/ml TNF-α (Calbiochem, CA, USA) or 100 ng/ml Salmonella typhimurium lipopolysaccharide (LPS; Sigma) in X-Vivo-15. IL-8 secretion was determined in the culture supernatants by ELISA and normalised to the numbers of viable cells in each well, determined with the Cell Counting Kit-8 (Alexis, CA, USA).(24) Bacterial injection

A fully antibiotic-sensitive clinical isolate of Escherichia coli was grown overnight in minimal citrate medium supplemented with 1.25 g/1 yeast extract (Oxoid), then killed by heating to 80⁰C for 30 min. Bacteria were washed twice in sterile PBS, aliquoted, centrifuged and the pellets snap-frozen and stored at -7O⁰Q Sterility was confirmed by culture. Bacteria for injection were thawed and resuspended at a protein concentration of 10 mg/ml in injection-grade normal saline. AB aliquot of this suspension (100 μl containing 1 mg of protein or 10⁹ organisms) was then injected subcutaneously into the volar aspect of each forearm.

Blood was collected prior to, and at 24 h and 48 h following inoculation, for full blood counts and measurements of serum C-reactive protein, serum amyloid A and. cytokines. Blood flow at the injection sites was determined by laser Doppler imaging (MoorLDI2; Moor Instruments Ltd., Devonshire, UK). Some healthy subjects and patients were treated with 50 mg sildenafil citrate (Pfizer, NY, USA) at either 24 h or 48 h (2 patients) following inoculation, and the perfusion monitored at 30 min intervals over the subsequent 90 min.

A further 3 healthy subjects were studied 24 h following inoculation to determine the effects on blood flow of intra-arterial norepinephrine (Clinalfa AG, Laeufelfingen,

Switzerland; 240 pmol/min) followed by NG-monomethyl-L-arginine acetate (1-

NMMA; Clinalfa AG; 4 μmol/min), with a normal saline washout between the two.

(Clapp et al. Circulation 2005 Mar 29; 111(12): 1530-6). These studies were conducted with the subject supine in a quiet, temperature-controlled laboratory. The brachial artery of the non-dominant arm was canulated with a 27-gauge needle inserted under local anaesthesia (2 ml of 1% lidocaine). Resting blood flow was allowed to normalise following needle insertion prior to the infusion of vasoactive agents. Drugs or saline were infused continuously at 0.5 ml/min for 15 min each.

Of the 12 CD patients, 5 (3 colonic and 2 ileal) were receiving 5-ASA, and of these 1 ileal patient also received azathioprine and 1 colonic patient received methotrexate and metronidazole. 1 of Hie UC patients was on 5-ASA. Statistical analysis

^"Statistical tests were performed using Graphpad Prism version 4.01 (Graphpad Software, CA, USA). The 2-tailed students' t-test was used for single comparisons, and Kruskal-Wallis Analysis of Variance (ANOVA) with Dunn post-tests or 2-way ANOVA with Bonferroni post-tests used for multiple comparisons, as appropriate. Significance values refer to comparison with healthy controls under the same conditions unless otherwise stated.

Example 1: Characterisation of the Defect in Crohn's Disease

Bowel biopsies

Figure 1 shows the effects of trauma on the intestinal mucosa. In rectal biopsies on 9 control subjects, an acute inflammatory response ensued with a large increase in numbers of neutrophils and IL-8-positive cells (Figure IA₅B)- Accumulation of both cell types were considerably reduced in traumatized CD rectum (8 patients, p=0.0003 and p=0.003 respectively). Of these patients, 4 were homozygous/compound heterozygous for NOD2/ CARD 15 polymorphisms (m/m) and 4 were wild-type (w/w): no significant differences, were observed between these two groups. Additional biopsies were conducted in the neo-terminal ileum of 3 CD patients and 2 control subjects with ileorectal anastomoses, to establish whether the impairment also applied to the small bowel; similar defects were observed (p<0.05). In contrast, samples taken from UC patients showed^" slightly raised numbers of neutrophils in the bowel in the- resting state, with elevation to levels found in controls following trauma.

Skin windows

To confirm whether this abnormal response in CD was localised to the bowel, we examined the consequences of trauma to the skin (Figure 2). Neutrophil efflux into skin windows has previously been shown to be low in CD after 8 hours.(20) It was also reduced after 24 hours in 13 CD subjects (p=0.2xl0^"δ) irrespective of NOD2/C4&D/J genotype (Figure 2A), but normal in subjects with other chronic inflammatory diseases, whether generalised (rheumatoid arthritis, RA) or localised to the bowel (UC). The- cytokine profile of skin window fluid was analysed using an antibody array. Of the 42 cytokines assayed, only IL-8 and BL- lβ were detected in any subject (Figure 2C). Their concentrations were considerably diminished in CD skin windows (IL-8: p=O.O7xlO^'3, IL-lβ: p=0.0005), regardless of NOD2/G4RDi5 genotype, but normal in RA and UC (Figure 2D,E). Levels of both were elevated by topical application of MDP in all groups (IL-8: p=0.0004, IL-lβ: p=Θ.Ol) except GD m/m subjects, validating in patients the prior in vitro and animal studies. Trauma applied in creating the windows was similar in all individuals, as evidenced by equivalent concentrations of C3a (measured as its rapidly generated stable conversion product C3a-desArg), histamine, PGE₂ and LTB₄ (Figure 2F), as well as albumin extravasation (Figure 2G).

Example 2: Use of BL-8 in treatment of impaired acute (local) inflammation

IL-8 is a potent neutrophil chemoattractant_} and we hypothesized that its reduced production might play a primary role in the failure of cellular migration. Consistent with this theory, addition of exogenous IL-8 to skin windows normalised neutrophil efflux (p=0.02, compared to saline alone) in 3 CD patients (2 w/w, 1 m/m; Figure 2B) indicating that cells were able to respond in the presence of an appropriate stimulus. Augmentation of endogenous IL-8 secretion by topical MDP was similarly effective in w/w patients (p=0.006, compared to saline alone) but not, as expected, in m/m subjects (Figure 2A).

These data help resolve the controversy surrounding the nature of the NOD2/GiRZ>i5 defect MDP was clearly pro-inflammatory in vivo in healthy subjects and this was abolished by the polymorphisms associated with CD. To confirm the cellular basis underlying this effect, cultured macrophages were exposed to MDP and the pattern of gene expression determined (Figure 3). Ih concordance with a similar prior study, a number of inflammation-related genes (including those for IL-8 and IL-lβ) were markedly up-regulated by normal macrophages (Figure 3Ai,3B). No such induction was seen in CD m/m cells (Figure 3Aii), whereas normal patterns of expression were observed in CD w/w macrophages from subjects with ileal (Figure 3Aiii) or colonic (Figure 3Aiv) disease. These transcripts were translated and secreted into the culture supernatant as reported previously- for IL-8 (Figure 3C-F).

Macrophage cultures Since NOD2/ CARDi 5 did not appear to cause the basal defect in IL-8 secretion, we sought alternative explanations. Since the cytokine is largely produced by macrophages, we-cultured these cells from peripheral blood monocytes. The formation of the skin window involves acute trauma to the skin. Consequently, we exposed cultured macrophages to wound fluid recovered from acute surgical incisions in healthy subjects undergoing inguinal hernia repair (Figure 4A; P=LSxIO^"6), as well as to two other mediators produced at acute inflammatory sites: C5a (Figure 4B; p=0.0005) and TNF-α (Figure 4C; p=0.07xl0^"3). In response to all these stimuli, CD macrophages secreted significantly less IL-8 than those from healthy controls or UC patients, although the response to LPS was normal (Figure 4D). IL-8 secretion was unrelated to NOD2/ CARDl 5 genotype for any stimulus. These results show that macrophages from CD subjects are constitutionally less able to produce IL-8 in response to pro-inflammatory agonists, even when removed from the body and placed in culture.

Example 3: Characterising the blood flow defect in Crohn's

Bacteriaϊ injection

Bowel diversion experiments have demonstrated the critical role of the luminal contents in the development of CD lesions, which typically arise at the sites of highest bacterial concentrations. The studies presented here suggest that reduced or delayed recruitment of neutrophils to sites at which bacteria penetrate the mucosa might lead to their persistence in the tissues, possibly within macrophages. Secondary secretion of pro-inflammatory cytokines, after the failure of initial clearance, could drive the development of chronic inflammation. To determine directly whether there was an abnormal response to the presence of bacteria in the tissues in CD, we injected heat- killed E. coli subcutaneously. This elicited a vigorous inflammatory response manifested by discomfort, erythema and swelling within 2-4 hours. By 24 hours, the erythema (Figure 5A,Ei) and swelling were more extensive, although the discomfort was reduced from 8^" hours. At 48 hours the discomfert and swelling had disappeared, although the erythema was more diffuse. In healthy controls, blood flow in the area of inflammation increased about 6-fold and 12-fold at 8 and 24 hours respectively, and had almost returned to baseline by 48 hours (Figure 5Bi₅C).

The response in CD- was very different. Although local appearances at the injection site were similar, elevations in blood flow were abnormally low at 8 and 24 hours (Figure 5Bu₅C), particularly in subjects with colonic disease (p=0.0003) but also in those with ileal disease (p=0.01). Of the 6 patients with ileal lesions, 3 carried two polymorphisms in NOD2/CARD15 and 1 was a simple heterozygote. Their response was indistinguishable from the wild-type ileal subjects. None of the patients with colonic disease carried the polymorphisms. Blood flow response was also unrelated to smoking status (4 CD and 5 HC were smokers).

Two patients with UC were also studied. In both cases the blood flow responses were higher than those in CD and did not show the normal resolution after 48 hours (p=0.6xl0^"6), clearly distinguishing it from the hypo-responsiveness characteristic of CD. The inflammatory response was so florid in one UC subject that we terminated these studies in this condition.

Example 4: Use of a PEE5 inhibitor in treatment of impaired acute inflammation

According to the above example, a primary defect underlying the impaired inflammation in Crohn's is the failure to induce locally increased levels of blood flow in response to the insult/injury inducing the initial inflammation. This leads to impaired acute (local) inflammation, which in turn leads to chronic or long-term symptoms of Crohn's. This example demonstrates the use of a PDE5 inhibitor in the restoration of the reactive blood flow response and thus the treatment of impaired acute inflammation, particularly acute local inflammation. Since LPS induces vasodilatation through release of nitric oxide (NO), we investigated the role of this mediator in the normal increase in blood flow induced by the killed bacteria. Intra-arterial infusion of 1-NMMA, a non-selective nitric oxide synthase (NOS) inhibitor rapidly reduced blood flow (p=0.008, compared to pre-treatment blood flow) by approximately 50% (Figure 4Di), whereas, norepinephrine had minimal effect. NO causes smooth muscle relaxation by increasing intracellular concentrations of cyclic GMP^' (cGMP). Consequently we examined the effect of sildenafil citrate (Viagra™), a phosphodiesterase-5 inhibitor and vasodilator, (Glossmann et al. Exp GerontoL 1999 Jun;34(3):305-18) to see ifϊt would correctihe deficient blood flow in CD. Oral administration of a 50 mg dose to 5 healthy subjects and 10 CD patients at 24 or 48 hours (2 patients) after bacterial injection resulted in marked elevation of blood flow (p=0.02, compared to pre-treatment blood flow) in most subjects (Figure 4Dii).

To quantify the systemic inflammatory response, we measured IL-lβ, IL-6, IL-8, IL- 10, IL-12, TNF-α and IFN-γ in the serum 24 hours after bacterial injection. Of these we could only consistently detect IL-6, a potent cytokine produced by macrophages that stimulates lymphocytes to proliferate and differentiate, and induces the secretion of acute phase proteins by the liver. Despite showing minimal elevation in forearm blood flow, patients with colonic CD demonstrated the highest levels of serum IL-6 (Figure 5Eiii) and C-reactive protein (Figure 5Eiv), and the greatest increase in peripheral blood neutrophil count (Figure 5Eii). This illustrates the principle that a weak local inflammatory reaction can engender a systemic pro-inflammatory state identical to that observed in active CD.

Summary

AU the different investigations described herein identify defective innate immunity in CD. They show reduced neutrophil accumulation and IL-8 production not only at sites of acute inflammation in the bowel but also in the skin, indicative of a general constitutional abnormality. This was confirmed by the deficient responses of CD macrophages, cultured in vitro for 5 days, to acute inflammatory mediators. The dynamic interplay of the myriad influences on the immune system that differentiate a robust acute inflammatory response from one that is more lethargic is discussed herein. The regulation is almost certainly polygenic and the effects will follow a normal Gaussian distribution. We propose that individuals at the lower end of this response are predisposed to CD. Lesions manifest most frequently in the gastrointestinal tract due to- its high commensal bacterial load, and within this organ at sites with the heaviest colonisation in the terminal ileum and colon. This is also consistent with the critical role demonstrated for the faecal stream, contents in the development of intestinal inflammation. It is also possible that CD patients might be more susceptible to other consequences of an impaired acute inflammatory response: in addition to developing arthritis, uveitis and aphthous ulceration, there could be a greater incidence of pyogenic infection. The latter would be difficult to identify in patients with varying levels of nutrition, immunosuppression and surgical intervention. Even patients with the most severe defect of neutrophil function, Chronic Granulomatous Disease (CGD), can go for years or even decades without infection. At least 20% of these patients develop a granulomatous colitis that is indistinguishable from CD.

Neutrophils are primarily responsible for the killing and digestion of bacteria, accomplished by the potent digestive enzymes released into the phagocytic vacuole from the cytoplasmic granules. They eradicate infecting microorganisms and are discharged as pus, or apoptose, resulting in resolution. Macrophages, by comparison, are also phagocytic but with less killing and digestive capacity. They play xaore of -a containing role, forming granulomata to wall off foreign material from the remainder of the body and secreting cytokines that prime and amplify the immune system; the latter results in florid local and systemic reactions. Although the mucosa provides a very effective barrier in health, insults such as infection and trauma allow the luminal contents access to the tissues of the bowel wall. Ih the absence of adequate numbers of neutrophils for the effective clearance of bacteria they will be taken up by macrophages to form the granulomata and foci of chronic inflammation characteristic of CD.

We disclosed that CD is associated with a failure of the translation of acute tissue damage into an effective neutrophil response. Whether this is a primary constitutive abnormality or a consequence of the disease or its treatment is difficult to dissect. The fact that it is present in patients with quiescent disease, is systemic involving the skin as well as the bowel, and is not seen with general systemic inflammation in rheumatoid arthritis or bowel inflammation in UC, all indicate that it constitutes the primary abnormality. The basic lesion appears to operate at the level of macrophages, since CD macrophages respond poorly to wound fluid and other inflammatory mediators. The fact that these cells were cultured for 5 days before stimulation also- makes suppression by some humoral factor or therapeutic agent highly unlikely.

Furthermore, CD macrophages produce less IL-8, a potent neutrophil chemoattractant, levels of which were also depressed in acutely traumatised bowel and skin in these patients. Neutrophil emigration normalised when IL-8 was applied according to the present invention to CD skin windows, confirming that there is nothing intrinsically wrong with the chemotaxis of these cells.

We further disclose that the underlying impairment of acute inflammation that predisposes to CD can be boosted by a second tier of immune enhancers. One of these, MDP, stimulates the pro-inflammatory NOD2/CARD15 pathway to cytokine production in macrophages. This leads to increased neutrophil migration into skin windows in CD, but not in subjects homozygous/compound heterozygous for polymorphisms shown to predispose to CD. These polymorphisms are only associated with disease of the ileum, a region with fluid contents where a-small soluble bacterial product such as MDP (that is present at high concentration-) is more likely to penetrate through a damaged mucosa than in the large bowel where contents are more solid. It is highly probable that similar compensatory stimuli and pathways operate in the large bowel, in which the triggering molecules and pathways remain to be identified. Toll- like receptor-4 (Franchimont et al. Gut 2004 Jul;53(7):987-92; Ouburg et al. Gut 2005 Mar;54(3):439-40) and CD14 (Klein et al Scand J Gastroenterol 2002 Feb;37(2):189- 91), receptors on macrophages which recognises LPS, have been linked to CD, and are strong candidates to play such a role. Thus the invention relates to the use of toll-like receptor-4 and/or CD 14, or agonists or antagonists thereof, in the treatment of CD. There is clearly a major problem in the handling of E. coli in the tissues, as evidenced by the very abnormal" vascular responses in CD. Unfortunately it was not possible to exhaustively define the differences in the handling of bacteria by the subjects into whom they were injected given the impracticality of serial skin biopsies. Without wishing to be bound by theory, one possible scenario is that in healthy subjects the E. coli evoked an efficient acute inflammatory response leading to their rapid phagocytosis, digestion and subsequent release of LPS, known to induce iNOS synthesis and vasodilatation. In CD, the intact bacteria may remain in the tissues as a consequence of suboptimal neutrophil destruction, to be phagocytosed instead into macrophages. The very high levels of circulating IL-6, secreted by macrophages, indicate that the organisms were recognised and engulfed by these. This is also consistent with the demonstration of bacterial DNA within Crohn's granulomata.

Causation of CD by failure of the acute inflammatory response fits well with the "hygiene hypothesis", in which the increased incidence of CD has been attributed to improved standards of sanitation. The inflammatory response to bacterial ingress would be much more effective if it were primed from a state of subclinical inflammatien induced by repeated mild infections or parasitic infestation than if it has to start de novo in a relatively unstimulated bowel. Other epidemiological risk factors for CD are the adverse effects of smoking and possibly stress on the incidence of the disease. Both reduce mucosal blood flow, which is closely related to rates of neutrophil emigration into the tissues. Smoking is also generally immunosuppressive as well as specifically reducing concentrations of mucosal IL-8.

Thus the invention provides more effective therapies for CD. Current treatments are immunosuppressive, but whilst they reduce symptoms by dampening the proposed secondary inflammation, they would seem to actually accentuate the underlying immunodeficiency. The invention relates to therapy by introducing IL-8 or other proinflammatory stimuli directly into acute lesions, either by direct enteral administration or through synthesis by genetically modified gut organisms (for example as taught in Farrar J Appl Microbiol 2005;98(5): 1191-7); this would be particularly advantageous as this cytokine would only penetrate the bowel wall through damaged mucosa. Agents that increase blood flow, such as long acting phosphodiesterase-5 inhibitors (eg. as disclosed in Seftel 2004 Clin Cardiol Apr;27(4 Suppl 1):I14-IΪ9) or other vasodilatatory or pro-inflammatory drugs, are useful in addressing the blood flow defect in CD and/or healing CD lesions according to the present invention.

AU publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and -system of the present invention will be apparent to those skilled in the art without departing from the scope of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the medical or related fields are intended to be within the scope of the following claims.

Claims

1. Use of a pro-inflammatory agent for the manufacture of a medicament for Crohn's disease.

2. Use according to claim 1 wherein said pro-inflammatory agent is IL-8 or muramyl dipeptide (MDP).

3. Use according to claim 1 or claim 2 wherein said pro-inflammatory agent is a neutrophil chemoattractant.

4. Use according to claim 2 or claim 3 wherein said pro-inflammatory agent is IL-8.

5. A method of treating Crohn's disease in a subject, said method comprising administering to said subject an effective amount of a pro-inflammatory agent.

6. A method according to claim 5 wherein said-pro-inflammatory agent is IL-8.

7. IL-8 for use in the treatment of Crohn's disease.

8. IL-8 for "use in the treatment of impaired acute inflammation.

9. Use ofJL-8 in enhancement of acute inflammation.

10. Use of IL-8 in the enhancement of acute inflammatory response in Crohn's disease.

11. Use of IL-8 as a topical therapy to aid healing following surgery.

12. Use of a neutrophil chemoattractant in the treatment of Crohn's disease.

13. Use of muramyl dipeptϊde (MDP) as a pro-inflammatory agent.

14. Use of muramyl dipeptide (MDP) to increase IL-8 production.

15. Muramyl dipeptide (MDP) ibr use in the treatment of Crohn's disease.

16. Muramyl dipeptide (MDP) for use in the- treatment of impaired acute inflammation.

17. A method for deteπriining whether a subject is likely to respond to MDP as a proinflammatory agent, said method comprising determining the CARD 15 genotype of said subject, wherein detection of at least one CARD 15 wild type allele indicates that the subject is likely to be responsive to MDP as a pro-inflammatory agent.

18. A composition comprising IL-8 and a PDEV inhibitor.

19. A composition according to claim 18 for use as a medicament.

20. A composition according to claim 18 for use in the treatment of Crohn's disease.

21. Use of a composition according to claim 18 for the manufacture of a medicament for Crohn's disease.

22. A method of treating Crohn's disease in a subject, said method comprising administering to said subject an effective amount of a PDE5 inhibitor and an effective amount of IL-8.

23. A composition according to claim 18, a use according to claim 21 or a method according to claim 22 wherein the PDE5 inhibitor is sildenafil citrate, vardenafϊl or tadalafil.

24. A composition according to claim 18, a use according to claim 21 or a method according to claim 22 wherein the PDE5 inhibitor is sildenafil citrate (Viagra™).

25. A kit comprising at least one dose of IL-8 and at least one dose of a PDEV inhibitor, together with instructions for their administration to a subject having

Crohn's disease.

26. A patch, dressing or swab comprising IL-8.

27. A cream, foam, enema or suppository comprising IL-8.