EP1397683A2

EP1397683A2 - Methods for identifying compounds for treatment of ibd

Info

Publication number: EP1397683A2
Application number: EP02724467A
Authority: EP
Inventors: Stefan Schreiber; Jochen Hampe; Monika Stoll
Original assignee: AstraZeneca AB
Current assignee: AstraZeneca AB
Priority date: 2001-05-12
Filing date: 2002-05-08
Publication date: 2004-03-17
Also published as: AU2002255159A1; GB0111639D0; WO2002093176A3; WO2002093176A2; JP2004534531A

Abstract

The invention provides a method of identifying a compound useful for treatment of IBD which comprises assaying the compound for its ability to modulate the activity or amount of AQP8. The assay is selected from measurement of AQP8 activity using a cell line which expresses AQP8 or using purified AQP8 protein, and measurement of AQP8 transcription or translation in a cell line expressing AQP8. The invention also provides a method of preparing a pharmaceutical composition, a diagnostic method for the determination of susceptibility to IBD, a method for the diagnosis of IBD or a predisposition thereto and use of a compound able to modulate the activity or amount of AQP8 in preparation of a medicament for the treatment of IBD.

Description

METHODS

IBD is characterised by a chronic relapsing intestinal inflammation of the gastrointestinal tract. It affects ~1/1,000 individuals in Western countries with the median age 5 of onset in early adulthood. To date, the etiology of this disease is unknown. Based on clinical and histopathological features, IBD is categorised into two main subtypes, Crohn's disease (CD) (On Line Mendelian Inheritance in Man, OMIM 266600) and ulcerative colitis (UC) (OMIM 191390). Although the cause of IBD is unknown, familial clustering of the disease and increased concordance in monozygotic twins shows a strong genetic susceptibility.

10 Estimates of sibling risk (λs) show a range of 10-50, suggesting that genetic factors play a significant role in the predisposition to IBD. Based on epidemiological data, IBD is considered to be a multifactorial disorder that is caused by multiple susceptibility genes as well as environmental factors. In the present context the term IBD is intended to include IBD, as well as Crohn's disease and ulcerative colitis.

15 Previous genome wide linkage analyses have identified a number of susceptibility locus for IBD, e.g. IBD1 (OMIM 266600) (Hugot et al.1996, Brant et al. 1998, Curran et al. 1998, Hampe et al. 1999a), IBD 2 (OMIM 601458) (Duerr et al 1988, Parkes et al 2000), IBD3 (OMIM 604519) (Hampe et al. 1999b), IBD7 (OMIM 605225) (Cho et al. 1998, Cho et al. 2000). The HBD1 locus is located on the pericentromeric region of human chromosome

20 16, mapped to the region 16p 12-q 13.

AQP8 belongs to the group of water channel proteins, the aquaporins. Aquaporins (AQP) are members of the major intrinsic super family of integral membrane proteins, which act as specialised channels to facilitate the passage of water through the cell membrane in animals, plants, and bacteria. So far ten AQP homologues named from 0 to 9 have been

25 cloned in mammals. They are widely distributed and more than one AQP can be present in the same cell (Echevarria and ϋundain, 1998). Several human AQP genes have been cloned and an increasing number of disturbances have been found associated to the abnormal function of these proteins (Echevarria and Ilundain, 1998).

Initially, the cDNA encoding mouse AQP8 was cloned by Ma et al. (1997a). Northern

30 blot analysis indicated expression in a number of tissue, including placenta, colon, liver, heart, pancreas, lung, kidney, testis, spleen, stomach and brain. The cDNA encoding human AQP8 was cloned in 1998 and was shown to be important for cell fluid transport (Koyama et al 1998). Expression of the gene was demonstrated by northern blot in human pancreatic and colonic tissue (Koyama et al 1998). Koyama et al (1999) demonstrated that in rat AQP8 is expressed in the absorptive columnar cells of the jejunum, proximal and distal colon by in situ hybridization and suggested that AQP8 has a role in water intake and/or outlet in the gastrointestinal tract. In mice the AQP3, AQP4 and AQP8 are expressed in the colon surface epithelium, suggesting a combined role of these aquaporins in fluid transport (Ma and Verkman 1999).

Expression, subcellular localisation and regulation of AQP8 in rat hepatocytes have been studied in more detail by Garcia et al. (2001). Recently, the expression of AQP8 in human colon was verified by Fischer et al. (2001) and further suggested to be a marker of normal proliferating colonic epithelial cells.

The aquaporins are small membrane-spanning very hydrophobic, intrinsic membrane proteins (monomer size -30 kDa) that are expressed at plasma membranes in many cells types involved in fluid transport. Basic features of aquaporin structure have been defined using mutagenesis, epitope tagging, and spectroscopic and freeze-fracture electron microscopy methods. Aquaporins appear to assemble in membranes as homotetramers in which each monomer, consisting of six membrane-spanning α-helical domains with cytoplasmically oriented amino and carboxy termini, contains a distinct water pore. The structure and function of the aquaporins have been reviewed by Verkman and

Mitra (2000) and King et al. (2000).

Humans with the rare Colton-null phenotype are deficient in AQP1 but have no obvious clinical phenotype (Preston et al. 1994), only a reduced capacity for urine concentration can be seen (Mathai et al. 1996. In contrast, AQP1 knock-out mice revealed a marked urinary concentration defect (Ma et al. 1998). Similarly, AQP3 knock-out mice develop a form of nephrogenic diabetes insipidus with a severe defect in the urinary- concentrating mechanism (Ma et al. 2000).

Mutations in AQP2 cause a dramatic clinical phenotype a rare form of hereditary nephrogenic diabetes insipidus (NDI) (Deen et al. 1994). NDI is a disease whose etiology is renal resistance to vasopressin, and whose clinical hallmark is excretion of large volumes of dilute urine. This also indicates that the expression and/or functional state of aquaporins, under certain circumstances, may be under hormonal influence. Patients with autosomal recessive NDI who have mutations in the AQP2 gene provide the first clear example that aquaporins can be rate-limiting for water transport. Acquired NDI is more common than the congenital form, and has a variety of causes. Lithium (Marples et al. 1995a), bilateral ureteral obstruction (Frøkiaer et al. 1996) and chronic hypokalemia (Marples et al. 1995b) - known causes of NDI- all produce marked reductions in AQP2 expression in animals, with a concomitant decrease in urinary concentrating ability. At the other end of the water imbalance spectrum, increased AQP2 expression has been demonstrated in conditions of fluid retention including congestive heart failure (Nielsen et al. 1997, Xu et al. 1997), cirrhosis (Fujita et al. 1995) and pregnancy (Ohara et al. 1998).

Disruption of the AQP4 gene in mice resulted in a mild urinary concentrating defect (Ma et al. 1997b) but had little or no effect on colonic fluid excretion or faecal dehydration (Wang et al. 2000).

Lung fluid transport in AQP1 and AQP4 knock-out mice were investigated by Bai et al. (1999). Despite a 10-fold reduction in airspace-capillary water permeability in AQP1 knockout mice compared with wild-type mice, the rates of isosmolar alveolar fluid absorption did not differ significantly. There was little effect of deletion of the airway water channel AQP4.

Ma et al (1999) reported that transgenic mice lacking AQP5 exhibit decreased saliva secretion, indicating a physiologic relevance of AQP5 in salivary gland function. Steinfeld et al. (2001) described the localisation of AQP5 in human salivary glands, and indicated an abnormal distribution of AQP5 in salivary glands from Sjδgren's Syndrome patients, which could be responsible for a loss of saliva secretion in these patients.

Two functional groups of mammalian aquaporins are now being recognized (Agre et al. 1998). The first, including AQP0, AQP1, AQP2, AQP4 and AQP5, are permeable only to water, as classically defined. A second group, including AQP3, AQP7 and AQP9, are highly permeable to water, but are also permeated by glycerol and other small molecules. The structural explanation and physiological relevance of these differences is not known. Although the sequence of AQP6 is similar to the water-selective group, the recent surprising observations of gated anion conductance indicates the functional distinction is more complex than previously thought (Yasui et al. 1999). The sequence of AQP8 is intermediate between the water-selective and the glycerol permeant groups, and functional definition is awaited (Koyama et al. 1997). Thus, it may be that aquaporins, including AQP8, play a profound role in gating water and possibly other small molecules in epithelia of the gut and of other tissues, that are thought to have high paracellular permeability. The present invention is based on the discovery of a genetic predisposition for IBD associated with genetic markers located in a region on chromosome 16 in close proximity to the location of the AQP8 gene. The use of compounds able to modulate the activity or amount of AQP8 has been identified as a new therapeutic concept for the treatment of IBD by the present inventors. The analysis of the sequence of the AQP8 gene will provide a new method for the diagnosis of susceptibility to IBD.

According to one aspect of the present invention there is provided use of compounds able to modulate the activity or amount of AQP8 for the treatment IBD. Modulation of the amount of AQP8 by a compound may be brought about for example through altered gene expression level or message stability. Modulation of the activity of AQP8 by a compound may be brought about for example through compound binding to AQP8 protein. In one embodiment, modulation of AQP8 comprises a compound able to reduce the activity or amount of AQP8. In another embodiment, modulation of AQP8 comprises a compound able to increase the activity or amount of AQP8. An example of a compound able to modulate the activity of AQP8 is an antibody.

Antibodies can be prepared using any suitable method. For example, purified polypeptide may be utilized to prepare specific antibodies. The term "antibodies" is meant to include polycional antibodies, monoclonal antibodies, and the various types of antibody constructs such as for example F(ab')₂, Fab and single chain Fv. Antibodies are defined to be specifically binding if they bind the allelic variant of SLC10A2 with a K_a of greater than or

7 1 equal to about 10 M^" . Affinity of binding can be determined using conventional techniques, for example those described by Scatchard et al., Ann. N.Y. Acad. Set, 51:660 (1949).

Polycional antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats, using procedures that are well-known in the art. In general, antigen is administered to the host animal typically through parenteral injection. The immunogenicity of antigen may be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immunizations, small samples of serum are collected and tested for reactivity to antigen. Examples of various assays useful for such determination include those described in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, see U.S. Patent Nos. 4,376,110 and 4,486,530. Monoclonal antibodies may be readily prepared using well-known procedures, see for example, the procedures described in U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), (1980). The monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 3: 1-9 (1990) which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, 7: 394 (1989).

Once isolated and purified, the antibodies may be used to detect the presence of antigen in a sample using established assay protocols, see for example "A Practical Guide to ELISA" by D. M. Kemeny, Pergamon Press, Oxford, England. According to another aspect of the present invention there is provided use of compounds able to modulate the activity or amount of AQP8 in preparation of a medicament for the treatment of IBD.

According to yet another aspect of the present invention there is provided a method of identifying a compound potentially useful for treatment of IBD which comprises assaying the compound for its ability to modulate the activity or amount of AQP8. Preferably the assay is selected from: i) measurement of AQP8 activity using a cell line which expresses AQP8 or using purified AQP8 protein; and _^ ii) measurement of AQP8 transcription or translation in a cell line expressing AQP8. The assay used to determine the effect of a compound to be tested on the activity of

AQP8 can be based on measurement of AQP8-mediated water-transport. Aquaporin-mediated water transport is generally measured using an osmotic swelling assay in Xenopus oocytes expressing an aquaporin cRNA. As originally described (Zhang et al. 1990), quantitative image analysis is used to deduce the time course of oocyte swelling in response to a sudden decrease in extracellular solution osmolality from 200 to 0-100 mosmol/kgH2O. Water transport in aquaporin-transfected mammalian cells has also been studied by a variety of biophysical approaches including stopped-flow light scattering (Ma et al. 1993), total internal reflection fluorescence microscopy (Farinas et al. 1995), laser interferometry (Farinas and Verkman 1996), and Fourier optics dark-field/phase-contrast microscopy (Farinas et al. 1997). Stopped-flow light scattering has also been used to assay aquaporin-mediated water transport in vesicles derived from yeast (Laize et al. 1995) and in reconstituted proteoliposomes containing purified aquaporin proteins (Van Hoek and Verkman 1992, Zeidel et al. 1992).

The assay used to determine the effect of a compound to be tested on the transcription or translation of AQP8 can be based on i) measurement of the amount of AQP8 mRNA formed using e.g. Northern blot analysis or quantitative real time PCR, ii) measurement of the amount of AQP8 protein formed using e.g. Western blot analysis, or immunochemical analysis such as ELISA, or iii) measurement of AQP8 activity as described above, in cells expressing AQP8.

The cells used in the assay can be cells naturally expressing an AQP8 or transfected cells expressing a recombinant AQP8. Preferably the AQP8 is the human recombinant AQP8. The AQP8 may be expressed in a variety of hosts such as bacteria, plant cells, insect cells, fungal cells and human and animal cells. Eukaryotic recombinant host cells are especially preferred. Examples include yeast, mammalian cells including cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including Drosophila and silkworm derived cell lines. Cell lines derived from mammalian species which may be used and which are commercially available include, L cells L-M(TK-) (ATCC CCL 1.3), L cells L- M (ATCC CCL 1.2), HEK 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) andMRC-5 (ATCC CCL 171). The expression vector comprising a nucleic acid encoding an AQP8 may be introduced into host cells to express a polypeptide of the present invention via any one of a number of techniques including calcium phosphate transformation, DEAE-dextran transformation, cationic lipid mediated lipofection, electroporation or infection.

The transfected host cells are propagated and cloned, for example by limiting dilution, and analysed to determine the expression level of recombinant AQP8. Identification of transformed host cells which express the AQP8 may be achieved by several means including immunological reactivity with antibodies and/or the detection of biological activity using the assays described herein. Recombinant human AQP1 or AQP2 have been expressed in yeast and were localised within secretory vesicles. Secretory vesicles containing AQP1 and AQP2 exhibited high water permeabilities and low activation energies for water flow, indicating expression of functional AQP1 and AQP2 (Coury et al. 1998). Expression of in yeast followed by measurement of water permeability of isolated yeast vesicles has also been used to study the function of AQP2 mutants (Shinbo et al. 1999).

Transcriptional regulation of gene expression is mediated by specific DNA elements in the promoter that directs binding of transcription factors, which thereby mediate transcription of the gene. Eukaryotic transcription factors can be divided in two main groups i) basal transcription factors that interact with promoter sequences proximal to the start of transcription, thereby initiating transcription upon recruitment of RNA polymerase II and ii) transcription factors that bind to specific distal promoter elements, thereby modulating the transcription upon contact with the basal transcription machinery. A fundamental physiological process in the eukaryotic organism is that cells can communicate with their environment and respond to extracellular stimuli through signalling molecules, such as hormones and growth factors. The final event for such signalling is the binding of transcription factors to specific distal promoter elements leading to for example up-regulated or tissue specific gene expression. Because of their regulatory role, promoter elements are putative targets for screening of therapeutic agents. The sequence of the human AQP8 promoter, disclosed in this patent application, makes it possible to screen for therapeutic agents selectively regulating transcription of AQP8.

Suitable host cells are cells known to express AQP8 or cells known to express transcription factors which can influence the transcription of AQP8. Host cells transfected with DNA encoding specific transcription factors can preferably be used to study the interaction with defined transcription factors and the AQP8 promoter.

The assay used to determine the effect of a compound to be tested on the transcription of AQP8 can also be based on measurement of the activity of the AQP8 promoter using a reporter gene system. The reporter gene system is an expression system comprising nucleic acid molecules constituting an AQP8 promoter, or fragments thereof, the expression system further comprising a reporter gene, the promoter and the reporter gene being positioned so that the expression of the reporter gene is regulated by the AQP8 promoter. The amount of reporter protein formed is used as an indication of the activity of the AQP8 promoter. Preferably the AQP8 promoter is the human AQP8 promoter, preferably the nucleotide sequence 1 - 1956 of SEQ ID NO: 1, or fragments thereof.

Suitable reporter genes that can be used for the construction of the reporter gene system are e.g. the firefly luciferase gene, the bacterial chloramphenicol acetyl transferase (CAT) gene, the β-galactosidase (β-GAL) gene, and the green fluorescent protein (GFP). According to another aspect of the present invention there is provided a method of preparing a pharmaceutical composition which comprises: i) identifying a compound as useful for treatment of IBD according to a method as described herein; and ii) mixing the compound or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable excipient or diluent.

In yet another aspect of the present invention there is provided a diagnostic method comprising the analysis of the sequence of chromosome 16, or a part thereof, in a DNA sample obtained from a patient, for the determination of susceptibility to IBD, further comprising determining the presence or absence of an allele of a polymorphic marker located on a chromosomal fragment corresponding to the BAC 504N19 (EMBL AF265340), BAC A- 249B10 (EMBL AC002288) or BAC CTD-2547G23 (EMBL AC008741). Preferably, the method comprises the analysis of the sequence of the AQP8 gene, or a part thereof, in a DNA sample obtained from a patient, for the determination of susceptibility to IBD, and further comprises determining the presence or absence of an allele of a polymorphic marker located in the AQP8 gene of the patient.

In another aspect of the invention there is provided a method for the modulation of a immune response to an auto-immune epitope on the AQP8 protein. Preferably this method comprises a method for the induction of immunologic tolerance. Immunologic tolerance can be induced e.g. by oral immunisation using the B subunits of cholera toxin (CT) from V. cholera or the heat labile toxin (LT) from E.coli in conjugation with the auto-antigen (Simmons et al. 2001). Autoimmune responses are known to be a cause of a number of inflammatory disease, such as rheumatoid arthritis, pancreatitis and multiple sclerosis.

In another aspect of the invention is there is provided a method for the diagnosis of IBD comprising the measurement auto-antibodies directed against the AQP8 protein. Abbreviations

The invention will now be illustrated with reference to the following non-limiting Examples in which Figure 1 represents a map of the chromosomal region of interest. AMPLLTAQ™ available from Perkin-Elmer Cetus, is used as the source of thermostable DNA polymerase.

General molecular biology procedures can be followed from any of the methods described in "Molecular Cloning - A Laboratory Manual" Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory, 1989) or in "Current Protocols in Molecular Biology Volumes 1-3 , edited by F M Asubel, R Brent and R E Kingston; published by John Wiley, 1998.

EXAMPLE 1

We have narrowed the region containing the susceptibility gene for 1BD1 to ~7Mb in physical distance using high-density genotyping with STS markers and yet unpublished single nucleotide polymorphisms, followed by standard procedures for linkage disequilibrium testing (Terwilliger, 1995). TDT-testing across the region flanked by STS markers, D16S401 and D16S409, respectively, with a total of 32 markers (13 STS and 19 SNPs) verified the initial linkage data and revealed linkage distortion at two sites. Our data indicate that the susceptibility gene for IBD1 is very likely located in the region flanked by D16S401 and D16S409. 1.1 Family ascertainment and pheno types:

Patients were recruited from IBD programs at Charite University Hospital (Berlin) and the 1st Department of Medicine at the Christian- Albrechts-University of Kiel. Kindreds with > 2 affected siblings diagnosed with IBD were sampled, leading to a more than 250 affected sib pairs included in the analysis. Written informed consent was obtained from all study participants. Recruitment protocols were approved by institutional review committees. The diagnosis of IBD and classification in CD and UC were determined by standard diagnostic criteria as described by Lennard- Jones (1989) and Podolsky (1991). Patients were directly examined by one or more of the principal investigators. Alternatively, two written records, containing a detailed disease history and results of all diagnostic procedures, were obtained for each patient and reviewed by the principal investigators. A venous blood sample was obtained from the affected siblings and their parents, if possible. 1.2 Genotyping:

Genomic DNA was prepared from whole blood samples with the Puregene system (Gentra Systems). The individual DNA samples were arrayed on 96-well microtiter plates and subjected to amplification by PCR, with individual marker amplicons. Microsatellite markers were genotyped with fluorescent methods, as described by Hall and Nanthakumar (1997). Data were collected using ABI377 automated sequencers and data analysis was performed using GENESCAN (Vs.2.1) and GENOTYPER (Vs. 1.1.1). Allele analysis and individual allele calling were performed as described by Hall and Nanthakumar (1997) and Idury and Cardon (1997). SNPs were identified by genomic sequencing using ABI3700 automated sequencers, cDNA selection and mutation detection methodologies. Genotyping of informative SNPs was performed by real-time PCR using the TAQMAN™ technology from PE Biosystems. PCR reactions were performed in an AB 19700, and fluorescence results were determined with the use of ABI7700 sequence-detector single-point measurement. Allele analysis and individual allele calling were performed as described by Hall and Nanthakumar (1997). Genetic maps were constructed with the automated mapping program MULT AP (Vs. 2.0, Matise et al 1994). The resulting distances between markers, determined by the Kosambi map algorithm, are given in centimorgans (cM). 1.3 Genetic analysis

Genetic analyses were conducted with the use of the two aforementioned standard diagnostic categories, CD and UC. A third category, ALL, contains CD/CD, UC/UC and CD/UC (mixed ASPs) and, therefore, represents IBD as a single phenotype for analysis. 5 Allele frequencies for each marker were calculated from the cohort genotyping data for all individuals. Association statistics were calculated by the TDTLIKE program from the ANALYZE software package (Terwilliger 1995). Tested alleles are restricted to > 10 observed transmissions. The program algorithm provides p-values corrected for the testing of multiple alleles. 0

Sequence information for markers (1STS, 3 SNPs) in the region between D16S401 and D16S409

5 D16S3068 EMBL HSA349ZH9

A-249BlO_27kb TTCCTGTTTATTTATTATcATTTTATGGAGAGTGTTGGTACAGAAA SEQ ID NO 5 TTCCTGTTTATTTATTATgATTTTATGGAGAGTGTTGGTACAGAAA SEQ ID NO 6 0 A-249B10_37kb TGGGAAAGACTTCAATAaGTTTACATTCCTAGATTCTCC SEQ ID NO

TGGGAAAGACTTCAATAtGTTTACATTCCTAGATTCTCC SEQ ID NO

A-249Bl0_53kb ACTTATTGAGGcACCCAGTTGACACCCATG SEQ ID NO 9 ACTTATTGAGGtACCCAGTTGACACCCATG SEQ ID NO 10 5

TDT-results for positive markers on 16p in 200 ASPs for IBD

0 a) TDTLIKE (Terwilliger, 1995)

b) PDT (Martin et al. 2000)

A strong association was seen for the D16S3068 marker as well as for three of the SNPs tested.

1.4 Bioinformatics

Three human BACs from chromosome 16, RP11-451N20 (EMBL AC060785), 504N19 (EMBL AF265340), A-249B10 (EMBL AC002288) comprising the marker D16S3068 as well as the BAC CTD-2547G23 (EMBL AC008741) partly overlapping the BAC A-249B10 were analysed for potential disease related genes. Sequences corresponding to the AQP8 cDNA were found on the BAC CTD-2547G23 (EMBL AC008741).

The sequence of this BAC, available in EMBL as accession no. AC008741, comprising 34 unordered fragments, were further analysed and one fragment was found to contain the AQP8 gene, including 1.9 kb of the promoter region. 1.5 Identification of SNPs in the AOP8 gene

PCR primers are constructed based on the DNA sequences flanking the exons as well as the promoter region of the AQP8 gene. After PCR amplification, the exons and the promoter region are sequenced from genomic DNA samples obtained from 23 IBD affected individuals as well as 20 normal controls. Sequence variants present in more than 10% of the individuals are identified and selected for SNP genotyping in the rest of the cohort using the TAQMAN™ technology described previously.

1.6 Method of diagnosing susceptibility to IBD in a patient

DNA samples are obtained from patients. The presence or absence of an allele of one or more polymorphic marker which is associated with increased susceptibility to IBD is determined, e.g. using the TAQMAN™ technology.

1.7 AOP8 assays

AQP8 activity is measured as water permeability of yeast secretory vesicles derived from yeast expressing recombinant AQP8.

Yeast strains andplasmid construction. The cDNA encoding AQP8 (SEQ ID NO: 1) is cloned into the yeast expression vector pYES2 (Invitrogen, San Diego, CA) by standard techniques (Sambrook et al. 1989). Yeast, strain SYl, Potenza et al (1992), is transformed e.g. by electroporation with a Bio-Rad gene pulser with the following settings: 1.5 kV, 200 ohms, and 25 μF. Transformed yeast is grown and maintained in defined media lacking uracil and containing raffinose as a carbon source. Yeast is transferred to rich yeast extract/peptone (YEP)-galactose medium [0.5% yeast extract (wt/vol), 1.0% bactopeptone (wt/vol), and 2.0% galactose (wt/vol)] at 25°C for 2-4 h to initiate protein expression and then switched to 37°C overnight to force accumulation of the secretory vesicles. Control studies is performed using vesicles prepared from the background strain or from yeast transformed with the pYES2 vector lacking the AQP8 insert. Immunoblotting. Samples are incubated at 60-80°C for 5 min and then separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using 4-20% continuous-gradient tris(hydroxymethyl)aminomethane (Tris)-Cl-glycine Ready Gels or 12% SDS-polyacrylamide slabs and transferred to nitrocellulose (Bio-Rad). The blots are blocked with blot buffer consisting of 1% powdered milk and 3% Tween 20 in phosphate-buffered saline (pH 7.25). The blots are incubated with anti-AQP8 antibody overnight at 4°C and then visualized e.g. by the enhanced chemiluminescence method (NEN or Amersham).

Vesicle preparation. The yeast is treated with 10 mM dithiothreitol (DTT) in 100 mM Tris-Cl, pH 9.4, and spheroplasts were generated by digesting the cell wall with bacterially expressed recombinant lyticase. The plasma membrane is cross-linked with concanavalin A to increase its density above that of the secretory vesicles. Spheroplasts are lysed in lysis buffer (0.8 M sorbitol, 10 mM triethanolamine, 1 mM EDTA, pH 7.2) containing 5,6-Carboxyfluorescein (CF) (7.5 mg/ml), and unlysed cells and concanavalin-cross-linked plasma membranes are pelleted at 11,000 revolutions/min (20,000 g) in a Sorvall GSA rotor for 10 min at 4°C. Vesicles are pelleted from the supernatant and washed to remove extravesicular CF by centrifugation at 29,000 revolutions/min (144,000 g) in a Sorvall TH-641 swinging-bucket rotor for l h at 4°C.

Water permeability. The integrity of the yeast vesicles that are loaded with CF during lysis is verified by first measuring their fluorescence intensity on a spectrofluorimeter, e.g. an SLM- AMINCO SPF-500C. Extravesicular CF is then quenched by addition of an anti-CF antibody, and vesicles are shrunk by successive additions of a high-osmolal solution using sucrose as the osmoticant. Yeast vesicles are loaded onto a stopped-flow apparatus, e.g. an Applied Photophysics SF7mv. The vesicles are subjected to an abrupt doubling of the osmolality, causing the vesicles to shrink and the CF to self-quench. The osmotic membrane water permeability is calculated from the initial rate of shrinkage of the yeast vesicles. The assay is designed to force a decrease in fluorescence due to shrinkage and CF self-quenching. The decrease in fluorescence is measured and correlated to the decrease in volume. Fluorescence is measured with incident light of 490 ± 1 nm and a cut-on filter that measures light emitted at wavelengths >510 nm.

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Claims

1 A method of identifying a compound useful for treatment of IBD which comprises assaying the compound for its ability to modulate the activity or amount of AQP8. 2 A method according to claim 1 wherein the assay is selected from: i) measurement of AQP8 activity using a cell line which expresses AQP8 or using purified AQP8 protein; and ii) measurement of AQP8 transcription or translation in a cell line expressing AQP8. 3. A method according to claim 2 wherein the assay used to determine the activity of AQP8 is based on measurement of AQP8-mediated water-transport

4 A method according to claim 2 wherein the measurement of transcription or translation of AQP8 is measured by any of the following: i) measurement of the amount of AQP8 mRNA ; ii) measurement of the amount of AQP8 protein formed; or iii) measurement of AQP8 activity in cells expressing AQP8.

5 A method according to any preceding claim wherein the AQP8 is human recombinant AQP8.

6 A method according to any preceding claim which measures the activity of the AQP8 promoter using a reporter gene system. 7 A method according to claim 6 wherein the AQP8 promoter is the human AQP8 promoter.

8 A method of preparing a pharmaceutical composition which comprises: i) identifying a compound as useful for treatment of IBD according to a method as defined in any one of claims 1-7; and ii) mixing the compound or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable excipient or diluent.

9 A diagnostic method for the determination of susceptibility to IBD comprising the analysis of a DNA sample obtained from a patient for presence or absence of an allele of a polymorphic marker located on a chromosomal fragment corresponding to the BAC 504N19 (EMBL AF265340), BAC A-249B10 (EMBL AC002288) or BAC CTD-2547G23 (EMBL AC008741).

10 A method for the diagnosis of IBD or a predisposition thereto comprising the measurement auto-antibodies directed against AQP8. 11 Use of a compound able to modulate the activity or amount of AQP8 in preparation of a medicament for the treatment of IBD.