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WO2008100217A1 - Polymorphismes des gènes de l'inflammasome cryopyrine et leurs utilisations dans le traitement des maladies inflammatoires - Google Patents

Polymorphismes des gènes de l'inflammasome cryopyrine et leurs utilisations dans le traitement des maladies inflammatoires Download PDF

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WO2008100217A1
WO2008100217A1 PCT/SE2008/050166 SE2008050166W WO2008100217A1 WO 2008100217 A1 WO2008100217 A1 WO 2008100217A1 SE 2008050166 W SE2008050166 W SE 2008050166W WO 2008100217 A1 WO2008100217 A1 WO 2008100217A1
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card8
ciasl
polymorphism
genes
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WO2008100217A8 (fr
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Peter Söderkvist
Per Eriksson
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of the treatment of inflammation, to the identification and selection of appropriate treatment for individual patients, to the prevention of disease progression and to the prevention of the development of related disorders and diseases due to tissue damage and degeneration resulting from inflammation.
  • Inflammatory diseases Inflammation is a heterogeneous process which occurs in response to tissue damage and can be a transient response to specific injury, infection, toxin, allergen or other temporary stimuli or can be a chronic condition, resulting from a myriad of single or combined underlying inherited and/ or environmental factors.
  • Chronic inflammation also appears to be an underlying causative factor in a number of cancers, with the processes of inflammation, such as the recruitment of leukocytes to sites of cell damage and subsequent induction and secretion of a number of cytokines and chemokines providing an environment of oxidative damage and increased risk of DNA mutation that is favorable for tumour progression.
  • Inflammation due to its inherent role in promoting cell growth in order to repair cell damage can be an exacerbating factor at all three stages of tumour development (initiation, progression and metastasis) and can also be a major contributory factor underlying the severity and time course of allergic reactions and responses.
  • Common inflammatory disorders include, but are not limited to; lung disorders such as asthma; joint and connective tissue diseases such as rheumatoid arthritis (RA), juvenile arthritis and ankylosing spondylitis, Crohn's disease, ulcerative colitis, inflammatory bowel and other digestive tract disorders; skin disorders such as psoriasis; diseases occurring in connection with inflammation associated with diabetes; inflammation in tissues and organs of the cardiovascular system and inflammation associated with bone disorders, such as osteopenia and osteoporosis.
  • RA rheumatoid arthritis
  • RA rheumatoid arthritis
  • Crohn's disease Crohn's disease
  • ulcerative colitis inflammatory bowel and other digestive tract disorders
  • skin disorders such as psoriasis
  • diseases occurring in connection with inflammation associated with diabetes inflammation in tissues and organs of the cardiovascular system and inflammation associated with bone disorders, such as osteopenia and osteoporosis.
  • inflammatory disorders and diseases are not uniform in nature and patients initially presenting with one type of immune problem often subsequently begin to display and suffer from other associated conditions, including cancers, as their disease progresses.
  • Underlying inflammatory disorders and diseases are the aberrant responses and stimulation of cells of the immune system.
  • Cytokines are messenger molecules that are produced by many of the cells of the body and which function to excite other immune cells and trigger initiation of diverse response signaling cascades and feedback regulatory mechanisms.
  • cytokines produced in large quantities, such as tumour necrosis factors (TNF) and interleukins (IL). Treatment of inflammation
  • anti-inflammatory treatment options available to the attending physician include steroid treatments such as corticosteroids (for example prednisone); non-steroidal anti-inflammatory drugs (NSAIDs); disease modifying anti-rheumatic drugs (DMARDs) such as methotrexate, leflunomide, hydroxychlorquinine an sulfasalazine and also biologic treatments such as antibodies and recombinant genetically engineered molecules which function as agonists or antagonists for specific receptors and ligands (for example TNF- pathway inhibitors and IL-pathway inhibitors - see below).
  • corticosteroids for example prednisone
  • NSAIDs non-steroidal anti-inflammatory drugs
  • DMARDs disease modifying anti-rheumatic drugs
  • biologic treatments such as antibodies and recombinant genetically engineered molecules which function as agonists or antagonists for specific receptors and ligands (for example TNF- pathway inhibitors and IL-pathway inhibitors - see below).
  • Inflammasome was coined by Tschopp and colleagues in 2004 (Agostini, Martinon et al. 2004). Inflammasomes are cytosolic protein complexes which have recently been shown to activate caspase-1 and thereby can be postulated to play a role in the regulation of IL- 1 ⁇ -formation (Martinon, Burns et al. 2002; Agostini, Martinon et al. 2004). There are at least two different types of inflammasome and these are denoted according to which NALP protein is involved in the complex.
  • cryopyrin inflammasome is a multi-protein complex consisting of cryopyrin (also known as NALP3 and PYPAFl), ASC (apoptosis-associated speck- like protein) and TUCAN (tumor-up-regulated CARD-containing antagonist of caspase-9; also known as CARD-8 or Cardinal).
  • cryopyrin also known as NALP3 and PYPAFl
  • ASC apoptosis-associated speck- like protein
  • TUCAN tumor-up-regulated CARD-containing antagonist of caspase-9; also known as CARD-8 or Cardinal
  • Cryopyrin which belongs to the NACTH-LRR/ CATERPILLAR protein - superfamily (Ting and Davis 2005), is encoded by the gene CIASl (cold-induced auto-inflammatory syndrome 1). This is composed of a pyrin (PYD) domain, a NACHT domain (also called NOD domain) and a LRR domain.
  • PYD pyrin
  • NACHT also called NOD domain
  • LRR LRR domain
  • the protein-protein interactions of the cryopyrin inflammasome result in the activation of two caspase-1 molecules, which convert pro-interleukin- 1 ⁇ (pro-IL-l ⁇ ) to its active form IL- l ⁇ (Petrilli, Papin et al. 2005; Drenth and van der Meer 2006; Ogura, Sutterwala et al. 2006) and mediates inflammation, defective apoptosis and persistent activation of neutrophils (Hawkins, Lachmann et al. 2004).
  • the expression patterns of inflammasome proteins remain to be elucidated, but it is thought that most immune cells express cryopyrin in their cytoplasm.
  • the invention relates to a method for treatment of a patient suffering from a disease related to polymorphisms in the CIASl and/or CARD8 genes characterized in that it comprises the steps
  • the method comprises treating said patient with an IL-blocking therapy if said patient has at least one polymorphism in both the CIASl and the CARD8 gene, respectively.
  • the polymorphisms may be Q705K in CIASl or ClOX in CARD8.
  • the IL-blocking therapy may be an IL- 1 -blocking therapy, such as an IL- l ⁇ -blocking therapy, e.g. administration of anakinra.
  • the disease related to polymorphisms in the CIASl and/ or CARD8 genes is an inflammatory disease, such as arthritis.
  • the patient to be treated has further been diagnosed with a cancer or an oncogenic lesion.
  • the invention relates to IL-inhibitors for use in a method for treatment of a patient suffering from a disease related to polymorphisms in the CIASl and/or CARD8 genes, wherein said method comprises the steps
  • the method comprises treating said patient by administration of an IL-inhibitor if said patient has at least one polymorphism in both the CIASl and the CARD8 gene, respectively.
  • the polymorphisms may be Q705K in CIASl or ClOX in CARD8.
  • said IL-inhibitor is an IL- 1 -inhibitor, preferably an IL-l ⁇ -inhibitor, such as anakinra.
  • said method is for treatment of a patient suffering from an inflammatory disease, such as arthritis.
  • the patient to be treated has further been diagnosed with a cancer or an oncogenic lesion.
  • the invention relates to IL-inhibitors for use in a method according to the first aspect. In a further aspect, the invention relates to the use of IL-inhibitors for production of a pharmaceutical composition for use in a method according to the first aspect.
  • the invention relates to a method of assessing if a patient suffering from, or being at risk of developing, a disease related to polymorphisms in the CIASl and/ or CARD8 genes is likely to respond to treatment with an IL-inhibitor, comprising obtaining information on polymorphisms in the CIASl and/ or CARD8 genes in the genome of said patient, wherein the presence of a polymorphism in at least one of the genes CIASl and CARD8 indicates that the patient is likely to respond to treatment with an IL-inhibitor.
  • the method comprises obtaining information on polymorphisms in both the CIASl and CARD8 genes in the genome of said patient.
  • the presence of the polymorphism Q705K in CIASl and/ or ClOX in CARD8 indicates that the patient is likely to respond to treatment with an IL-inhibitor.
  • the IL-inhibitor is an IL-I inhibitor and may be an IL-l ⁇ -inhibitor, such as anakinra.
  • the patient to be treated has further been diagnosed with a cancer or an oncogenic lesion.
  • the invention relates to a kit for detection of gene polymorphisms, comprising means for detecting a polymorphism Q705K in CIASl and/ or means for detecting a polymorphism ClOX in CARD8.
  • said means for detecting a polymorphism Q705K in CIASl comprise PCR-primers having the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 and a SNP-specific primer having the sequence of SEQ ID NO: 3 and/ or said means for detecting a polymorphism ClOX in CARD8 comprise PCR-primers having the sequences of SEQ ID NO: 4 and SEQ ID NO: 5 and a SNP-specific primer having the sequence of SEQ ID NO: 6.
  • said means for detecting a polymorphism comprises:
  • Q705K in CIASl comprise an molecule specifically binding to the Q705K-CIAS1 protein and means for detecting said molecule and/ or said means for detecting a polymorphism Q705K in CIASl comprise an molecule specifically binding to the C10X-CARD8 protein and means for detecting said molecule.
  • the molecule specifically binding to the Q705K-CIAS1 protein or C10X-CARD8 protein may be an antibody or a binding fragment thereof, as commonly used in the art. It may also be any other molecule that naturally binds specifically to said proteins or can be modified to bind specifically to them.
  • the inflammatory disease referred to in the above aspects may be any inflammatory disease susceptible to treatment with IL-inhibitors. Such inflammatory diseases are further discussed under the section "Inflammatory diseases" in the background section of this specification.
  • the inflammatory disease may be inflammatory disorders of joints and connective tissue such as rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, polymyalgia rheumatica and temporal arthritis; Crohn's disease and other inflammatory disorders of the gastrointestinal tract; inflammatory disorders of the skin, such as, but not limited to psoriasis; inflammatory disorders of the circulatory system and inflammatory bone disorders, such as osteoporosis and osteopenia.
  • a disease related to polymorphisms in a specified gene or genes shall be construed as a disease caused, aggravated or ameliorated by polymorphisms in said gene or genes.
  • CIASl cold-induced auto-inflammatotry syndrome 1
  • NLRP3 is the gene encoding cryopyrin, also known as NALP3 and PYPAFl .
  • CARD8 is the gene encoding TUCAN (tumour up-regulated CARD- containing antagonist of caspase-9), also known as Cardinal.
  • CIAS1 /CARD8 -/- is used to denote the presence of at least one variant allele in both CIASl and CARD8 genes, respectivelty.
  • obtaining information on polymorphisms in the genome of a patient shall be construed broadly.
  • the information may be obtained directly by sequencing of the genome or individual genes of interest in a sample from the patient, but may also be obtained from sequence data previously obtained from the patient or relatives of the patient.
  • CRP C-reactive protein; normal value: ⁇ 10 mg/L
  • WBC white blood count normal range: 3.5-8.8 xlO 9 /L
  • the figure shows baseline or microbe-challenged caspase- 1 activity and IL- 1 ⁇ release in the patient compared to healthy control persons.
  • C Mean IL- l ⁇ levels in response to live S.
  • CIASl is present on chromosome Iq44.
  • mutations in CIASl have been shown to be implicated in familial periodic fever syndromes, like Muckle-Wells syndrome (MWS), familial cold auto-inflammatory syndrome (FCAS) and neonatal-onset multisystem inflammatory disease (NOMID; also known as chronic infantile neurologic cutaneous articular (CINCA) syndrome) (Agostini, 2004 see above).
  • FCAS familial cold auto-inflammatory syndrome
  • NOMID neonatal-onset multisystem inflammatory disease
  • Recurrent fever, joint pain, skin rashes and systemic inflammation are common features to these disorders, but differences in disease severity are often seen (reviewed in(Simon and van der Meer 2006).
  • FCAS represents the mildest form and is precipitated by exposure to cold, while CINCA is characterized by papilledema and arthropathy with premature ossification and overgrowth. More than 50 different familial mutations in cryopyrin have been reported so far. The majority of these mutations are present in exon 3 (http://frnf.igh.cnrs.fr/infevers/), while single cases have been reported to occur in exons 4 and 6 (Frenkel, van Kempen et al. 2004; Matsubayashi, Sugiura et al. 2006).
  • the mutations described above are considered to be familial mutations which are activating and dominant, since they are present in heterozygous form in affected individuals, and depending on the mutation all lead to different grades of disease severity.
  • CIASl knock-out mice show a clear deficiency in caspase-1 mediated IL- 1 ⁇ -activation (Kanneganti, Ozoren et al. 2006; Mariathasan, Weiss et al. 2006; Martinon, Petrilli et al. 2006; Sutterwala, Ogura et al. 2006) and the exact mechanism of CIAS 1 in the activation of interleukins remains to be clearly documented.
  • cryopyrin-inflammasome Le. the genes encoding ASC and TUCAN, have not to date been addressed in the context of many inflammatory disorders, such as, for example, periodic fever syndromes (or so called cryopyrinopathies) involving CIAS 1.
  • TUCAN was identified as an anti-apoptotic member of the human CARD family of proteins that is overexpressed in some types of cancer (for example colon cancer) and selectively binds and inhibits activation of procaspase- 1 , -8 and -9(Pathan, Marusawa et al. 2001 ; Razmara, Srinivasula et al. 2002; Yamamoto, Torigoe et al. 2005).
  • the ClOX polymorphism of CARD8 (rs204321 1) introduces an early stop codon, truncating the normally 643 amino acid protein, and is thus highly likely to be of functional importance.
  • a connection to inflammatory disorders was only recently identified when genetic variants in the TUCAN gene were found to be associated with Crohn ' s disease (McGovern, Butler et al. 2006).
  • the present invention provides the first disclosure of the association of the ClOX polymorphism with a response to treatment, for a range of inflammatory disorders, comprising administration of IL-inhibitors.
  • the present invention provides the first disclosure of the association of the combination of polymorphic positions in the two independent genes, CIASl and CARD8, and their use in the identification of individuals who will respond to IL-inhibitors for the prevention and treatment of inflammatory disorders.
  • the present invention also discloses a more general utility for the individual genetic components of the cryopyrin inflammasome complexes and highlights in particular a more widespread role for the CIASl and CARD 8 genes especially in general diseases where interleukins are upregulated or are otherwise implicated.
  • CIASl -QK/ CARD8-CX compound polymorphic individuals i.e. individuals heterozygous for CIASl and CARD8 polymorphisms, as denoted in the above table as CIASl -QK/ CARD8-CX, display high IL- l ⁇ levels and have a worse prognosis in terms of inflammatory disease progression.
  • IL-inhibitors such as Anakinra.
  • CIASl -QK/ CARD8-CX individuals who have suffered from inflammatory disorders for a number of years and who have been treated with a range of anti-inflammatory medications, including corticosteroids (for example prednisone); non-steroidal anti-inflammatory drugs (NSAIDs); disease modifying anti-rheumatic drugs (DMARDs) such as methotrexate, leflunomide, hydroxychlorquinine an sulfasalazine and also biologic treatments such as TNF- inhibitors, without significant alleviation of inflammation have been found by us to demonstrate superior response to IL-inhibitor treatment and alleviation of symptoms immediately following initiation of IL-inhibitor treatment.
  • corticosteroids for example prednisone
  • NSAIDs non-steroidal anti-inflammatory drugs
  • DMARDs disease modifying anti-rheumatic drugs
  • TNF- inhibitors without significant alleviation of inflammation have been found by us to demonstrate superior response to IL-inhibitor treatment and alleviati
  • inflammatory responses and symptoms have reoccurred.
  • many cancers arise in the region of sites of infection, chronic irritation and inflammation (Coussens L. M. and Werb Z., Nature (2002), 420 (6917):860-7) with the risk of development of disorders such as Crohn's disease, ulcerative colitis and other cancers of the digestive tract being particularly associated with chronic inflammation.
  • Other disorders known to have a major inflammatory component include central nervous system (CNS) disorders and the pathological and physiological processes of inflammation are also of particular interest for example in multiple sclerosis (Ransohoff R. M., et al., Cytokine Growth Factor Rev.
  • TNF-alpha Tumor necrosis Factor- ⁇
  • cytokine IL- 1 ⁇ In addition to activation of the cytokine IL- 1 ⁇ by a pathway involving the CIASl and CARD8 genes, production of this cytokine is also known to be regulated by the product of the NF- ⁇ gene. Leukocyte infiltration and the subsequent activation of cytokines and of particular chemokines has been reported to be able to activate anti-apoptotic pathways in some types of cancer cells (Rollins B.J., Eur. J. Cancer, (2006), 42 (6): 760-7). The coupling of inflammatory processes and apoptosis inhibition is intriguing in the context especially of the CARD8 gene which has previously described above as a suppressor of apoptosis in some circumstances.
  • RA Rheumatoid arthritis
  • TNF tumour necrosis factor alpha
  • IL-I interleukin 1
  • the TNF- blocking biologies available today etanercept, infliximab, and adalimumab
  • IL-I is still regarded as a key mediator of long-term injury to cartilage and bone (Smeets, Barg et al. 2003).
  • RA is a heterogeneous disease, it cannot be excluded that distinct cytokine patterns in different clinical subgroups, may relate to different responsiveness to anti-rheumatic therapy (Ulfgren, Andersson et al. 2000; Ulfgren, Grondal et al. 2000).
  • the formation of biologically active IL- l ⁇ is dependent on cleavage of its inactive precursor pro-IL ⁇ by the cysteine protease caspase-1 , also called IL- l ⁇ converting enzyme or ICE (Thornberry, Bull et al. 1992).
  • inflammasomes cytosolic protein complexes called "inflammasomes” were shown to activate caspase-1 and thereby regulate the amount of IL-I ⁇ -formed (Martinon, Burns et al. 2002; Agostini, Martinon et al. 2004).
  • inflammasomes There are at least two different types of inflammasomes, usually denoted according to the NALP protein involved (Petrilli, Papin et al. 2005).
  • NALP3 cryopyrin
  • ASC proteins 'apoptosis-associated speck-like protein'
  • CARD-containing antagonist of caspase-9 also known as cardinal
  • IL-I blocking therapy has proven highly effective in these patients (Hawkins, Lachmann et al. 2003; Goldbach-Mansky, Dailey et al. 2006).
  • the study population comprised 174 patients (70% females, mean age 56 years) enrolled in a prospective multi-centre early RA cohort (the Swedish TIRA project) in south-east Sweden between 1996 and 1999 (Kastbom, Strandberg et al. 2004).
  • the patients fulfilled either >4/7 of the 1987 American College of Rheumatology (ACR) classification criteria (Arnett, Edworthy et al. 1988) (95% of the patients), or had morning stiffness > 60 minutes, symmetrical arthritis, and small joint arthritis (proximal interphalangeal/metacarpo- /metatarsophalangeal joints /wrists).
  • the symptom duration onset of joint swelling was ⁇ 12 months and > 6 weeks.
  • rheumatoid factor Agglutinating rheumatoid factor (RF) was present in 63% of the patients and anti-CCP in 68%.
  • the patients were followed regularly during three years and disease activity was assessed by C- reactive protein (CRP), erythrocyte sedimentation rate (ESR), physician's global assessment of disease activity (PGA) on a 4-degree ordinal scale, and the 28- joint disease activity score (DAS28) (Prevoo, van 't Hof et al. 1995).
  • CRP C- reactive protein
  • ESR erythrocyte sedimentation rate
  • PGA global assessment of disease activity
  • DAS28 28- joint disease activity score
  • Pharmaceutical therapy with disease-modifying anti-rheumatic drugs (DMARDs), analgesics and corticosteroids were instituted as judged appropriate by the patient's rheumatologist.
  • Anti-TNF therapy became available during the study period, and was prescribed to patients that had experienced unsatisfactorily response to conventional DMARD therapy including
  • the controls comprised 360 individuals (63% women, mean age 57 years) from a population-based reference material, randomly selected from the same geographical area as the patients. Individuals reporting rheumatic disease were exluded.
  • Autoantibodies RF was measured by latex particle agglutination at the local laboratories of the participating hospitals.
  • Anti-CCP was analyzed by a second generation enzyme-linked immunosorbent assay kit (Immunoscan RA CCP2; Eurodiagnostica, Arnhem, the Netherlands), where > 25 units was regarded as a positive test. Genotyping
  • the Q705K polymorphism of CIASl was detected by a commercially available MegabaceTM SNuPe genotyping kit (GE Healthcare, Bucks, UK).
  • DNA from peripheral blood was amplified using the primers 5'-tcctctttggcctggtaaac-3' (SEQ ID NO: 1) and 5'-caagaagaagctggcgaggaa-3' (SEQ ID NO: 2) with an initial denaturation step of 2 min at 94°C, followed by 35 cycles of 94°C for 20 s, 57°C for 20 s, 72°C for 20 s and a final extension step of 72°C for 5 min.
  • the PCR product was treated with exonuclease I and shrimp alkali phosphatase (Exo-SAP-IT®, GE Healthcare, Bucks, UK) for 15 minutes at 37°C followed by inactivation for 15 min at 80 0 C .
  • Purified DNA was combined with SNuPe premix and a single SNP-specific primer (5'-gagcttgggaggacacact-3', SEQ ID NO: 3) and thermally cycled at: 96°C for 10 seconds, 57°C for 5 seconds, 60 0 C for 10s, for a total of 26 cycles.
  • the product was finally combined with a formamide loading solution and a multi-injection marker for detection on MegaBACETM 1000 DNA sequencing system (GE Healthcare, Bucks, UK).
  • CARD8-C10X was similarly detected, using the primers 5'- tgctatcatcaggcacctacc-3' (SEQ ID NO: 4) and 5'-gagcttgggaggacact-3' (SEQ ID NO: 5) in a PCR reaction with an initial denaturation step of 2 min at 94°C, followed by 35 cycles of 94°C for 30 s, 56 0 C for 30 s, 72 0 C for 30 s and a final extension step of 72 0 C for 5 min.
  • the SNP-specific primer used was 5'- agaggcagagccattattg-3' (SEQ ID NO: 6). SE was defined and analyzed as previously described (Kastbom, Ahmadi et al. 2005).
  • Genotype frequencies were compared by the chi square test with Yates correction or Fisher's exact test, where appropriate. Odds ratios (OR) or relative risks (RR) and 95% confidence intervals (CI) were calculated for 2 x 2 contingency tables. Disease activity measures were compared by the Mann- Whitney U-test. Two-sided p-values less than 0.05 were regarded as significant. Results
  • Genotype distributions of CIASl and CARD 8 in patients and controls are detailed in Table 1. None of the polymorphisms showed significant deviations from the Hardy- Weinberg equilibrium among patients or controls. Compared to CIASl-QQ, genotypes with the variant allele present in CIASl (CIASl-QK or CIASl-KK) were slightly more common among patients than in controls, but the difference was not significant (OR 1.2, 95% CI 0.7-2.1). Similar ORs were seen when CARD8-CX and CARD8-XX was compared to CARD8-CC (OR 1.4, [0.9-2.1] and 1.3, [0.7-2.3], respectively).
  • CIAS1 /CARD8 -/- CIAS1 /CARD8 +/ + .
  • DAS28, ESR, PGA and CRP were significantly higher in CIAS1 /CARD8 -/- patients at several points of measurement (Fig. 1).
  • DMARD or oral glucocorticoid therapy did not differ significantly between the groups, although there was a trend toward CIAS1 /CARD8 -/- patients more often receiving a combination of >2 DMARDs (data not shown) .
  • NALP3 inflammasome proteins in the synovial cells of the RA patients remains to be investigated.
  • recent work have revealed that most immune cells express NALP3 in the cytoplasm, and it has been reported that RA patients express higher NALP3 levels in the synovium than do osteoarthritis patients (Rosengren, Hoffman et al. 2005; Kummer, Broekhuizen et al. 2006).
  • the polymorphisms investigated in this study were selected on the basis of their presence in a patient with a severe periodic fever syndrome and who experienced dramatic improvement on IL-I blocking therapy.
  • the CIAS1 /CARD8 compound polymorphism shows stronger association with the SE+/anti-CCP+ phenotype of RA. Apart from contributing to a more aggressive disease course (Kastbom, Strandberg et al. 2004; van Gaalen, van Aken et al. 2004), this raises interesting questions regarding an aetiopathogenetic importance of this SNP complex in RA. Whether or not proinflammatory cytokines are involved in the loss of self-tolerance has not been established in human autoimmune disease.
  • IL- l ⁇ may induce expansion of autoreactive T cells and promote their survival at peripheral check- points such as CD4 + CD25 + FoxP3 + regulatory T cells (O'Sullivan, Thomas et al. 2006).
  • Adopting the notion that IL- l ⁇ promotes an immune response towards self-antigens in the context of RA could provide an explanation to the increased risk of developing SE+/anti-CCP+ RA in CIAS1/CARD8 -/- individuals.
  • the presence of autoantibodies towards citrullinated proteins is highly specific for RA (Schellekens, Visser et al.
  • This example describes the successful use of anakinra, an IL- 1 receptor antagonist, in a patient with arthritis and chronic febrile illness.
  • a mutation previously described not to be associated with disease, in the patient's CIASl .
  • CARD8 which gives a premature, truncated protein was identified.
  • Monocytes isolated from the patient displayed increased spontaneous caspase-1 activity and IL- l ⁇ - production, indicating that the mutations result in a constitutively active NALP3 inflammasome.
  • the mutations are predicted to occur simultaneously (compound polymorphisms) in 4% of the Swedish population, indicating a role as susceptibility factors for chronic inflammatory disorders.
  • Genomic DNA was isolated from peripheral leukocytes using the Wizard Genomic DNA purification kit [Promega Inc.].
  • the PCR-products were sequenced using DYEnamic ET, Dye Terminator cycle sequencing kit for MegaBACE, [GE Healthcare].
  • SNP:s single nucleotide polymorphisms
  • non-adherent cells e.g. lymphocytes
  • Caspase-1 activity was measured in monocytes using a fluorometric caspase-1 assay [R&D systems]. The fluorescence was recorded at 535 nm after excitation at 450 nm in a Chameleon multi-label detection platform [Hidex OY]. IL- l ⁇ concentrations were measured in plasma or in the supernatants of stimulated monocytes using a LINCOplex kit [LINCO Research Inc.]. The samples were examined using Luminex® 100TM system. The data was analyzed using the software program StarStation 2.0 [Applied Cytometry Systems].
  • Staphylococcus aureus (S. aureus), strain Wood46 [American Type Culture Collection], were cultured for 18h (Zheng et al., 2004). Live bacteria were given to the monocytes (bacteria-to-cell ratio of 20: 1) for 3h. In some cases the monocytes were/ had been pre-activated over night with lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • NF- ⁇ detection monocytes, differentiated on glass cover slips for 9-1 1 days, were stimulated with LPS or left untreated. After fixing and permeabilization, the cells were stained with an anti-NF- ⁇ antibody [sc-8008, Santa Cruz], and an Alexa594-conjugated anti-mouse antibody [Molecular Probes]. The samples were analyzed with a Zeiss microscope equipped with a Canon G3 digital camera. The staining intensity of the nucleus vs. the cytoplasm was measured in at least 50 cells using Scion Image software, and NF- ⁇ B activity was expressed as the ratio of nuclear vs. cytoplasmic staining.
  • Characteristic signs of NOMID, FCAS and MWS such as urticaria, bony overgrowth of joints, aseptic meningitis and other CNS manifestations, chronic papilledema, sensorineural hearing loss, and short stature, were absent.
  • the patient has been investigated repeatedly, inter alia by colonoscopy, leukocyte scintigraphy, X-ray (small intestine) and CT (thorax and abdomen), but without definitive diagnosis.
  • CT of sacroiliac joints has shown bilateral sacroiliitis.
  • ANA, ANCA, RF and complements have been negative.
  • ferritin was significantly elevated (1572 ⁇ g/L) with corresponding findings of C-reactive protein (CRP) 1 17 mg/L, erythrocyte sedimentation rate (ESR) 80 mm/h, and white blood cell count (WBC) 27 xlO 9 /L (patient's maximum value 89 xlO 9 /L; Fig. 2).
  • CRP C-reactive protein
  • ESR erythrocyte sedimentation rate
  • WBC white blood cell count
  • Still's disease was considered, but excluded by other rheumatic diseases according to the Yamaguchi criteria ((Yamaguchi, Ohta et al. 1992)).
  • Yamaguchi criteria (Yamaguchi, Ohta et al. 1992)
  • azathioprine, sulfasalazine, methotrexate, cyclosporine A, colchicine, intravenous administration of immunoglobulin, moderate doses of cortisone, and NSAIDs was ineffective.
  • Infliximab produced side effects, while the effects of adalimumab and etanercept have been inadequate.
  • the prevalence rates of the mutations of NLRP3 and CARDS were assessed in a population of 806 randomly selected individuals. These point mutations were found to be common polymorphisms, present at allele frequencies of 6.5% for Q705K in NLRP3 and 34% for ClOX in the CARDS gene.
  • the half-life of anakinra is 4-6 hours.
  • the baseline caspase-1 activity of monocytes was therefore investigated 4, 24 and 34h after administration of the drug.
  • the patient demonstrated similar spontaneous caspase-1 activity and IL- l ⁇ plasma levels as the control persons (Fig. 4A-B).
  • the IL- l ⁇ level was elevated (Fig. 4B), and at 34 hours, high spontaneous caspase-1 activity in the patient's monocytes (Fig. 4A) and elevated levels of plasma IL- l ⁇ (Fig. 3B) were evident.
  • NF- ⁇ B NF- ⁇ B.
  • LPS induced a transient increase of NF- ⁇ B activity, which was 1.4 fold over the unstimulated cells at 30 min and decreased to basal levels at 6h (not shown). However, no difference between the controls and the patient could be observed.
  • This example concerns a patient with a long history of inflammatory disease resulting from excessive IL- l ⁇ production.
  • the patient was found to be a heterozygous carrier of two common polymorphisms in the NLRP3 and CARDS genes; both components of the NALP3 inflammasome. Administration of anakinra proved effective in treating this patient.
  • the Q705K mutation in NLRP3 found in the patient has previously been described as a low penetrance mutation found at an allele frequency of 3% in the a Spanish population(Hoffman, Gregory et al. 2003). However, in a randomly selected population of 806 individuals from the southeastern region of Sweden, the Q705K polymorphism was found to be more prevalent (allele frequency 6.5%). Sequencing of other components of the patient's NALP3 inflammasome revealed that ASC was wild type, while a codon change from cysteine to a premature stop codon was found in the CARDS gene.
  • the increased spontaneous caspase-1 activity and the elevated IL- l ⁇ levels observed in the patient after withdrawal of anakinra is likely to be the consequence of the polymorphisms in either NLRP3 or CARDS genes, or as a combined, synergistic effect of both mutations. Since the predicted fraction of individuals carrying this compound polymorphism is -4%, it may represent a significant susceptibility factor for several chronic inflammatory disorders. This is partly supported by our unpublished study (included herein as Example 1), where these two SNP: s are shown to represent a genetic susceptibility factor for rheumatoid arthritis with a more severe disease course.
  • the drug has been shown to affect IL-I production by decreasing the expression of pro-IL-l ⁇ and its target genes ((Goldbach-Mansky, Dailey et al. 2006)), whereas withdrawal of IL-I receptor antagonists in NOMID-patients increases the expression of these genes ((Goldbach-Mansky, Dailey et al. 2006)).
  • the levels of NF- ⁇ B activity in resting and stimulated cells from the patient were similar to the levels found in cells from control subjects, suggesting that this pro-inflammatory signaling pathway is not important for his disease.
  • NALP3 and related proteins have been suggested to be sensors of intracellularly encountered microbial motifs and "danger signals" ((Martinon, Burns et al. 2002)).
  • Polymorphisms in these genes in combination with an exogenous exposure, e.g. infection may elicit an inflammatory response that develops into a chronic condition.
  • an exogenous exposure e.g. infection
  • the patient Prior to the outbreak of the disease, the patient suffered from a streptococcal infection, which might have triggered his disease.
  • the patient is also HLA B27- positive.
  • HLA B27 is commonly found in patients with reactive arthritis and other subgroups of spondyloarthropathy where bouts of arthritis often are preceded by infections.
  • the interrelation between HLA B27, the gene polymorphisms of the inflammasome, and exogenous agents may be of importance in the pathogenesis of rheumatic diseases or other chronic inflammatory conditions.
  • Still's disease is also characterized by elevated levels of IL- l ⁇ , and treatment with anakinra has been efficient also in these patients ((Fitzgerald, Leclercq et al. 2005)). Still's disease has been considered in the present case, but if diagnostic criteria are used strictly, other rheumatic diseases exclude a diagnosis of Still's disease ((Yamaguchi, Ohta et al. 1992)). However, the combination of clinical, genetic, and experimental data should be emphasized, and we believe that these combined data may improve accuracy of disease definitions in the future.
  • Peripheral human blood was drawn from patient and control individuals (after obtaining ethical approval and informed consent) and collected in heparin- containing "vaccutainer” tubes.
  • Neutrophils and monocytes were isolated by sucrose gradient centrifugation means (Le. LymphoprepTM layered over
  • the mononuclear cells were plated in DMEM (supplemented with 25 mM Hepes, 100 U/ml of penicillin, and 100 ⁇ g/ml streptomycin; l-2h, 37°C, 5 % CO2), washed to eliminate non-adherent cells (e.g. lymphocytes) and cultured overnight in DMEM supplemented with 10% human serum.
  • DMEM supplied with 25 mM Hepes, 100 U/ml of penicillin, and 100 ⁇ g/ml streptomycin; l-2h, 37°C, 5 % CO2
  • IL- l ⁇ concentrations were measured in plasma, or in the supernatants of stimulated neutrophils and monocytes, using a LINCOplex kit [LINCO Research Inc.], as per the manufacturer's instructions. The samples were analyzed using the Luminex® 100TM system. Data was analyzed using the software program "StarStation” 2.0 [Applied Cytometry Systems, Sheffield, UK] and IL- l ⁇ concentrations were determined and compared with standards run in parallel.
  • DNA samples were obtained from a colon cancer patient cohort. Individual DNA samples were subsequently additionally studied for the presence of a deletion polymorphism in the promoter of the NF- ⁇ gene.
  • NF- ⁇ activation had previously been examined in patients found to be compound heterozygotes for the CIASl and CARD8 genes.
  • NF- ⁇ activity was analysed as follows:
  • Monocytes were differentiated on glass cover slips for 9-1 1 days, and stimulated with lipopolysaccharide (LPS) . After fixing, the cells were stained with an antibody directed towards the p65-subunit of NF ⁇ : ⁇ [Santa Cruz, sc-8008], which translocates into the nucleus upon activation, and an Alexa594- conjugated anti-mouse antibody [Molecular Probes].
  • the preparations were mounted in mounting medium [Dako], and analyzed with a Zeiss microscope equipped with a camera. The staining intensity of the nucleus vs. the cytoplasm was measured in at least 50 cells using Scion Image software, and NFKB activity was expressed as the ratio of nuclear vs. cytoplasmic staining.
  • EXAMPLE 5 Spontaneous apoptosis of neutrophils in patients who are compound heterozvgotes for CIASl and CARD8 polymorphisms 4 hours, 24 hours and 34 hours after treatment with Anakinra.
  • the degree of spontaneous apoptosis was determined in the patient's neutrophils after 6h of incubation and was found to be significantly reduced compared to controls (see Figure 5).
  • Neutrophils were isolated from compound CIASl and CARD8 heterozygous patients 4h, 24h and 34h after withdrawal of Anakinra (black), or from healthy, age and gender-match controls (grey) and were kept at 37°C in cell culture medium for 6 h, after which the degree of apoptosis (Le. spontaneous apoptosis) was determined.
  • the cells were stained with FITC-conjugated annexin V/propidium iodide.
  • the binding of FITC-annexin V (FLl) and propidium iodide (FL2) was measured in at least 10 000 cells/ sample by FACS using forward and side scatter to exclude cell debris.
  • the maesurments were analyzed using CellQuest software and nonspecific binding of annexin V to the cells was excluded.
  • CARD-8 protein a new CARD family member that regulates caspase-1 activation and apoptosis.
  • WBC white blood count
  • CRP C-reactive protein

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Abstract

Cette invention se rapporte à un procédé de traitement d'un patient souffrant d'une maladie associée aux polymorphismes des gènes CIAS1 et/ou CARD8, caractérisé en ce qu'il comprend les étapes consistant à obtenir des données sur les polymorphismes des gènes CIAS1 et/ou CARD8 dans le génome dudit patient ; à traiter ledit patient avec un traitement bloquant l'IL si ledit patient présente au moins un polymorphisme dans les gènes CIAS1 et/ou CARD8. L'invention concerne par ailleurs des inhibiteurs d'interleukine (IL) utilisables dans ces procédés, des procédés permettant de déterminer si un patient est sensible au traitement avec les inhibiteurs d'IL, ainsi que des kits utilisables dans ces procédés.
PCT/SE2008/050166 2007-02-12 2008-02-12 Polymorphismes des gènes de l'inflammasome cryopyrine et leurs utilisations dans le traitement des maladies inflammatoires Ceased WO2008100217A1 (fr)

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US20040038224A1 (en) * 2001-10-05 2004-02-26 Richard Kolodner Isolated cryopyrins, nucleic acid molecules encoding these, and use thereof
US20050267101A1 (en) * 2004-05-27 2005-12-01 Randle John C Treatment of diseases using ICE inhibitors

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040038224A1 (en) * 2001-10-05 2004-02-26 Richard Kolodner Isolated cryopyrins, nucleic acid molecules encoding these, and use thereof
US20050267101A1 (en) * 2004-05-27 2005-12-01 Randle John C Treatment of diseases using ICE inhibitors

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DAMIANO J.S. ET AL.: "CARD Proteins as Therapeutic Targets in Cancer", CURRENT DRUG TARGETS, vol. 5, no. 4, 2004, pages 367 - 374 *
FLEISCHMANN R.M. ET AL.: "Anakira, a Recombinant Human Interleukin-1 Receptor Antagonist (r-metHuIL-1ra), in Patients With Rheumatoid Arthritis", ARTHRITIS & RHEUMATISM, vol. 48, no. 4, April 2003 (2003-04-01), pages 927 - 934, XP008041551 *
KASTBOM A. ET AL.: "Genetic variation in proteins of the cryopyrin inflammasome influences susceptibility and severity of rheumatoid arthritis (The Swedish TIRA project)", RHEUMATOLOGY, vol. 47, 2008, pages 415 - 417 *
KASTBOM A.: "Autoantibodies and genetic variation in rheumatoid arthritis", LINKÖPING UNIVERSITY MEDICAL DISSERATIONS, FACULTY OF HEALTH SCIENCES, no. 990, 2007, pages 1 - 73 *
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