WO2006122723A1

WO2006122723A1 - Method for the prevention and/or treatment of autoimmune disease or allogeneic transplant rejections

Info

Publication number: WO2006122723A1
Application number: PCT/EP2006/004491
Authority: WO
Inventors: Alexander Flügel; Veronika Grau; Reinhard Zeidler
Original assignee: Vaecgene Biotech Gmbh; Max-Planck-Gesellschaft Zur Förderung Der Wissenschaften
Priority date: 2005-05-19
Filing date: 2006-05-12
Publication date: 2006-11-23

Abstract

The present invention relates to a method to use lycoricidinol (narciclasine) in order to suppress the immune system of mammals. Consequently, lycoricidinol can be used for the prevention and/or treatment of autoimmune disease as well as for the prevention and/or treatment of allogeneic transplant rejections and episodes of severe sepsis or septic shock. In another aspect thereof, the present invention relates to a method to avoid or treat multiple sclerosis (MS). In autoimmune encephalomyelitis (EAE), an experimental model related to multiple sclerosis (MS), the development of EAE is achieved by inhibiting the protein complex, S100A8/S100A9. Inhibition of the S100A8/S100A9 complex can be achieved either with a specific antibody or an inhibitor, like the alkaloid narciclasine.

Description

Method for the prevention and/or treatment of autoimmune disease or allogeneic transplant rejections

Field of Invention

For the purposes of the present invention, all references as cited herein below are incorporated by reference in their entireties.

Background of the invention

The S100A8/S100A9 complex - S100A8 (MRP8, Calgranulin A) and S100A9 (MRP 14, Calgranulin B) are members of the family of the SlOO protein family. S100A8/A9 are predominantly expressed in myeloid cells, which belong to the so-called innate immunity. Both proteins form heteromers and homomers but may also exert specific functions as monomers [I]. In granulocytes, they constitute up to 40% of the cytosolic proteins [I]. The myeloid expression of S100A9 and its association with a number of inflammatory diseases led to the assumption that this molecule together with S100A8 is involved in the body's defense against inflammation [2]. Indeed, the first cells, which infiltrate acute inflammatory lesions, often express S100A8/A9: phagocytes expressing S100A8 and S100A9 are readily detectable in rheumatoid arthritis, allograft rejection, and inflammatory bowel disease and lung diseases [3]. In addition, inflammatory disorders are often associated with elevated serum levels of S100A8/A9 [4]: follow-up studies in patients with rheumatoid arthritis revealed a good correlation of serum concentrations of S100A8/S100A9 with the clinical course of this disease [5], making the proteins a circulating surrogate marker for rheumatoid arthritis [6], juvenile rheumatoid oligoarthritis [7], systemic lupus erythematosus [8], polyneuropathies [9], Sjogren syndrome, polymyositis, dermatomyositis [8], multiple sclerosis [10], and allograft rejection [1 1].

In myeloid cells, expression of S100A9 is restricted to a distinct stage of differentiation. For instance, the protein is present in circulating monocytes but absent from differentiated tissue macrophages. In myeloid cells it is generally co-expressed with S100A8 but both proteins may be independently expressed in inflammatory conditions. Usually it is claimed that both proteins are absent from lymphocytes. However, their expression in germinal center B-cells has recently been described [12]. S100A9 can also be found in keratinocytes in psoriasis and microglia, where its expression was observed after cerebral infarction [13, 14].

In monocytes, S100A8 and S100A9 are predominantly localized in the cytoplasm. Upon activation of the cells, they are translocated to the cytoskeleton and, at a later time point, they also appear at the cell surface and are secreted [15]. It is the membrane-associated form of the proteins, which, up to now, remains enigmatic. In contrast, the secreted form, which appears at elevated levels in patients with cystic fibrosis, rheumatoid arthritis, and sarcoidosis, has growth-inhibitory and antimicrobial activities [16] like growth inhibition of Candida [17, 18]. In addition, S100A8 and S100A9 may be involved in leukocyte trafficking into inflammatory lesions. It has been shown that S 100A9-positive monocytes bind to extracellular matrix [19]. Newton und Haug demonstrated that S100A9 triggers neutrophil adhesion by stimulating the β2-integrin, Mac-1, whereas S100A8 may have regulatory properties [20]. In another study, S100A9 and S100A8/A9 complexes function as regulatory molecules in transendothelial migration of monocytes [21]. However, the receptors that bind S100A8 and S100A9 have not been biochemically characterized so far. In summary, there exist a number of hypotheses, the exact functions of both proteins remain unknown, but recent investigations indicate a prominent role in leukocyte trafficking [22], arachidonic acid metabolism [23], and activation of NADPH-oxidase [22].

S100A8/A9 in Allograft Rejection - Immunohistochemical analysis of biopsy specimens obtained from kidney and heart allografts after acute rejection revealed parallel expression of S100A8 and S100A9 in infiltrating monocytes [3, 24]. In addition, elevated serum levels preceded acute rejection episodes by a median of five days [1 1], making S100A8/S100A9 early markers for allograft rejection.

ATG Fresenius S is an immunosuppressive drug used in transplant patient in order to avoid or treat allogeneic graft rejection. ATG Fresenius S is produced by immunizing rabbits with the human T-leukemia cell line Jurkat [25]. Upon immunization, animals generate antibodies against antigens derived from Jurkat cells. ATG is thus a polyclonal immunoglobulin mixture, consisting of Jurkat-specific antibodies and of antibodies already present in the rabbits before immunization. The composition and the mode of action of ATG Fresenius S are only partially understood. The inventor has started to analyze ATG Fresenius S at the molecular level and identified (among others) S100A9-specific antibodies as a component. Hence, S100A9 can in principle contribute to the clinical activity of ATG.

The S100A8/A9-inhibiting drug Narciclasine - In 1995, Yui and co-workers purified and characterized the S100A8/S100A9-complex from exudate cells of the peritoneum of rats and demonstrated its apoptosis-inducing activity against the murine breast cancer cell line MM46 [26] and a panel of other human and murine cell lines [27] as well as normal human fibroblasts [28]. Since inflammatory processes like rheumatic arthritis are characterized by apoptosis and necrosis of synovial cells, the group of Yui hypothesized that the S100A8/S100A9 complex might causally be involved in this process [29]. Hence, they were investigating substances isolated from different plants, for their potential, to reduce or inhibit the pro-apoptotic activity of the S100A8/S100A9 complex and identified, among others, lycorine [29] and later on lydoricidinol, also termed narciclasine [30]. Structure of narciclasine, taken from Hudlicky et al. [31 ]

narciclasiriG (3)

Narciclasine is an isocarbostyril alkaloid with antimitotic activity present in narcissa bulbs like Narcissus spp., Haemanthus kalbreyeri und Lycoris lonituba [32]. The substance also inhibits the formation of peptide bonds in eukaryotic ribosomes. [33, 34], thus having, in principle, antitumor activity [34, 35]. Narciclasine also exhibits a wide range of inhibitory effects on plant growth. Narciclasine interacts with different hormones in various physiological concentrations, such as blocking the promotion of wheat coleoptile elongation by indole-3 -acetic acid, the enhancement of excised radish cotyledons by 6- benzylaminopurine, and α-amylase activity of wheat seeds induced by gibberellic acid [36]. The same biological activities may be exhibited by derivates of Narciclasine, which are derived by chemical modifications like they are described in the publication by G. Pettit [35], in WO 2004/052298 (Synthesis of Pancrastistatin, inventors: G. Pettit & M. Noeleen), or in WO 2004/052298 (Narcistatin Prodrugs, inventors G. Pettit & M. Noeleen).

Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis - Multiple sclerosis (MS) is the prototype inflammatory autoimmune disorder of the central nervous system and, with a lifetime risk of one in 400, potentially the most common cause of neurological disability in young adults. As with all complex traits, the disorder results from interplay between as yet unidentified environmental factors and susceptibility genes. Together, these factors trigger a cascade of events, involving engagement of the immune system, acute inflammatory injury of axons and glia, recovery of function and structural repair, post- inflammatory gliosis, and neurodegeneration. The sequential involvement of these processes underlies the clinical course characterized by episodes with recovery, episodes leaving persistent deficits, and secondary progression. Although the disease course is highly variable, 50% of patients will not be able to walk independently within 15 years of onset [37, 38]. The aim of treatment is to reduce the frequency, and limit the lasting effects, of relapses, relieve symptoms, prevent disability arising from disease progression, and promote tissue repair. Current treatments for MS include immunomodulatory and immunosuppressive drugs. Interferon beta and glatiramer acetate reduce the number of relapses, but if these therapies are not successful or the disease develops into a progressive phase there are no effective treatments for modification of the course of the disease [39]. Mitoxantrone slowed the clinical progression of secondary progressive MS in a randomized clinical trial, although its long-term clinical effect is unknown [40]. The limited effectiveness of these treatments justifies the assessment of alternative therapeutic strategies in patients with MS with aggressive clinical course.

Experimental autoimmune encephalomyelitis (EAE) has proven to be a valuable model for studying the immunological reactions involved in the pathogenesis of demyelinating diseases, in particular MS. Disease is induced in genetically susceptible mice or rats by immunization with myelin proteins or peptides or by the transfer of myelin-specific CD4+ T-lymphocytes, which leads to an infiltration of leukocytes into the CNS. EAE has been subjected to investigations of genetic susceptibility to disease development. By the identification of genes predisposing to EAE, the hope is to get clues as to what genetic elements are also important in MS. Upon induction of EAE, animals develop severe clinical symptoms like weight loss, paralysis, and fever. EAE is rarely lethal and the animals recover spontaneously after 10-15 days.

Today, allogeneic organ transplantation, including kidney transplantation, is a routine intervention in patients with severe organ complications or cancer. In order to avoid episodes of organ rejections, transplanted patients are heavily immunosuppressed with drugs like ATG or calcineurin inhibitors, such as cyclosporine or FK506, which mainly target T-lymphocytes. However, unspecific suppression of T-lymphocytes significantly increases the risk for the development of different kinds of cancer like skin cancer and Epstein-Barr-Virus-associated B-cell lymphoma in transplant patients.

Severe Sepsis - Severe sepsis is a systemic inflammatory response to bacterial invasive infection associated with coagulatopathy, multiple organ failure, and death. Despite significant advances in intensive care therapy, the overall mortality due to severe sepsis is about 30%. The principal active agent involved in the pathogenesis of sepsis is bacterial lipopolysaccharide (LPS) of the surface of gram-negative bacteria. LPS exerts its toxic effects by potently activating macrophages and endothelial cells, and inducing the expression of inflammatory cytokines such as tumor necrosis factor a (TNF α) and interleukin 6 (IL-6). In many cases, this inflammatory cascades is refractory to treatment and proceeds to septic shock associated with multi-organ failure and, eventually, death.

As mentioned above, the S100A8/S100A9 complex is only present in a subpopulation of monocytes/macrophages. Hence, inhibition of the complex by suitable means may constitute a much more specific immune suppression compared to conventional immunosuppressive drugs like cyclosporine, FK506, methotrexate, or steroids. This hypothesis is supported by data published by N. Hogg and colleagues: in an a S100A9 knock-out mouse model, they observed normal myeloid functions like apoptosis, phagocytosis, and oxidative burst [41]. Manitz et al. demonstrated in a similar animal model a slightly reduced sensitivity of myeloid cells for chemotactic stimuli, but a normal capacity of leukocytes to invade the peritoneum and the skin [42]. These data implicate that S100A9 is dispensable for many immunological functions.

It is thus an object of the present invention to present a method for the prevention or treatment of inflammatory conditions characterized by elevated S 100 A8 and/or S 100A9 serum levels and/or S100A8 and/or S100A9 expression in immune effector cells in the diseased tissues as they appear in autoimmune diseases like MS and acute graft rejections or graft versus host disease in transplant patients.

Summary of the invention

In one aspect thereof, the object of the present invention is solved by a method for the prevention or treatment of a disease in a patient in need thereof, wherein said disease can be prevented or treated by administering an effective amount of an inhibitor of the protein complex S 100A8/S 100A9 to said patient. Preferably, said inhibitor of the protein complex S100A8/S100A9 is selected from an S100A8/S100A9 specific antibody or an isocarbostyril alkaloid. More preferred, said S100A8/S100A9 specific antibody is selected from S100A8 and/or S 100A9-specific antibodies, in particular S32.2 and said isocarbostyril alkaloid is lycoricidinol (narciclasine).

Preferably said disease to be prevented or treated is selected from autoimmune diseases and inflammatory conditions, that are, more preferably, further characterized by elevated S 100 A8 and/or S100A9 serum levels and/or S100A8 and/or S100A9 expression in immune effector cells in the diseased tissues.

In another aspect thereof, the object of the present invention is solved by a method for the prevention or treatment of an autoimmune disease selected from demyelinating diseases, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, juvenile rheumatoid oligoarthritis, systemic lupus erythematosus, Sjogren syndrome, cystic fibrosis, polymyositis, dermatomyositis, psoriasis, and sarcoidosis wherein said disease is prevented or treated by administering an effective amount of an inhibitor of the protein complex S100A8/S100A9 to a patient in need of said prevention and/or treatment.

In another aspect thereof, the object of the present invention is solved by a method for the prevention or treatment of an inflammatory condition is selected from allograft rejection, acute graft rejections, graft versus host disease, sepsis, and polyneuropathies wherein said condition is prevented or treated by administering an effective amount of an inhibitor of the protein complex S100A8/S100A9 to a patient in need of said prevention and/or treatment.

Preferably, the effective amount for the prevention and/or treatment is a concentration in vivo of 1 ng/ml for narciclasine or 2 μg/ml for an S100A9-specific antibody.

The present invention is furthermore related to a pharmaceutical composition, comprising an effective amount of an inhibitor of the protein complex S100A8/S100A9, together with suitable additives or excipients. This pharmaceutical composition can be further characterized in that the inhibitor of the protein complex S100A8/S100A9 is present in form of a depot substance together with a suitable, pharmaceutically acceptable diluents or carrier substance. Respective formulations are known to the person of skill and can, amongst others, found in textbooks, such as Remington's Pharmaceutical Sciences, 19th ed., in particular part 8.

Brief Description of the Drawings

Figure 1 shows that an S100A9-specific antibody, S32.2, and narciclasine both reduce the adhesion of monocytes/macrophage to TNFa-activated HUVEC cells by approximately 50% (from 5.66% to 2.80% and 2.68%, respectively).

Figure 2 shows that an S100A9-specific antibody, S32.2, reduced phagocytosis by phagocytes of opsonized, FITC-labeled Escherichia coli. Only 3.21% of phagocytes present in a preparation of fresh PBMCs revealed phagocytosis in the presence of S32.2 (left) in contrast to 22.46% in the presence of an irrelevant control antibody, targeting the nuclear protein MYC (right).

Figure 3 shows that narciclasine and an S100A9-specific antibody, S32.2, inhibit the release of IFN -γ by allogeneic PBMCs

Figure 4 shows that narciclasine inhibits the release of TNF-α by allogeneic PBMCs.

Figure 5 shows that in Lewis rats, narciclasine inhibits the development of EAE after transfer of MBP-specific T-cells. Clinical score: 0, no disease; 1, flaccid tail; 2, gait disturbance; 3, complete hind limb paralysis; 4, tetraparesis; and 5, death.

Figure 6 shows that primary PBMCs were lysed and proteins were immunoprecipitated with ATG or pre-ATG, coupled to sepharose beads. Precipitated proteins were separated on a conventional polyacrylamide gel, blotted onto a membrane and hybridized to the S100A9- specific antibody S32.2. As can been seen, S100A9 precipitated with ATG but not with pre- ATG.

Figure 7 shows that in Lewis rats, established EAE after transfer of MBP-specific T-cells can be treated. Narciclasine treatment was started at day 3 after transfer of MBP-specific T-cells, when initial clinical symptoms became obvious. Clinical score: 0, no disease; 1, flaccid tail; 2, gait disturbance; 3, complete hind limb paralysis; 4, tetraparesis; and 5, death.

Figure 8 shows that in Lewis rats, Narciclasine prevents the migration of different classes of immune effector cells into the central nervous system (CNS) after transfer of MBP-specific T- cells.

Fig 9 shows that Narciclasine treatment at a concentration of 0.5mg/kg/day for days followed by 0.2 mg/kg/day for 5 days of allogeneic kidney-transplanted Lewis rats significantly prolongs survival. Treatment was initiated one day after transplantation.

The present invention now shall be described in more detail based on the following data and the accompanying figures without being limited thereto. The data have been obtained from several experiments that all support the hypothesis that the inhibition of the S100A8/A9 complex constitutes a suitable method for the prevention and/or therapy of the above mentioned diseases and conditions.

mRNA and protein sequence of human S100A8 and SlOO A9:

S100A8 (MRP-8): mRNA sequence (accession No.: NM 002964) (SEQ ID No. 1)

AUGUCUCUUGUCAGCUGUCUUUCAGAAGACCUGGUGGGGCAAGUCCGUGGGCA

UCAUGUUGACCGAGCUGGAGAAAGCCUUGAACUCUAUCAUCGACGUCUACCAC

AAGUACUCCCUGAUAAAGGGGAAUUUCCAUGCCGUCUACAGGGAUGACCUGAA

GAAAUUGCUAGAGACCGAGUGUCCUCAGUAUAUCAGGAAAAAGGGUGCAGAC

GUCUGGUUCAAAGAGUUGGAUAUCAACACUGAUGGUGCAGUUAACUUCCAGG

AGUUCCUCAUUCUGGUGAUAAAGAUGGGCGUGGCAGCCCACAAAAAAAGCCAU

GAAGAAAGCCACAAAGAGUAGCUGAGUUACUGGGCCCAGAGGCUGGGCCCCUG

GACAUGUACCUGCAGAAUAAUAAAGUCAUCAAUACCUCAAAAAAAAAAAAAA

AAAAAAA

Protein sequence (accession No.: NP 002955) (SEQ ID No. 2) MLTELEKALNSIIDVYHKYSLIKGNFHAVYRDDLKKLLETECPQYIRKKGADVWFKE LDINTDGAVNFQEFLILVIKMGVAAHKKSHEESHKE

SlOO A9 (MRP- 14): niRNA sequence (accession No.: NM_002965) (SEQ ID No. 3)

AAACACUCUGUGUGGCUCCUCGGCUUUGGGACAGAGUGCAAGACGAUGACUUG

CAAAAUGUCGCAGCUGGAACGCAACAUAGAGACCAUCAUCAACACCUUCCACC

AAUACUCUGUGAAGCUGGGGCACCCAGACACCCUGAACCAGGGGGAAUUCAAA

GAGCUGGUGCGAAAAGAUCUGCAAAAUUUUCUCAAGAAGGAGAAUAAGAAUG

AAAAGGUCAUAGAACACAUCAUGGAGGACCUGGACACAAAUGCAGACAAGCAG

CUGAGCUUCGAGGAGUUCAUCAUGCUGAUGGCGAGGCUAACCUGGGCCUCCCA

CGAGAAGAUGCACGAGGGUGACGAGGGCCCUGGCCACCACCAUAAGCCAGGCC

UCGGGGAGGGCACCCCCUAAGACCACAGUGGCCAAGAUCACAGUGGCCACGGC

CAUGGCCACAGUCAUGGUGGCCACGGCCACAGGCCACUAAUCAGGAGGCCAGG

CCACCCUGCCUCUACCCAACCAGGGCCCCGGGGCCUGUUAUGUCAAACUGUCU

UGGCUGUGGGGCUAGGGGCUGGGGCCAAAUAAAGUCUCUUCCUCCAA

Protein sequence (accession No.: NP_002956) (SEQ ID No. 4):

MTCKMSQLERNIETIINTFHQYSVKLGHPDTLNQGEFKELVRKDLQNFLKKENKNEK VIEHIMEDLDTNADKQLSFEEFIMLMARLTWASHEKMHEGDEGPGHHHKPGLGEGT P

Material and Methods

Adhesion assay - Primary human peripheral blood mononuclear cells (PBMCs) were obtained from healty donors by Ficoll centrifugation according to standard protocols and resuspended in Dulbecco's modified eagle medium (DMEM) containing 10% fetal calf serum. Cells were stained with the vital fluorescent dye 5-chloromethylfluoresceine diacetate (CMFDA; Molecular Probes Inc., Eugene, OR 97402-0469) according to the manufacturers protocol. In brief, cells were incubated with the dye for 20 min at 37°C and washed twice with phosphate- buffered saline (PBS). Confluent human umbilical vein endothelial cells (HUVEC) were pre- activated with 10ng/ml tumor necrosis factor-alpha (TNFa) for 3 hours in 6-well cluster plates. lxlOeό stained PBMCs were added to each well and cells were co-incubated for 30 min at 37°C in an atmosphere containing 5% CO₂. After cultivation, non-adherent cells were removed by extensive washing and adherent cells were counted by fluorescence-activated cell sorting (FACS) analysis using a FacsCalibur device (Becton Dickinson). Mononuclear cells were easily distinguishable from HUVEC cells by their green fluorescence after CMFDA staining. Narciclasine, (stock solution of lOmg/ml dissolved in DMSO, dilutions thereof prepared in PBS) at a final concentration of 1 ng/ml or an S100A9-specific antibody (Clone S32.2, BMA Biomedicals AG, Rheinstrasse 28-32, CH-4302 Augst) at a final concentration of 2 μg/ml were added to some of the cultures. For control purposes, similar amounts of DMSO or an irrelevant non-specific antibody was used.

Phagocytosis assay - Phagocytic activity by polymorphonuclear neutrophils and monocytes was investigated using the PHAGOTEST® assay according to the manufacturers protocol (Orpegen Pharma, D-69115 Heidelberg, Germany). In brief, heparinized peripheral blood was co-incubated with an opsonized FITC-labeled E.coli suspension for 10 min at 37°C. Thereafter, A quenching solution was added for suppressing fluorescence of the bacteria attached to the outside of cells and cells were analyzed for green fluorescence by FACS, using the blue-green excitation light (488nm argon-ion laser, FacsCalibur). DNA staining was performed to discriminate cells and E.coli.

Mixed lymphocyte reaction (MLR) - 2,5x10e5 PBMCs were co- incubated with 5.000 - 10.000 allogeneic target cells (Epstein-Barr- Virus-immortalized B cell lines or W138 human fibroblasts) for 1-3 days at 37°C in standard cell culture medium. Thereafter, supernatants were removed analyzed for the presence of cytokines (see below). ELISA assay - Elisa assays for interferon-gamma and TNFD were performed according to the manufacturers protocol (Mabtech AB, D-20251 Hamburg, Germany). In brief, a high protein binding ELISA plate was coated with the first antibody (final concentration 2μg/ml) in PBS over night at 4°C. Plates were washed twice and the plate was blocked with DMEM/10% FCS for 1 hours. Thereafter, plates were washed and samples were added for 2 hours. Plates were washed again and the second, biotin-labeled antibody was added (1 hour, final concentration lμg/ml). After additional washing, streptavidine-alkaline phosphatase was added for 1 hour and the plate was developed with p-nitrophenyl-phosphate and the optical density was measured at 405 run in an ELISA reader.

Experimental Autoimmune Encephalitis (EAE) - EAE was induced in Lewis rats (6-8 weeks of age) by adoptive transfer of retrovirally labeled green fluorescent protein- expressing myelin basic protein (MBP)-reactive T cells (T_MBP-_GFP cells, essentially as described in the publication by Flϋgel et al. (Nature Medicine, 5:843 [1999]). The dose of T cells injected was adjusted to 5 x 10e6— 1 x e7 T cell blasts/animal. Animals were monitored daily by measuring weight and examining disease scores. Starting from day 1 (T cell transfer) to day 11, three out of six animals received a daily intraperitoneal injection of narciclasine (resolved in DMSO, diluted in PBS) at a concentration of 2 mg/kg body weight. Control animals were treated with identical amounts of DMSO, only.

Analysis of immune effector cells in EAE Lewis rats

Single cell suspensions from organs were prepared as described previously (Flϋgel et al. Immunity 14, 574-560 [2001]). Cytofiuorometric analysis was performed with FACS-Calibur operated by Cell Quest software (Becton Dickinson). The following monoclonal antibodies were used for surface membrane analysis: W3/25 (anti-CD4), 0X33 (CD45RA, B cells), CD8α, CDl Ib (integrin αM chain), CDl Ic (integrin αX chain) (all Becton Dickinson, Heidelberg, Germany). Allophycocyanin-labeled anti-mouse antibody (Invitrogen, Karlsruhe, Germany) was used as secondary antibody. The numbers of T_MBP-_GFP cells and the recruited cells were determined using flow cytrometry as described (Kawakami et al. J. Immunol. 175, 69-81 [2005]). Allogeneic kidney transplantation Renal transplantation was performed in a fully allogeneic rat strain combination (Brown Norway to Lewis). After recipient nephrectomy on the left side, kidneys of heparinized donors were transplanted orthotopically. An end-to-end anastomosis of the renal arteries, veins and of the ureter was performed. Total ischemic times remained below 30 min. The contralateral kidney was removed at the end of surgery. Hence the survival of the graft recipient depends on the function of the transplanted kidney. The rats received one prophylactic dose of 30 μg ampicillin (Ratiopharm, UIm, Germany) i.p.. No immunosuppression was applied. Allograft recipients were treated s.c. with at a concentration of 0.2 mg Narciclasine/kg/day for 2 days followed by 0.5 mg Narciclasine/kg/day for 5 days.

EXAMPLE 1: Blocking of SlOO A9 reduced the capacity of human mononuclear cells to adhere to endothelial cells

Adhesion of immune effector cells to endothelial cells is a prerequisite for extravasation, migration into inflammatory lesions, and thus for proper immune function. Interaction of immune and endothelial cells involves a plethora of adhesion and secondary molecules.

Primary human peripheral blood mononuclear cells (PBMCs) were stained with the vital dye 5-chloromethylfluoresceine diacetate (Molecular Probes Inc., Eugene, OR 97402-0469) and cultivated for 30 min at 37°C on human umbilical vein endothelial cells (HUVEC) pre- activated with 10ng/ml tumor necrosis factor-alpha (TNFα) for 3 hours. After cultivation, non-adherent cells were removed by extensive washing and adherent cells were counted by fluorescence-activated cell sorting (FACS) analysis. Mononuclear cells were easily distinguishable from HUVEC cells by their green fluorescence after CMFDA staining. Addition of narciclasine (1 ng/ml) or an S100A9-specific antibody (2 μg/ml; Clone S32.2, BMA Biomedicals AG, Rheinstrasse 28-32, CH-4302 Augst) reduced the number of adherent by approx. 50%.

EXAMPLE 2: An Sl 00 A9 -specific Antibody inhibits Phagocytosis of Opsonized E.coli by Monocytes/Macrophages and Granulocytes For results and details, see Figure 2. EXAMPLE 3: Narciclasine inhibits secretion of IFN- γ and TNF- a by allogeneic immune effector cells

Activation of immune effector cells like monocytes/macrophages and T-cells upon recognition of allogeneic cells or tissues, infective agents like bacteria or viruses, or even self molecules as in autoimmune diseases is accompanied by the release of pro-inflammatory cytokines like IFN-γ and TNF-α, and the upregulation of surface molecules like CD25 and CD69 on T-lymphocytes. For instance, elevated IFN-γ and TNF-α levels are detectable in acute allogeneic organ rejection episodes.

Human embryonic fibroblasts (Wi38) were seeded at 5.000 cells/well into a 96-well plate. 2,5x10e5 peripheral blood mononuclear cells were added to each well and cells were co- incubated for 4 days in the presence of different concentrations of narciclasine or, as a control, without narciclasine. After cultivation, supernatants were tested for the presence of the pro-inflammatory cytokines interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), using standard ELISA assays (Mabtech). As demonstrated in figure 4, narciclasine inhibited IFN-g and TNF-a production in a dose-dependant manner, narci = narciclasine; S32.2 = S100A9-specific antibody;

EXAMPLE 4: Narciclasine prevents experimental autoimmune encephalomyelitis in a rat model

In Lewis rats, the adoptive transfer of activated myelin basic protein (MBP)- specific T cells triggers a severe, potentially lethal neurological disease associated with the invasion of the central nervous system (CNS) by T cells and large numbers of activated macrophages. In this model system, clinical and histological changes do not develop in the CNS immediately upon cell transfer but only after a latency period of at least 3 days. We transferred encephalitogenic T cells, known to transfer strong monophasic clinical EAE, into Lewis rats by intraperitoneal injection. The dose of T cells injected was adjusted to 5 x 10e6-le07 T cell blasts/animal. Animals were monitored daily by measuring weight and examining disease scores. Starting from day 1 (T cell transfer) to day 1 1 , three out of six animals received a daily intraperitoneal injection of narciclasine (resolved in DMSO) at a concentration of 2 mg/kg body weight. Control animals were treated with identical amounts of DMSO, only. As demonstrated in Fig 5, control animals developed severe EAE as expected (green bars), showing all clinical symptoms. In contrast, narciclasine-treated animals (brown bars) developed only very weak symptoms (2 out of 3 animals) or developed no clinical symptoms at all (1 out of 3 animals). Also, narciclasine-treated animals only lost approximately 13g of weight (red line), whereas control animals lost up to 4Og (blue line). Of interest, animals of the narciclasine group stayed healthy even after cessation of treatment and did not develop relapse (not shown).

EXAMPLE 5: S100A9-specific antibodies are a component of the immunosuppressive drug, ATG Fresenius-S.

ATG-Fresenius S (ATG) is a medicinal product that is used in the clinic in order to suppress acute graft rejection after solid organ transplantation. ATG is produced and sold by Fresenius AG. ATG is generated by immunizing rabbits with the human cell line, Jurkat, derived from a T cell leukemia [25]. After immunization, rabbit sera are pooled and the immunoglobulins (Igs) present are purified. Thus, ATG is of a polyclonal composition, containing both Igs induced by the immunization process but also Igs, which have already been present in the animals. In addition, the specificities of the Igs present in ATG are not known.

Investigating the specificity of the Igs using a proprietary technology, AMiDA [43], we detected antibodies against S100A9 in ATG, which were not present in the animals prior to immunization (pre-ATG, Figure 6). In brief, IgGs present in ATG and pre-ATG were coupled to sepharose. Primary PBMCs were lysed in proteins were immunoprecipitated with ATG and pre-ATG, respectively. Precipitated proteins were separated on two-dimensional polyacrylamide gels and visualized with silver. Proteins that exclusively precipitated with ATG were excised from the gel, digested with trypsine, and analyzed by mass spectrometry using a MALDI-ToF device. Among others, we identified S100A9 as a protein that precipitated with ATG but not with pre-ATG. Hence, Jurkat cells induce the generation of S 100A9-specific antibodies in rabbits.

EXAMPLE 6: Renal transplantation of fully allogeneic kidneys in the Brown Norway to LEW rat strain combination results in acute graft rejection and recipient death within 8 to 10 days. Treatment with low 0.5 mg/kg Narciclasine for 2 days followed by 0.2 mg/kg for 5 days resulted in a significant delay of allograft rejection. For details of the survival times see Fig. 9. Listing of Literature as cited

1. Nacken, W., Roth, J., Sorg, C, and Kerkhoff, C. (2003). S100A9/S100A8: Myeloid representatives of the SlOO protein family as prominent players in innate immunity. Microsc Res Tech 60, 569-580.

2. Bhardwaj, R.S., Zotz, C, Zwadlo-Klarwasser, G., Roth, J., Goebeler, M., Mahnke, K., FaIk, M., Meinardus-Hager, G., and Sorg, C. (1992). The calcium-binding proteins MRP8 and MRP 14 form a membrane-associated heterodimer in a subset of monocytes/macrophages present in acute but absent in chronic inflammatory lesions. Eur J Immunol 22, 1891-1897.

3. Goebeler, M., Roth, J., Burwinkel, F., Vollmer, E., Bocker, W., and Sorg, C. (1994). Expression and complex formation of SlOO-like proteins MRP8 and MRP 14 by macrophages during renal allograft rejection. Transplantation 58, 355-361.

4. Brun, J.G., Jonsson, R., and Haga, HJ. (1994). Measurement of plasma calprotectin as an indicator of arthritis and disease activity in patients with inflammatory rheumatic diseases. J Rheumatol 21, 733-738.

5. Berntzen, H.B., Munthe, E., and Fagerhol, M.K. (1989). A longitudinal study of the leukocyte protein Ll as an indicator of disease activity in patients with rheumatoid arthritis. J Rheumatol 16, 1416-1420.

6. Berntzen, H.B., Fagerhol, M.K., Ostensen, M., Mowinckel, P., and Hoyeraal, H.M. (1991). The Ll protein as a new indicator of inflammatory activity in patients with juvenile rheumatoid arthritis. J Rheumatol 18, 133-138.

7. Frosch, M., Strey, A., Vogl, T., Wulffraat, N.M., Kuis, W., Sunderkotter, C, Harms, E., Sorg, C, and Roth, J. (2000). Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis. Arthritis Rheum 43, 628- 637.

8. Kuruto, R., Nozawa, R., Takeishi, K., Arai, K., Yokota, T., and Takasaki, Y. (1990). Myeloid calcium binding proteins: expression in the differentiated HL-60 cells and detection in sera of patients with connective tissue diseases. J Biochem (Tokyo) 108, 650-653.

9. Kiefer, R., Kieseier, B.C., Bruck, W., Hartung, H.P., and Toyka, K.V. (1998). Macrophage differentiation antigens in acute and chronic autoimmune polyneuropathies. Brain 121 (Pt 3), 469-479.

10. Bogumil, T., Rieckmann, P., Kubuschok, B., Felgenhauer, K., and Bruck, W. (1998). Serum levels of macrophage-derived protein MRP-8/14 are elevated in active multiple sclerosis. Neurosci Lett 247, 195-197.

1 1. Burkhardt, K., Radespiel-Troger, M., Rupprecht, H.D., Goppelt-Struebe, M., Riess, R., Renders, L., Hauser, I. A., and Kunzendorf, U. (2001). An increase in myeloid-related protein serum levels precedes acute renal allograft rejection. J Am Soc Nephrol 12, 1947-1957.

12. Husson, H., Carideo, E.G., Neuberg, D., Schultze, J., Munoz, O., Marks, P. W., Donovan,

J. W., Chillemi, A.C., O'Connell, P., and Freedman, A.S. (2002). Gene expression profiling of follicular lymphoma and normal germinal center B cells using cDNA arrays. Blood 99, 282- 289.

13. Akiyama, H., Ikeda, K., Katoh, M., McGeer, E.G., and McGeer, P.L. (1994). Expression of MRP14, 27E10, interferon-alpha and leukocyte common antigen by reactive microglia in postmortem human brain tissue. J Neuroimmunol 50, 195-201.

14. Postler, E., Lehr, A., Schluesener, H., and Meyermann, R. (1997). Expression of the S-100 proteins MRP-8 and -14 in ischemic brain lesions. GHa 19, 27-34.

15. Roth, J., Burwinkel, F., van den Bos, C, Goebeler, M., Vollmer, E., and Sorg, C. (1993). MRP8 and MRP 14, S-100-like proteins associated with myeloid differentiation, are translocated to plasma membrane and intermediate filaments in a calcium-dependent manner. Blood 82, 1875-1883. 16. Hilt, D.C., and Kligman, D. (1991). The S-IOO protein family: a biochemical and functional overview. In Novel calcium-binding proteins. Fundamentals and clinical implications., CW. Heizman, ed. (Heidelberg: Springer Verlag), pp. 65-103.

17. Okutomi, T., Tanaka, T., Yui, S., Mikami, M., Yamazaki, M., Abe, S., and Yamaguchi, H. (1998). Anti-Candida activity of calprotectin in combination with neutrophils or lactoferrin. Microbiol Immunol 42, 789-793.

18. Sohnle, P.G., Hunter, M.J., Hahn, B., and Chazin, WJ. (2000). Zinc-reversible antimicrobial activity of recombinant calprotectin (migration inhibitory factor-related proteins 8 and 14). J Infect Dis 182, 1272-1275.

19. Mahnke, K., Bhardwaj, R., and Sorg, C. (1995). Heterodimers of the calcium-binding proteins MRP8 and MRP 14 are expressed on the surface of human monocytes upon adherence to fibronectin and collagen. Relation to TNF-alpha, IL-6, and superoxide production. J Leukoc Biol 57, 63-71.

20. Newton, R. A., and Hogg, N. (1998). The human SlOO protein MRP- 14 is a novel activator of the beta 2 integrin Mac-1 on neutrophils. J Immunol 160, 1427-1435.

21. Eue, I., Konig, S., Pior, J., and Sorg, C. (2002). S100A8, S100A9 and the S100A8/A9 heterodimer complex specifically bind to human endothelial cells: identification and characterization of ligands for the myeloid-related proteins S100A9 and S100A8/A9 on human dermal microvascular endothelial cell line-1 cells. Int Immunol 14, 287-297.

22. Kerkhoff, C, Nacken, W., Benedyk, M., Dagher, M.C., Sopalla, C, and Doussiere, J. (2005). The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2. Faseb Journal express article 10.1096/fj.04-2377 jje. Published online January 10, 2005.

23. Kerkhoff, C, Klempt, M., Kaever, V., and Sorg, C. (1999). The two calcium-binding proteins, S100A8 and S100A9, are involved in the metabolism of arachidonic acid in human neutrophils. J Biol Chem 274, 32672-32679.

24. Mues, B., Brisse, B., Zwadlo, G., Themann, H., Bender, F., and Sorg, C. (1990). Phenotyping of macrophages with monoclonal antibodies in endomyocardial biopsies as a new approach to diagnosis of myocarditis. Eur Heart J 11, 619-627.

25. Schneider, U., Schwenk, H.U., and Bornkamm, G. (1977). Characterization of EBV-genome negative "null" and "T" cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma. Int J Cancer 19, 621-626.

26. Yui, S., Mikami, M., and Yamazaki, M. (1995). Purification and characterization of the cytotoxic factor in rat peritoneal exudate cells: its identification as the calcium binding protein complex, calprotectin. J Leukoc Biol 58, 307-316.

27. Yui, S., Mikami, M., and Yamazaki, M. (1995). Induction of apoptotic cell death in mouse lymphoma and human leukemia cell lines by a calcium-binding protein complex, calprotectin, derived from inflammatory peritoneal exudate cells. J Leukoc Biol 58, 650-658.

28. Yui, S., Mikami, M., Tsurumaki, K., and Yamazaki, M. (1997). Growth-inhibitory and apoptosis-inducing activities of calprotectin derived from inflammatory exudate cells on normal fibroblasts: regulation by metal ions. J Leukoc Biol 61, 50-57.

29. Yui, S., Mikami, M., Kitahara, M., and Yamazaki, M. (1998). The inhibitory effect of lycorine on tumor cell apoptosis induced by polymorphonuclear leukocyte-derived calprotectin. Immunopharmacology 40, 151-162.

30. Mikami, M., Kitahara, M., Kitano, M., Ariki, Y., Mimaki, Y., Sashida, Y., Yamazaki, M., and Yui, S. (1999). Suppressive activity of lycoricidinol (narciclasine) against cytotoxicity of neutrophil-derived calprotectin, and its suppressive effect on rat adjuvant arthritis model. Biol Pharm Bull 22, 674-678.

31. Hudlicky, T., Rinner, U., Gonzalez, D., Akgun, H., Schilling, S., Siengalewicz, P., Martinot, T.A., and Pettit, G.R. (2002). Total synthesis and biological evaluation of Amaryllidaceae alkaloids: narciclasine, ent-7-deoxypancratistatin, regioisomer of 7-deoxypancratistatin, 1 Ob- epi-deoxypancratistatin, and truncated derivatives. J Org Chem 67, 8726-8743. 32. Ceriotti, G. (1967). Narciclasine: an antimitotic substance from Narcissus bulbs. Nature 213, 595-596.

33. Jimenez, A., Santos, A., Alonso, G., and Vazquez, D. (1976). Inhibitors of protein synthesis in eukarytic cells. Comparative effects of some amaryllidaceae alkaloids. Biochim Biophys Acta 425, 342-348.

34. Carrasco, L., Fresno, M., and Vazquez, D. (1975). Narciclasine: an antitumour alkaloid which blocks peptide bond formation by eukaryotic ribosomes. FEBS Lett 52, 236-239.

35. Pettit, G.R., Melody, N., Simpson, M., Thompson, M., Herald, D.L., and Knight, J.C. (2003). Antineoplastic agents 500. Narcistatin. J Nat Prod 66, 92-96.

36. Bi, Y.R., Yung, K.H., and Wong, Y.S. (1998). Physiological effects of narciclasine from the mucilage of Narcissus tazetta. Plant Sciences 135, 103-108.

37. Compston, A., and Coles, A. (2002). Multiple sclerosis. Lancet 359, 1221-1231.

38. Noseworthy, J.H., Lucinetti, C₅ Rodriguez, M., and Weinshenker, B.G. (2000). Multiple sclerosis. N Engl J Med 343, 938-952.

39. O'Connor, P. (2002). Key issues in the diagnosis and treatment of multiple sclerosis: an overview. Neurology 59, S 1-33.

40. Hartung, H. P., Gonsette, R., and Konig, N. (2002). Mitoxantrone in progressive multiple sclerosis: a placebo-controlled, double-blind, randomised multicentre trial. Lancet 360, 2018- 2025.

41. Hobbs, J.A., May, R., Tanousis, K., McNeill, E., Mathies, M., Gebhardt, C, Henderson, R., Robinson, M.J., and Hogg, N. (2003). Myeloid cell function in MRP-14 (S100A9) null mice. MoI Cell Biol 23, 2564-2576.

42. Manitz, M.P., Horst, B., Seeliger, S., Strey, A., Skryabin, B.V., Gunzer, M., Frings, W., Schonlau, F., Roth, J., Sorg, C, and Nacken, W. (2003). Loss of SlOO A9 (MRP 14) results in reduced interleukin-8-induced CDl Ib surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants in vitro. MoI Cell Biol 23, 1034-1043.

43. Gires, O., Munz, M., Schaffrik, M., Kieu, C₅ Rauch, J., Ahlemann, M., Eberle, D., Mack, B., Wollenberg, B., Lang, S., Hofmann, T., Hammerschmidt, W., and Zeidler, R. (2004). Profile identification of disease-associated humoral antigens using AMIDA, a novel proteomics- based technology. Cell MoI Life Sci 61, 1 198-1207.

Claims

1. Use of an effective amount of an inhibitor of the protein complex S 100A8/S 100A9 for the manufacture of a pharmaceutical composition for the prevention or treatment of a disease in a patient in need thereof.

2. Use according to claim 1, wherein said inhibitor of the protein complex S 100A8/S 100A9 is selected from an S100A8/S100A9 specific antibody or an isocarbostyril alkaloid.

3. Use according to claim 2, wherein said S100A8/S100A9 specific antibody is selected from S100A8 and/or S 100A9-specific antibodies, in particular S32.2.

4. Use according to claim 2, wherein said isocarbostyril alkaloid is lycoricidinol (narciclasine).

5. Use according to any of claims 1 to 4, wherein said disease is selected from autoimmune diseases and inflammatory conditions.

6. Use according to any of claims 1 to 5, wherein said disease is further characterized by elevated S100A8 and/or S100A9 serum levels and/or S100A8 and/or S100A9 expression in immune effector cells in the diseased tissues.

7. Use according to any of claims 1 to 6, wherein said autoimmune disease is selected from demyelinating diseases, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, juvenile rheumatoid oligoarthritis, systemic lupus erythematosus, Sjogren syndrome, cystic fibrosis, polymyositis, dermatomyositis, psoriasis, and sarcoidosis.

8. Use according to any of claims 1 to 6, wherein said inflammatory condition is selected from allograft rejection, acute graft rejections, graft versus host disease, sepsis, and polyneuropathies.

9. Use according to any of claims 1 to 8, wherein said effective amount for the prevention and/or treatment is a concentration in vivo of 1 ng/ml for narciclasine or 2 μg/ml for an S100A9-specific antibody.

10. Method for the prevention or treatment of a disease in a patient in need thereof, wherein said disease can be prevented or treated by administering an effective amount of an inhibitor of the protein complex S100A8/S100A9 to said patient.

11. Method according to claim 10, wherein said inhibitor of the protein complex S100A8/S100A9 is selected from an S100A8/S100A9 specific antibody or an isocarbostyril alkaloid.

12. Method according to claim 11, wherein said S100A8/S100A9 specific antibody is selected from S100A8 and/or S100A9-specific antibodies, in particular S32.2.

13. Method according to claim 11, wherein said isocarbostyril alkaloid is lycoricidinol (narciclasine).

14. Method according to any of claims 10 to 13, wherein said disease is selected from autoimmune diseases and inflammatory conditions

15. Method according to any of claims 10 to 14, wherein said disease is further characterized by elevated S100A8 and/or S100A9 serum levels and/or S100A8 and/or S100A9 expression in immune effector cells in the diseased tissues.

16. Method according to claim 14 or 15, wherein said autoimmune disease is selected from demyelinating diseases, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, juvenile rheumatoid oligoarthritis, systemic lupus erythematosus, Sjogren syndrome, cystic fibrosis, polymyositis, dermatomyositis, psoriasis, and sarcoidosis.

17. Method according to claim 14 or 15, wherein said inflammatory condition is selected from allograft rejection, acute graft rejections, graft versus host disease, sepsis, and polyneuropathies.

18. Method according to any of claims 10 to 17, wherein said effective amount for the prevention and/or treatment is a concentration in vivo of 1 ng/ml for narciclasine or 2 μg/ml for an S 100 A9-specific antibody.