DE102007004909A1 - Use of pancreatic zymogen granule polypeptide GP2 as a medicament, especially for treating autoimmune diseases by plasmapheresis - Google Patents

Abstract

Pancreatic zymogen granule polypeptide GP2 is used as a medicament : Pancreatic zymogen granule polypeptide GP2 having SEQ ID NO:1 (a defined sequence of 527 amino acids given in the specification) is used as a medicament. Independent claims are also included for: (1) treating inflammatory bowel diseases by preparing a column to which specific ligands for human immunoglobulins are coupled, passing plasma from a patient through the column to remove immunoglobulins, and returning the plasma to the patient; (2) kit for diagnosis of autoimmune diseases, comprising GP2 and optionally instructions for combining the contents of the kit and/or preparing a formulation; (3) chromatography device comprising GP2; (4) treating an autoimmune disease by binding and/or removing autoantibodies using GP2 immobilized on a solid phase. ACTIVITY : Immunosuppressive; Antiinflammatory; Hepatotropic. No biological data given. MECHANISM OF ACTION : Plasmapheresis.

Description

The The invention relates to a method for the detection of antibodies from body fluids through an immune reaction with GP2 from zymogenic granules of the pancreas, whose immunoreactive Sequences or analogues excluding tissue sections.

The Method can be used for diagnosis or therapy control of diseases serve, which are associated with an immune response to these substances. The invention is therefore in particular the use of GP2, its immunoreactive sequences or analogues for diagnosis or Therapy control of chronic inflammatory or autoimmune diseases, in particular Crohn's disease (MC) and chronic pancreatitis (CP).

The The present invention is based on the recognition that GP2 is an autoantigen of immune processes favored by MC and CP.

MC and ulcerative colitis (CU) represent the two main inflammatory Intestinal diseases (EDE). They are caused by chronic, recurrent, Tissue destructive inflammatory processes in the digestive system characterized. The etiology and pathogenesis of MC like also CU are so far unclear.

While in the CU the inflammation especially in the mucosa and submucosa of the colon and rectum occurs, MC's are wall-penetrating, granulomatous inflammatory processes of the whole Gastrointestinal tract characteristic.

genetic as well as environmental factors seem to play a crucial role in the Training EDE. The connection between mutations in the NOD2 gene and occurrence of MC is in several cohorts as assured to watch. A clear association also exists of the MC in the terminal ileum. A reference between genetic markers and therapy could so far for no treatment (including the anti-TNF therapy) are established.

The Incidence of MC in Europe is 5.6 per 100,000 per year. The Prevalence of MC in Germany is 1/500 to 1/800 specified.

First Disease symptoms of MC occur on average relatively early at 30 years old. MC patients are thus in their professional life affected, causing corresponding socioeconomic effects after pulls. Similar to the CU is in MC patients with Crohn's Colitis and long-term course, the carcinoma incidence elevated.

The Symptoms include abdominal pain, diarrhea, malabsorption, Abscesses, fistulas, gallstone complications, kidney stones and their Complications.

MC Patients may have a number of extraintestinal manifestations with 3.5% pancreatitis being relatively rare in MC Patient occurs. Hyperamylasemia and hyperlipidemia without signs of acute pancreatitis, however, in 8-17% Patients are observed, indicating a higher rate indicative of silenter pancreatitis. Isolated are changes in the pancreatic duct and limitations of pancreatic function been described. The amount of hyperamylasemia and hyperlipidemia correlates with activity from MC. In 4.6% of MC patients, changes are found simultaneously in bile and pancreatic duct similar to patients with Primary sclerosing cholangitis. Chronic pancreatitis at However, MC patients generally differ from that in CU, which more often involve bile duct involvement, Weight loss and pancreatic ductal stenosis. It becomes the existence idiopathic chronic pancreatitis associated with MC is discussed. In contrast to CU associated chronic Pancreatitis is more common in MC patients with intestinal symptoms before the appearance of pancreatic findings. The frequent exocrine pancreatic insufficiency in MC is easy attributed to pronounced acinar degeneration, which is associated with dense inflammatory infiltrates in the parenchyma.

When Therapy is recommended the administration of 5-aminosalicylic acid, although in different studies only limited and occasionally no effects were achieved. The application, however, appears in patients with mild to moderately severe Thrust justified on the basis of existing data, though to initiate a change of therapy in case of inefficiency In severe episodes without complications is the administration of prednisolone equivalents to consider. There are frequent relapses (≥ 2 / year) may additionally azathioprine or Be given 6-mercaptopurine.

The Total costs of a patient with MC in Germany to 20,000 EUR per year and case estimated. The expenses for MC patients including indirect costs amount in Germany to an estimated 2 billion EUR, in The US will receive $ 2.6 billion for both EDE socio-economic costs indicated.

Anti-TNFalpha Preparations are effective in MC and induce remission the chronic disease. They are however due to the potential Side effects as reserve medications depending on the clinical situation recommended. MC patients with active spondylathropathy as an extraintestinal complication seem to be of an anti-TNFalpha Therapy to benefit in terms of both complaints.

For adequate therapy and follow-up of these patients a clear diagnosis is necessary. Clinical diagnostics of the MC contains ileocolonoscopy as an essential component with segmental biopsies, which also before selective bowel surgery is indexed. However, it is not regular in the course for every acute symptom or before a new anti-inflammatory Therapy required. An upper endoscopic diagnosis should be done in primary care in each patient.

in the Diagnostics include histological examinations of mucosal biopsies an important building block. That's what biopsies are all about from grossly conspicuous and inconspicuous Taken from areas. To the possibilities of histopathological Being able to use differential diagnosis efficiently will be biopsies from at least five different anatomical segments of the entire colon including the rectum, from the terminal Ileum and from the upper gastrointestinal tract recommended.

Of the transabdominal ultrasound as an imaging method serves as Sensitive method for the detection of inflammatory bowel wall changes and the detection of abscesses, fistulas and stenoses in MC patients. A detectable increased blood flow in both the mesenteric arteries as well as in the intestinal wall is with the presence of acute inflammation associated. Endorectal ultrasound and magnetic resonance imaging (MRI) of the lesser pelvis are considered equally sensitive procedures for the diagnosis and classification of anorectal fistulas and abscesses accepted.

In Laboratory diagnostics of MC represent the determination of C-reactive Protein (CRP), platelets, hemoglobin (Hb) / hematocrit and leukocytes are the basic diagnostic. Other parameters such as The differential blood picture and the albumin can make a meaningful Be supplement. In the acute phase of the MC are the above Parameters such as CRP and leukocyte count as well as acute phase proteins increased in many patients and will also be for the follow-up is recommended.

In In the acute phase there is an increase in intestinal permeability, alphal-antitrypsin clearance and excretion of calprotectin in the chair.

The However, collection of clinical and histological data does not allow the clear distinction between MC and CU and leads frequently for the definition of a colitis indeterminata (CI). Furthermore you can Intestinal infections as well as functional diseases similar Develop symptoms and complicate the differential diagnosis. at 10 to 15% of patients with EDE are classified by CD or MC biopsy data and a certain overlap of clinical Symptoms in the area of the colon difficult. Patients with CI seem more frequent complications and anastomotic leak after surgical procedures as patients with CU. The distinction whether CI patients are prognostic in direction of a MC or a CU, however, has a significant Influence on prognosis and course of disease as well as the choice of the medicinal Therapy and timing of surgical procedures. In the course of the disease can often be later based on make an assignment to further clinical data. The distinction by MC and CU is for example the basis for the decision whether in the patient an ilioanal pouchanastomosis into consideration can be pulled. For MC patients with predominant Infestation of the colon (Crohn's Colitis) is this surgical procedure only displayed in very rare cases while CU, this method is more often indicated. MC patients have a significantly higher rate of anastomotic leakage so that every surgical intervention is thoroughly considered must become.

in the The differential diagnosis of EDE is a series of antibodies which has been described with endogenous and food antigens react. These antibodies do not appear pathogenetic To play a role and not to map the disease activity. However, the serological antibody diagnostics a provide crucial help with the diagnosis and above all in the case of indeterminate colitis essential for the treatment decision be.

Autoantibodies against cytoskeletal proteins have been described in biopsy-confirmed MC patients ( Mayet et al., 1990 ). Among other autoantibodies against cytokeratin 18, actin, vimentin, desmin and tropomyosin were found. Although cytokeratin 18 autoantibodies correlated with disease activity, they probably did not become established in routine diagnostics of EDE because of its low specificity.

For MC patients, autoantibodies to exocrine pancreatic tissue (PAH) and antibodies to mannan from Saccharomyces cerevisiae (ASCA) have been identified as pathognomonic ( Stoecker et al., 1987; Main et al., 1988 ). Autoantibodies to human neutrophil granulocytes (ANCA) and goblet cells (BAC) are found preferentially in Cu patients.

The Determination of PAH, ANCA and ASCA is for diagnosis considered helpful in CI.

EDE specific autoantibodies against pancreatic tissue, goblet cells and human neutrophil granulocytes are still present today Help of immunofluorescence technique (IFT) due to the unknown, intended for the immune reaction responsible autoantigens. Thus, with this technique, 27-39% of MC patients were Autoantibodies found against pancreatic antigens. about half of MC patients (68%) with extraintestinal Complications may include PAH.

For the MC also specific ASCA are reinforced detected in enzyme immunoassay (EIA), as this method is less subjective in the evaluation and can be automated.

today are the individual antibody determinations for the serological diagnosis of MC is considered too insensitive. The combination of different antibody specificities can the diagnostic sensitivity or specificity however, for the differential diagnosis of EDE extraordinarily improve and allow for CI a forecast.

For the combination of parameters is a common technology platform as the EIA's extremely beneficial. That sets however, advance knowledge of the auto-antigen of the PAH which of the PAHs in the pancreatic tissue sections of different species is specifically recognized.

There have been various approaches in the past to the identification of autoantigens in MC, with pancreatic antigens being the focus of interest due to the already stated relatively high sensitivity of PAHs. PAHs were detected by tissue sections of different species (human, rat, monkey) in the IFT. This suggests phylogenesis of conserved epitopes recognized by PAHs. Fricke et al. described a protein complex consisting of several PAK-reacting subunits with a molecular weight (MW) greater than 800 kDa ( Fricke et al., 1999 ). The authors, however, were unable to sequence and thus identify either the corresponding protein or immunoblot PAK-reactive subunits of MW 16, 18, 19, 24, 27, 29, 31, and 34 kDa. It has been suggested that the protein recognized by PAK appears to be a large protein complex with multiple subgroups. Due to inhibition experiments with different glycoproteins, reactivity of the PAHs with carbohydrate chains of the putative autoantigens was excluded.

Seibold et al. described the reactivity of PAK against a pancreatic juice purified macromolecular antigen with a MW greater than 10 ⁶ Da (1000 kDa), which lost its PAK reactivity after trypsin treatment ( Seibold et al., 1991 ). In the ELISA with various pancreatic proteins such as amylase, lipase, phospholipase A and C, enterokinase, carboxypeptidase A and B, chymotrypsin A and B, chymotrypsinogen, elastase, trypsin, trypsin inhibitor, lactoferrin and kallekrein, no reactivity with PAH could be detected.

The necessity of pending identification of the autoantigen (s) of the pancreas was expressly pointed out in order to elucidate the importance of autoimmune processes in the pathogenesis of MC and to support the discrimination of unclear EDE cases by appropriate laboratory diagnostics ( Fricke et al., 1999, Seibold et al., 1991 ). However, until now it has not been possible to identify the corresponding pancreatic antigen (s) ( Bossuyt, 2006 ).

This Problem becomes by the characterization of GP2 as autoantigen for MC as well as the associated chronic pancreatitis and the use of GP2 in the diagnosis of EDE according to the Claims solved, wherein advantageous embodiments the invention of the subclaims.

The Invention accordingly relates to a molecule according to the sequence SEQ ID NO: 1 for the Use as a medicine.

zur Prophylaxe, Diagnose, Therapie oder Nachbehandlung von Autoimmunerkrankungen.Another aspect of the invention is the use of the molecule GP2 according to SEQ ID No. 1

for the prophylaxis, diagnosis, therapy or aftercare of autoimmune diseases.

The Invention accordingly relates to the surprising Teaching that the sequence according to the invention or the they used encoding nucleic acid in therapy in particular, to treat autoimmune diseases inflammatory bowel disease, most preferably Crohn's disease, chronic pancreatitis and ulcerative colitis.

In a preferred embodiment of the invention is the Autoimmune disease selected from the group of inflammatory Bowel disease and / or autoimmune liver disease.

In a further preferred embodiment of the invention is the inflammatory bowel disease Crohn's disease or a chronic pancreatitis.

• • Gartenschlauchphänomen: Durch Fibrosierung verursachte Segmentstenosen
• • Pflastersteinphänomen: Entzündete Schleimhaut wechseln sich mit tiefen Ulzerationen ab, wodurch ein pflastersteinartiges Aussehen entsteht.
• • Entzündlicher Konglomerattumor: Verschiedene Darmabschnitte verkleben miteinander.

Crohn's disease belongs to the group of chronic inflammatory bowel diseases. It is a suspected autoaggressive, chronic granulomatous inflammation that can occur throughout the gastrointestinal tract from the oral cavity to the anus. Preference is given to the lower small intestine (terminal ileum, around 40% of the infestation) and colon, less commonly the esophagus and the mouth. Characteristic for Crohn's disease is the discontinuous, segmental involvement (so-called "skip lesions") of the intestinal mucosa, ie it can be affected simultaneously several sections of the intestine, which are separated by healthy sections.Other names for the disease are regional Crohn's disease, ileitis terminalis , Regional enterocolitis and sclerosing chronic enteritis, or the abbreviations MC, CD (Crohn's Disease) and overall as IBD (Inflammatory Bowel Disease) Crohn's disease according to the invention is therefore any condition which can be characterized macroscopically by the following changes:

• • Garden hose phenomenon: segmental stenosis caused by fibrosis
• • Cobblestone phenomenon: Inflamed mucosa alternate with deep ulcerations, creating a cobblestone appearance.
• • Inflammatory conglomerate tumor: Different intestinal sections stick together.

Histologically (fine tissue) one recognizes above all an accumulation of lymphocytes, (eosinophils) Granulocytes and histiocytes in the biopsy of the inflamed intestinal tissue. Adjacent lymph nodes are usually enlarged. Frequently, granulomas form, which can be divided into two types: epithelial cell granulomas and microgranulomas (smaller and without central necrosis).

• • Appendizitis: meist ein sich rasch entwickelnder Schmerz im rechten Unterbauch. Häufig eine Temperaturdifferenz > 1°C zwischen rektaler und axillärer Messung.
• • Divertikulitis: tastbare Resistenzen bei meist linksseitigem Unterbauchschmerz.
• • Yersiniose: Erregernachweis aus dem Stuhl oder aus dem Biopsiematerial, Anstieg des Antikörpertiters.
• • Darmtuberkulose: In Mitteleuropa mittlerweile sehr selten. Die Darmtuberkulose geht häufig mit Beteiligung der Lunge einher. Es finden sich „verkäsende" epitheloidzellige Granulome im Biopsiematerial.
• • jede andere invasive infektiöse Colitis (Salmonellenenteritis, pseudomembranöse Colitis etc.)

However, Crohn's disease according to the invention can also be characterized diagnostically. In this case, Crohn's disease according to the invention is a state in which at least one of the following features is detectable:

• • Appendicitis: usually a rapidly developing pain in the right lower abdomen. Often a temperature difference> 1 ° C between rectal and axillary measurement.
• • Diverticulitis: palpable resistance in usually left-sided pelvic pain.
• • Yersiniosis: pathogen detection from the stool or biopsy material, increase in antibody titer.
• • Intestinal tuberculosis: In Central Europe meanwhile very rare. The intestinal tuberculosis is often associated with the involvement of the lungs. There are "Verkäsende" epitheloidzellige granulomas in the biopsy material.
• • any other invasive infectious colitis (salmonella enteritis, pseudomembranous colitis etc.)

pancreatitis is within the meaning of the invention an inflammation of the pancreas (Pancreas), which may be acute or chronic.

In In most cases, the pancreatitis is caused by a Activation of pancreatic enzymes within the organ. Since it is the The task of these enzymes is to digest proteins and fats, begins a self-digestion of the organ. This self-digestion leads for inflammation of the pancreas. In severe cases can cause bleeding, serious tissue damage, infection and cysts arise. An inflamed gland can cause enzymes to enter the bloodstream and so reach the lungs, the heart and the kidneys, where further damage may occur. Acute pancreatitis arises when the pancreas suddenly ignites, but then recovered. Some patients suffer several times acute pancreatitis, but can be complete each time recover. Acute pancreatitis occurs suddenly and can be a serious, life-threatening disease that causes numerous Causes complications, usually patients recover but from acute pancreatitis. The incidence is about five to ten new cases per 100,000 inhabitants per year.

• 1. ödematöse Pankreatitis: blander Verlauf mit Schwellung des Organs und geringen Nekrosen im Fettgewebe der Umgebung
• 2. hämorrhagisch – nekrotisierende Pankreatitis: ausgedehnte Nekrosen und Blutungen im Pankreas und in die Umgebung; durch ihr fulminates Krankheitsbild oft auch als Pankreasapoplexie bezeichnet

It occurs in two forms:

• 1. edematous pancreatitis: blander course with swelling of the organ and low necrosis in the fatty tissue of the environment
• 2. hemorrhagic necrotizing pancreatitis: extensive necrosis and hemorrhage in the pancreas and surrounding area; often referred to as pancreatic apoplexy due to her fulminate clinical picture

The morphological evaluation of the pancreas, especially the differentiation between edematous and necrotizing pancreatitis works best with the contrast-enhanced computed tomography. To classify the severity of the Balthazar score has been proven (0 to 10 points).

• • Als Nebenwirkung von Medikamenten (z. B. Asparaginase, Azathioprin, Furosemid, Glukokortikoide, Antibiotika (Tetrazykline, Sulfamethoxazol, Trimethoprim), Antikonvulsiva (Valproat, Carbamazepin), Propofol und andere
• • Infektionen z. B. Mumps, Coxsackie-Virus, Hepatitis, HIV, Zytomegalie-Virus
• • Erhöhter Blutkalziumwert, z. B. bei Nebenschilddrüsenüberfunktion
• • Stark erhöhte Blutfette (Triglyzeride)
• • iatrogen nach ERCP
• • genetisch: Zystische Fibrose

Acute pancreatitis can have several causes. The most common are gallstones, which are stuck in the mouth of the bile duct in the duodenum, which is also the mouth of the pancreatic duct, temporarily or longer. (about 45% of acute pancreatitis). Another common cause is chronic alcohol abuse (about 35%). In about 15% of those affected no concrete trigger can be found, in these cases one speaks of idiopathic genesis. In addition, more rare causes such as:

• • As a side effect of medications (eg asparaginase, azathioprine, furosemide, glucocorticoids, antibiotics (tetracyclines, sulfamethoxazole, trimethoprim), anticonvulsants (valproate, carbamazepine), propofol and others
• • Infections z. Mumps, coxsackie virus, hepatitis, HIV, cytomegalovirus
• • Increased blood calcium, eg. B. in parathyroid hyperfunction
• • Highly elevated blood lipids (triglycerides)
• Iatrogen after ERCP
• • Genetic: Cystic fibrosis

Acute pancreatitis is initially characterized by pain in the epigastrium (epigastrium), which radiates into the chest, and disappears after a few days. The pain is often very intense and sometimes persistent. The pain can be sudden and intense, or begin as a slight pain and become worse after taking food (by stimulating the pancreas to form pancreatic enzymes to digest the food). Of the Belly can be swollen and very sensitive. Characteristic in the physical examination are a painful abdomen and a so-called rubber belly, which is due to meteorism and (moderate) defense tension. It can also cause pain in the lower part of the thoracic spine. This pain is initially similar to a slight lumbago, but develops more in the episode to a "Durchstochenwerden", from the back to the area of the pancreatic head on the ventral side.

patients with acute pancreatitis usually look very ill and feel that way too. Other symptoms include nausea, Vomiting, constipation, fever, increased heart rate. In heavy Jaundice (icterus) (when the bile ducts are relocated), Ascites (when laying the portal vein system), Pleural effusions and shock and sepsis signs added. laboratory diagnosis may be an increased white blood cell count (leukocytosis) as well Increase in the concentration of pancreatic enzymes (eg trypsin, amylases, Lipase). Also the calcium, magnesium, sodium, Potassium, Eicarbonat-, sugar or fat levels in the blood can be elevated. About 20% of acute pancreatitis cases are serious. The patient may dehydrate and have low blood pressure develop. Sometimes it comes to heart, lung or kidney failure. In the worst cases, acute pancreatitis results bleeding, shock and sometimes death.

To The current Atlanta classification will be the mild of the heavy acute pancreatitis. An obsolete classification divided an edematous form (early stage), a Hemorrhagic form with local or generalized hemorrhage and an acute necrotizing form.

A Chronic pancreatitis has many causes, but 70 to 80% are attributed to chronic alcohol abuse. It occurs more often in men than in women and often develops between the 30th and the 40th year of life. Chronic pancreatitis can also be characterized develop an acute inflammation if the cause is not is eliminated or damaged the execution passage is.

Some Chronic pancreatitis is hereditary. These are based on an abnormality of the enzymes formed by the pancreas, which damage the tissue. Other forms of the disease have their causes in external factors such. As the tobacco smoking.

In In the early stages of pancreatitis, the doctor may frequently Do not decide whether it is an acute or a chronic Form acts. The symptoms can be the same.

A Chronic pancreatitis often causes chronic pain. In some cases, chronic pancreatitis leaves the pain subsides as the disease progresses. she leads also to a hypofunction of the pancreatic activity, which leads to weight loss and indigestion. The insufficient digestion and absorption leads for dispensing fat and protein over the stool. When the endocrine cells (islets of Langerhans) in the pancreas are damaged diabetes can develop.

A Diagnosis of chronic pancreatitis is difficult, however, stand Some advanced medical techniques are available. Pancreatic function blood tests can help decide whether the pancreas is still able to digest enough digestive enzymes manufacture. However, they have hardly prevailed in practice.

abnormalities The pancreas can also be detected by sonography, ERCP and computed tomography be recognized.

In more advanced stages of chronic pancreatitis, though Diabetes and faulty absorption may occur, the doctor may as well Blood, urine and stool tests to make a diagnosis to deliver.

A Chronic pancreatitis is caused by prescribing painkillers and treated a diet change. Patients can Reduce the loss of fat and protein by taking medicines that contain pancreatic enzymes. This will be an improved Diet and gain weight. Sometimes Insulin or other medications are prescribed to lower the blood sugar level to control.

In Some cases of chronic pancreatitis becomes a surgical one Surgery done to relieve the pain by having an enlarged and congested pancreatic duct is relieved.

In a further preferred embodiment of the invention, the liver disease is primary sclerosis inducing cholangitis or autoimmune enteritis.

cholangitis, also cholangiitis in the sense of the invention, denotes an inflammation intrahepatic bile ducts. This can be done by different Causes are triggered, among other things by blockages bile ducts through gallstones, stenoses, tumors or parasitic infestation. One distinguishes between acute purulent cholangitis and non-adherent destructive cholangitis and chronic sclerosing cholangitis.

acute purulent cholangitis: Acute cholangitis is usually caused by an infection when colonized by bacteria, usually Escherichia coli, Enterococcus or Klebsiella species. The symptoms are one-sided Pain in the upper abdomen, fever and chills. A severe purulent cholangitis also occurs to shock states, disorders of the central nervous system as well as kidney dysfunction. The treatment includes endoscopic interventions on the bile ducts like the endoscopic retrograde Cholangiopancreatography (ERCP) or percutaneous transhepatic Cholangiodrainage (PTCD), which restores the bile flow, and usually a lens.

Not Purulent destructive cholangitis: This form of cholangitis runs Chronic and is also called primary biliary cirrhosis designated. 95% of those affected are women, with the frequency peak between the 40th and 60th year of life. Diagnostically one finds in most patients antimitochondrial antibodies (AMA) in the blood, which is why an autoimmune genesis was adopted becomes. Patients are clinically characterized by itching, jaundice and hypercholesterolemia. Later in the patient can only by means of Liver transplantation will be helped.

Chronic sclerosing cholangitis: Chronic sclerosing cholangitis is the rarest bile duct inflammation and gets into one primary and a secondary sclerosing form. The primary form is caused by infections in existing ones genetic disposition (it became an association with the antigen HLA-B8 detected) and affects men twice as often like women. In up to 90% of cases, p-ANCA is found. The secondary form arises on the floor of existing ones Immunodeficiency syndromes. As with the destructive cholangitis also here in the final stage a liver transplant necessary.

Autoimmunenteritiden Within the meaning of the invention is any form of enteritis, in particular those that are essentially chronic inflammatory Intestinal diseases are caused. Within the meaning of the invention Autoimmune enteritis but also those caused by Salmonella, E. Coli, cholera or Typhuserreger be evoked, or else by fungi, protozoa, toxic substances, but also any allergic conditional enteritis or any form of actinic enteritis, the Yersinia enteritis or bacterial dysentery.

In a further preferred embodiment of the invention the amino acid sequence is at least 60%, preferably 70%, preferably 80%, most preferably 90% homologous to the Sequence according to SEQ ID No. 1. That is, the invention refers to all peptides which are preferably 60%, 70%, 80%, most preferably 90% homology to the sequence according to SEQ ID No. 1. Of course you can these homologs by deletion, addition, substitution, translocation, Inversion and / or insertion may be modified. This modification specifically relates to homologous peptides that are functionally analogous. Functional analogues are the peptides according to the invention, if they with autoantibodies associated with the o. g. Diseases associated are, interact specifically.

Inventive subject matter Accordingly, the disclosed peptide and the Homologues that can be used in a functional analogue as well as their use; d. H. also the disclosure of earmarked materials for the first medical indication, as well as the use in research and for other medical applications. The person skilled in the art is familiar with homologs / functional analogs, that he changes by additions, deletions or substitutions without substantially altering the polypeptide becomes. Not significantly altered is the modified amino acid sequence, if it performs the same function as the sequence according to SEQ ID NO: 1 and that essentially on the same way, leading to the same result. That is, a modified amino acid sequence according to the invention is any altered sequence, their application being none significant effect on the solution of the invention Has problems and if this is obvious to the skilled person is the skilled artisan recognizes that the inventors are not intend to base their teaching on the wording of the claims - d. H. to the sequence according to SEQ ID No. 1 - to restrict.

oder SEQ ID Nr. 3

oder SEQ ID Nr. 4

oder aber SEQ ID Nr. 5

sein. Die genannten Peptide werden für die Verwendung in der Forschung, aber auch für medizinische Anwendungen, insbesondere für die oben ausgeführten Immunerkrankungen beansprucht. Die genannten Sequenzen erfüllen im Wesentlichen dieselbe Funktion auf im Wesentlichen demselben Weg und bringen im Wesentlichen dasselbe Ergebnis hervor wie die Sequenz SEQ ID Nr. 1. Sie werden daher von der erfindungsgemäßen Lehre, der Verwendung des Moleküls GP2 für die Verwendung als Arzneimittel, insbesondere zur Prophylaxe, Diagnose, Therapie und/oder Nachbehandlung von Immunerkrankungen, erfasst.Functionally analogous peptides can be, for example, SEQ ID NO. 2

or SEQ ID NO: 3

or SEQ ID NO: 4

or else SEQ ID No. 5

be. The said peptides are claimed for use in research, but also for medical applications, in particular for the above-mentioned immune disorders. The said sequences fulfill substantially the same function in substantially the same way and produce substantially the same result as the sequence SEQ ID No. 1. They are therefore the teaching of the invention, the use of the molecule GP2 for use as a medicament, in particular to Prophylaxis, diagnosis, therapy and / or treatment of immune diseases.

The Amino acid sequences, d. H. the peptides in the sense of Invention, so much more amino acids, Spacers or other structures include that they are suitable with the antibodies, preferably autoantibodies, to interact, preferably so that they are an epitope for represent these. The sequence according to the invention is therefore not limited to the peptides, which concern antibody epitopes, but it refers on the molecule and all fragments of it, specifically interacting with autoantibodies. The terms Epitope and peptide as well as amino acid sequence can therefore in preferred embodiments according to the invention be used synonymously.

Various possibilities for the production of functionally analogous peptides are disclosed in the prior art. Peptides which are designed starting from the peptides according to the invention starting from such methods are covered by the teaching according to the invention. One way of generating functionally analogous peptides is, for example, in PNAS USA 1998, Oct. 13; 9521: 12179-84, WO 99/6293 and or WO 02/38592 described; These teachings are included in the disclosure of the invention. That is, all peptides, peptide fragments, or structures comprising peptides conjugated to said Ver starting - were generated from the peptides according to the invention, are peptides in the context of the invention, provided that they solve the object of the invention, in particular interact with the disease-causing autoantibodies. These autoantibodies may be, for example, agonistic autoantibodies that activate receptors.

In a further preferred embodiment of the invention the molecule comprises a linker or spacer selected from the group α-aminocarboxylic acids and their Homo- and hetero-oligomers; , Ω-aminocarboxylic acids α as well as their branched homo- or hetero-oligomers; other amino acids and the linear and branched homo- or hetero-oligomers; Amino-oligoalkoxy-alkylamines; Maleimidocarboxylic acid derivatives; Oligomers of alkylamines; 4-alkylphenyl derivatives; 4-oligoalkoxyphenyl or 4-oligoalkoxyphenoxy derivatives; 4-oligoalkylmercaptophenyl or 4-oligoalkylmercaptophenoxy derivatives; 4-oligoalkylaminophenyl or 4-oligoalkylaminophenoxy derivatives; (Oligoalkylbenzyl) phenyl or 4- (oligo-alkylbenzyl) phenoxy derivatives and 4- (oligoalkoxybenzyl) phenyl or 4- (oligoalkoxybenzyl) phenoxy derivatives; Trityl derivatives; Benzyloxyaryl or benzyloxyalkyl derivatives; Xanthene-3-yl-oxyalkyl derivatives; (4-alkylphenyl) - or ω- (4-alkylphenoxy) -alkanoic acid derivatives; Oligoalkyl-phenoxylalkyl or oligoalkoxy-phonoxyalkyl derivatives; Carbamate derivatives; amines; Trialkylsilyl or dialkylakoxysilyl derivatives; Alkyl or aryl derivatives or combinations thereof.

In a further preferred embodiment of the invention the molecule becomes GP2 for the production of a drug used for the treatment of autoimmune diseases.

In a further preferred embodiment of the invention the molecule GP2 may be soluble or solid phase bound for direct or indirect autoantibody detection in Body fluids, especially blood or serum, be used.

In a further preferred embodiment of the invention be in addition to the soluble or solid phase bound Molecule GP2 uses nonspecific adsorber molecules, selected from the group comprising protein A, protein G, anti-human immunoglobulins or L-tryptophan.

Prefers becomes the application sequence or fragments used therefrom as a therapeutic agent. The usage as a therapeutic agent in the context of the invention means the application of Amino acid sequence or the peptides formed from this can be, in the entire field of medicine.

• (i) Quervernetzung: Bei der Quervernetzung werden die Peptide miteinander fixiert, ohne dass ihre Aktivität nachteilig beeinflusst wird. Sie sind vorteilhafterweise durch die Quervernetzung nicht mehr löslich.
• (ii) Bindung an einen Träger: Die Bindung an einen Träger erfolgt zum Beispiel durch Adsorption, Ionenbindung oder kovalente Bindung. Dies kann auch innerhalb von mikrobiellen Zellen bzw. Liposomen oder anderen membranhaltigen geschlossenen bzw. offenen Strukturen erfolgen. Die Peptide werden durch die Fixierung vorteilhafterweise nicht in ihrer Aktivität beeinflusst. Die Peptide können mit Vorteil zum Beispiel in der Klinik in Diagnose oder Therapie trägergebunden mehrfach oder kontinuierlich eingesetzt werden.
• (iii) Einschluss: Der Einschluss erfolgt im Sinne der Erfindung insbesondere an eine semipermeable Membran in Form von Gelen, Fibrillen oder Fasern. Gekapselte Peptide sind durch eine semipermeable Membran so durch die umgebende Probenlösung getrennt, dass sie vorteilhafterweise noch mit den Autoantikörpern oder mit Fragmenten dieser interagieren können. Für die Immobilisierung stehen verschiedene Verfahren zur Verfügung, wie beispielsweise die Adsorption an einen inerten oder elektrisch geladenen anorganischen oder organischen Träger. Solche Träger können beispielsweise poröse Gele, Aluminiumoxid, Betonid, Agarose, Stärke, Nylon oder Polyacrylamid sein. Die Immobilisierung erfolgt hierbei durch physikalische Bindungskräfte, oft unter Beteiligung von hydrophoben Wechselwirkungen und ionischen Bindungen. Derartige Methoden sind vorteilhafterweise einfach zu handhaben und sie beeinflussen die Konformation der Peptide nur in geringem Umfang. Durch elektrostatische Bindungskräfte zwischen den geladenen Gruppen der Peptide und dem Träger kann die Bindung vorteilhafterweise verbessert werden, zum Beispiel durch die Verwendung von Ionenaustauschern, insbesondere Sephadex.

In a further preferred embodiment of the invention, in particular the sequence according to the application or peptides which can be generated therefrom are immobilized. For the purposes of the invention under immobilization, various methods and techniques for fixing the peptides on certain carriers, for example according to the WO 99/56126 or the WO 02/26292 Understood. The immobilization can serve, for example, to stabilize the peptides, as a result of which they are not reduced or adversely modified in their activity, in particular during storage or in the case of a single batch batch, due to biological, chemical or physical effects. Immobilization of the peptides allows repeated use under routine technical or clinical conditions; Furthermore, a sample - preferably blood constituents - can be continuously reacted with at least one of the peptides according to the invention. This can be achieved, in particular, by various immobilization techniques, wherein the binding of the peptides to other peptides or molecules or to a carrier takes place such that the three-dimensional structure, in particular at the center, which mediates the interaction with the autoantibodies, of the corresponding molecules, in particular the peptides, does not change. Advantageously, the specificity to the autoantibodies of the patients is not lost by the immobilization. For the purposes of the invention, three basic methods of immobilization can be used:

• (i) Crosslinking: In crosslinking, the peptides are fixed with each other without adversely affecting their activity. They are advantageously no longer soluble by the crosslinking.
• (ii) Binding to a Carrier: Binding to a carrier occurs, for example, by adsorption, ionic bonding or covalent bonding. This can also take place within microbial cells or liposomes or other membrane-containing closed or open structures. The peptides are advantageously not affected by the fixation in their activity. The peptides can advantageously be used repeatedly or continuously in the hospital in diagnosis or therapy carrier-bound.
• (iii) Inclusion: In the sense of the invention, the inclusion takes place in particular on a semipermeable membrane in the form of gels, fibrils or fibers. Encapsulated peptides are separated by a semipermeable membrane by the surrounding sample solution so that they can advantageously still interact with the autoantibodies or with fragments of them. For the immobilization are various Ver available, such as adsorption to an inert or electrically charged inorganic or organic support. Such supports may be, for example, porous gels, alumina, concrete, agarose, starch, nylon or polyacrylamide. The immobilization takes place here by physical binding forces, often involving hydrophobic interactions and ionic bonds. Such methods are advantageously easy to handle and affect the conformation of the peptides only to a limited extent. By electrostatic binding forces between the charged groups of the peptides and the carrier, the binding can be advantageously improved, for example by the use of ion exchangers, in particular Sephadex.

One Another method is covalent bonding to support materials. The carriers may have reactive groups for this purpose, the homopolar bonds with amino acid side chains received. Suitable groups in peptides are carboxy, hydroxy and sulfide groups and in particular the terminal amino groups of lysines. Aromatic groups offer the opportunity for diazo couplings. The surface of microscopic Porous glass particles can be obtained by treatment with silanes activated and then reacted with peptides. Hydroxy groups of natural polymers can be used for Example with bromocyane activated and then with peptides be coupled. With polyacrylamide resins, numerous Peptides advantageously enter into direct covalent bonds. When included in three-dimensional networks, the peptides become in ionotropic gels or other structures known to those skilled in the art locked in. The pores of the matrix are in particular so that the peptides are retained and an interaction with the target molecules is possible. In the cross-linking The peptides are synthesized by crosslinking with bifunctional agents converted into polymeric aggregates. Such structures are gelatinous and easily deformable and especially for use suitable in different reactors. By adding other inactive Components, such as gelatin, in the networking can advantageously improves the mechanical and binding properties become. In microencapsulation, the reaction space of the peptides becomes bounded by membranes. The microencapsulation can for example, as interfacial polymerization become. By immobilizing in the microencapsulation be the peptides insoluble and thereby reusable. in the For the purposes of the invention, immobilized peptides are all peptides which are in a state that allows their reuse. The limitation of mobility and solubility the peptides by chemical, biological or physical means leads advantageously to low process costs, especially in the elimination of autoantibodies from blood components.

In a further preferred embodiment of the invention is the peptide or the entire amino acid sequence to a Hard phase bound. The binding of the peptide or the entire amino acid sequence to the solid phase can be done via a spacer. As a spacer all chemical compounds can be used, for the function of the spacer the appropriate structural and functional requirements, as long as they are not Modify binding behavior such that binding of the autoantibody is adversely affected with the peptide.

The Pharmaceutical composition can be used especially as a medicine be used. For this it is possible, for example, the peptides or the complete amino acid sequence Cyclization or other methods known to those skilled in the art, that they through body-own peptide-degrading structures, such as serum proteases, are not destroyed can. By using the invention Peptides or the protein (SEQ ID NO: 1) it is possible to neutralize the autoantibodies in or ex vivo. at In vivo neutralization, the drugs become the patient administered directly, in an ex vivo neutralization, for example the blood over a loop - for example in Form of a hose circuit - out of the body following, contacted with the drug and after the completed neutralization of the autoantibodies again in the Organism, especially the patient, attributed. For the purposes of the invention, pharmaceuticals include both such pharmaceutical Compositions suitable for therapeutic and prophylactic Purposes, as well as those pharmaceutical compositions, which can be used as a diagnostic agent.

Medicaments or pharmaceutical compositions used interchangeably herein are, according to the invention, substances and preparations of substances which are intended to heal, alleviate or prevent diseases, suffering, bodily injury or pathological complaints by application to or in the human body. According to the invention, medical adjuvants are substances which are used for production as active ingredients of medicaments. Pharmaceutical-technical excipients serve to properly formulate the drug or pharmaceutical composition and may even be removed, if needed only during the manufacturing process, or may be part of the pharmaceutical composition as pharmaceutically acceptable carriers. Examples for pharmaceutically acceptable carriers are listed below. The drug formulation or formulation of the pharmaceutical composition is optionally in combination with a pharmaceutically acceptable carrier and / or diluent. Examples of suitable pharmaceutically acceptable carriers are known to those skilled in the art and include, for example, phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of detergents, sterile solutions, etc. Drugs or pharmaceutical compositions comprising such carriers , can be formulated by known conventional methods. These drugs or pharmaceutical compositions may be administered to an individual at a suitable dose, for example, in a range of from 1 μg to 10 g of peptides or protein per day and patient. Doses of 1 mg to 1 g are preferred. Preference is given to administering as few and as low doses as possible, and more preferably a single dose. Administration may be by various routes, for example, intravenous, intraperitoneal, intrarectal, intragastrointestinal, intranodal, intramuscular, local, but also subcutaneous, intradermal or on the skin or mucous membranes. The administration of nucleic acids coding for the peptide according to the invention can also be carried out in the form of gene therapy, for example via viral vectors. The type of dosage and the route of administration may be determined by the attending physician according to the clinical factors. It is well known to those skilled in the art that the type of dosage will depend on various factors, such as size, body surface area, age, sex or general health of the patient, but also on the particular agent being administered Duration and route of administration and other medications that may be administered in parallel. Furthermore, it is known to the person skilled in the art that he can first diagnose the concentration of the autoantibodies with the peptides according to the invention in order to determine the necessary concentration of the medicament.

The pharmaceutical compositions or the drug in particular a pharmacological substance containing one or more peptides of the invention or the protein and / or these coding nucleic acid molecules in one suitable solution or administration form. These can either alone with the appropriate under Medicaments or pharmaceutical compositions described Excipients or in combination with one or more adjuvants, for example QS-21, GPI-0100 or other saponins, water-oil Emulsions such as montanides, adjuvants, polylysine, Polyarginine compounds, DNA compounds such as CpG, Detox, bacterial vaccines such as typhoid or BCG vaccines, salts such as calcium phosphates and / or a other suitable substance for enhancing the effect become; preferably immunostimulatory molecules, such as Interleukins, for example IL-2, IL-12, IL-4 and / or growth factors, for example GM-CSF. These are in known methods with the peptides or recognition molecules according to the invention mixed and administered in a suitable formulation and dosage. Formulations, dosages and suitable components are those skilled in the art known.

The pharmaceutical composition or the drug can of course also a combination of two or more of the invention pharmaceutical compositions or pharmaceuticals, as well as a combination with other medicines, such as antibody therapies, chemotherapies or radiotherapies that work together in a timely manner or administered separately. The production the pharmaceutical or pharmaceutical compositions according to known methods.

The Invention also relates to a kit comprising the nucleic acid molecule, the vector, the peptide and / or the protein, optionally with a guide or information for pharmaceutical provision or for the therapeutic treatment method. The information can for example, a leaflet or another medium, what informing the user about which therapeutic procedure the substances mentioned are to be used. The leaflet contains in particular detailed and / or essential information about the healing process. Of course it is not mandatory requires that the information be a leaflet, it is possible that this information is transmitted via, for example the internet is communicated.

The The invention also relates to a device for chromatography, the comprises the peptides of the invention.

In In a preferred embodiment, the peptides are within of the chromatography system bound to a solid phase.

The device according to the invention can be used, in particular, for the autoantibodies to eliminate from a patient's fluids or to neutralize the autoantibodies. This method is known to the person skilled in the art by the term immunoadsorption and apheresis therapy. Immunoadsorption removes immunoglobulins from the patient's blood.

advantageously, This immunoadsorbent treatment can be inpatient and outpatient be performed. It can be provided that the Device, in particular the so-called adsorber, part of a extracorporeal blood circulation is. This is the patient from a larger body vessel, in particular a arm vein, continuous or discontinuous Taken from blood and by filtration or centrifugation in individual Ingredients, such as the cellular and the humoral components, separated. An essential component of the blood which is thereby obtained is in particular blood plasma. The blood plasma can advantageously by the inventive Device passed and after adsorption of autoantibodies together with the previously separated blood components, in particular the cellular components returned to the patient especially through another arm or leg vein. It can continue be provided that the peptides immobilized on a Sepharose matrix are. This matrix can be placed in a container which has a volume of 10 to 400 ml. The blood plasma of the patient can then be directed across this matrix, with the autoantibodies bind and can be eliminated from the blood plasma.

the Professional are known various ways such provide solid phase-fixed peptides, for example in the form of (i) regenerable adsorption columns, in shape. of (ii) double columns and in the form of (iii) Once columns. The various rinsing and eluting solutions, which allow a high efficiency of treatment be determined by the skilled person easily by routine experimentation. By providing the teaching according to the invention, in particular the peptides according to the invention are the expert various possibilities. revealed this in vivo, ex vivo and in vitro, for prophylaxis, diagnosis, therapy and for post-treatment of cold-induced, autoantibody-mediated diseases use.

In a further preferred embodiment of the invention The molecule GP2 is used to immunize mammals for obtaining poly-, monoclonal or anti-idiotypic autoantibodies used.

• a) ein Molekül aufweisend eine Aminosäuresequenz, die eine ausreichende Homologie zu dem Molekül GP2 aufweist, um zu diesem funktionsanalog zu sein,
• b) ein Molekül nach a), welches durch Deletion, Addition, Substitution, Translokation, Inversion und/oder Insertionen modifiziert wurde und zu dem Molekül gemäß a) funktionsanalog ist.

In a further preferred embodiment of the invention, the molecule GP2 is selected from the group comprising:

• a) a molecule having an amino acid sequence which has sufficient homology to the molecule GP2 in order to be functionally analogous to it,
• b) a molecule according to a) which has been modified by deletion, addition, substitution, translocation, inversion and / or insertions and is functionally analogous to the molecule according to a).

In a further preferred embodiment of the invention the molecule indicated under b) is at least 40% homologous to the molecule indicated under a).

In a further preferred embodiment of the invention the molecule indicated under b) is at least 60%, preferably 70%, preferably 80%, most preferably 90% homologous to the under a) specified molecule.

In a further preferred embodiment of the invention the molecule GP2 is linear or in cyclic form before, wherein the peptide cyclization in the presence of two cysteines by disulfide bond formation or by amide cyclization, which optionally via the side chains, on the terminal C and N or by a combination of these possibilities he follows.

In a further preferred embodiment of the invention is the solid phase-bound molecule GP2 attached to organic, bonded inorganic, synthetic or mixed polymers, preferably Agarose, cellulose, silica gel, polyamides or polyvinyl alcohols.

The The invention also relates to a pharmaceutical composition comprising at least one molecule GP2 possibly together with a pharmaceutical acceptable carrier.

In a preferred embodiment of the invention is provided that the pharmaceutical carrier is selected from the group comprising fillers, disintegrants, binders, Humectants, diluents, dissolution inhibitors, Absorption accelerator, wetting agents, absorbents and / or Lubricant.

The The invention relates in a further aspect to the use of a specific ligands for human immunoglobulins for production a column coupled to this ligand for treatment of inflammatory bowel disease, taking this treatment passing a patient's plasma over the column comprises conditions are selected which a effective binding of the specific ligand to the immunoglobulins allow in the patient's plasma, causing a significant amount the immunoglobulins are removed from the patient's plasma and returned the plasma thus obtained to the patient becomes.

• a) Bereitstellung einer Säule, an welche spezifische Liganden für humane Immunglobuline gekoppelt sind,
• b) Passieren von Plasma des Patienten über die Säule unter Konditionen, welche eine effektive Bindung des spezifischen Liganden an die Immunglobuline im Plasma des Patienten erlauben, wodurch eine signifikante Menge der Immunglobuline aus dem Plasma des Patienten entfernt werden und
• c) Rückführung des so gewonnenen Plasmas in den Patienten.

The invention also relates to a method for the treatment of inflammatory bowel disease comprising the steps of:

• a) providing a column to which specific ligands for human immunoglobulins are coupled,
• b) passing plasma of the patient across the column under conditions which permit effective binding of the specific ligand to the immunoglobulins in the patient's plasma thereby removing a significant amount of the immunoglobulins from the patient's plasma and
• c) returning the plasma thus obtained to the patient.

In Another aspect of the invention relates to a method for the treatment of inflammatory bowel disease, wherein the specific ligand selected is selected from the group polyclonal anti-human immunoglobulin antibodies, monoclonal anti-human immunoglobulin antibodies, fragments this antibody, recombinant molecules of the Antibody Idiotypes, Synthesized Peptides, Protein A and / or protein B.

In a preferred embodiment of the invention recognizes the specific ligand directed against intestinal tissue autoantibodies.

In a further preferred embodiment of the invention the specific ligand is an antigen-mimicking molecule, selected from the group consisting of polyclonal and monoclonal anti-idiotypic antibodies, fragments this or synthetic peptides.

In a further preferred embodiment of the invention the specific ligand is a synthesized peptide, which is a Sequence of a structure mimicked by GP2.

The The invention also relates to a diagnostic test kit (kit) for determination of autoimmune diseases.

In In another aspect, the invention relates to an immunogenic agent, containing at least one GP2 molecule.

• – Abkehr vom technisch Üblichen
• – neue Aufgabenstellung
• – Vorliegen eines seit langem ungelösten dringenden Bedürfnisses für die Lösung des mit der Erfindung gelösten Problems
• – bisheriges vergebliches Bemühen der Fachwelt
• – die Einfachheit der Lösung spricht für erfinderische Tätigkeit, insbesondere da sie kompliziertere Lehren ersetzt
• – Entwicklung der wissenschaftlichen Technik ging in eine andere Richtung
• – entwicklungsstraffende Leistung
• – Fehlvorstellungen der Fachwelt über die Lösung des entsprechenden Problems (Vorurteil)
• – technischer Fortschritt, wie z. B.: Verbesserung, Leistungssteigerung, Verbilligung, Ersparnis an Zeit, Material, Arbeitsstufen, Kosten oder schwer beschaffbaren Rohstoffen, erhöhte Zuverlässigkeit, Beseitigung von Fehlern, Qualitätshebung, Wartungsfreiheit, größere Effektivität, höhere Ausbeute, Vermehrung der technischen Möglichkeiten, Bereitstellung eines weiteren Mittels, Eröffnung eines zweiten Weges, Eröffnung eines neuen Gebietes, erstmalige Lösung einer Aufgabe, Reservemittel, Alternativen, Möglichkeit der Rationalisierung, Automatisierung oder Miniaturisierung oder Bereichung des Arzneimittelschatzes
• – glücklicher Griff, da aus einer Vielzahl von Möglichkeiten eine bestimmte gewählt wurde, deren Ergebnis nicht vorausgesagt werden konnte, daher handelt es sich um ein patentwürdigen glücklichen Griff)
• – Irrtum in Entgegenhaltungen
• – junges Gebiet der Technik
• – Kombinationserfindung, d. h. mehrere bekannte Elemente werden zu einer Kombination zusammengeführt, die einen überraschenden Effekt aufweist
• – Lizenzvergabe
• – Lob der Fachwelt und
• – wirtschaftlicher Erfolg.

The teaching according to the application is characterized by the following features:

• - departure from the technical usual
• - new task
• - There is a long unresolved urgent need for the solution of the problem solved by the invention
• - Past futile efforts of the experts
• The simplicity of the solution speaks to inventive step, especially as it replaces more complicated teachings
• - Development of scientific technology went in a different direction
• - development-firming achievement
• - misconceptions of the experts about the solution of the corresponding problem (prejudice)
• - technical progress, such as B .: improvement, performance increase, cheapening, saving time, material, work stages, costs or difficult-to-procure raw materials, increased reliability, elimination of errors, quality improvement, freedom from maintenance, greater effectiveness, higher yield, increase in technical possibilities, providing a further means, Opening up a second path, opening a new territory, solving a task for the first time, reserve resources, alternatives, possibility of rationalization, automation or miniaturization or enrichment of the drug
• - happy grip, because of a variety of possibilities was chosen a particular, the result of which could not be predicted, so it is a patentable lucky grip)
• - Error in citations
• - young field of engineering
• - Combination invention, ie several known elements zusammenge together to a combination leads, which has a surprising effect
• - Licensing
• - praise the experts and
• - economic success.

These Properties relate in particular to the preferred embodiments the invention.

in the The invention is based on an example closer are explained without being limited to this example to be.

methods

Purification of GP2 from rat pancreas

Obtaining the zymogen granule (ZG) and Cleaning the ZG membranes

All The following steps are in the ice bath or under cooling been carried out at 4 ° Celsius. The pancreas of four adult Wistar rats (about 2.4 g tissue) became mechanical crushed and in ten times the volume of ice-cold 0.3 M sucrose solution unlocked in the POTTER homogenizer (2 strokes at 1000 Rpm and 2 strokes at 1300 rpm). Subsequently The digest was filtered through a gauze cloth. By Centrifugation at 500 g for 10 min was cell debris and the cores are separated. From the supernatant were through Centrifugation at 3000 g for 10 min the zymogen granules (lower, white solid pellet) and the mitochondria (overlying, loose brownish pellet). The mitochondria are by buffer A (10 mM - morpholinepropanesulfonic acid (MOPS), pH 6.8) and the zymogen granules in 2 ml of 0.1 M sodium carbonate solution, 1 mM diisopropylfluorophosphate (DFP), resuspended by vortexing. The lysis of the granules took place for 1 h in an ice bath. The lysis approach was over a layered sucrose gradients (0.3 M / 1 M) and centrifuged at 200,000 g for 90 min. The membrane fraction accumulated as a band in the density boundary layer. The gang was filtered off and the resulting solution 0.3 M of sodium bromide set. By centrifugation at 200,000 g for 60 min, the membranes were sedimented.

Solubilization of the GP-2

The recovered membrane pellet was dissolved in 0.5 ml buffer B (20 mM morpholineethanesulfonic acid (MES), pH 7.0; 80 mM KCl; 45 μg / ml saponin) by means of ultrasound resuspended and after addition of phosphatidylinosole-specific Phospholipase C (B. cereus) by incubation for 1 h at Cleaved 37 ° C GP2 from the membrane. The membranes were pelleted by centrifugation (200,000 g, 60 min) and the supernatant with the soluble GP-2 by ultrafiltration about 1 to 5 concentrated.

Enzyme linked immosorbent assay (ELISA) for the determination of GP2 antibodies

microtiter plates (Maxisorb, Nunc, Roskilde) were 50 μl / well a solution of 10 μg / ml rat GP2 in coating buffer (100 mM Na carbonate, pH 9.6) overnight at 4 ° C coated. After a single wash of the microtiter plate with a Wash Buffer (10mM Na Phosphate, 150mM NaCl, 0.1% Tween20, pH 7.4) The wells were filled with 300 μl of the blocking solution (Wash Buffer, 1% bovine serum albumin (RSA), pH 7.4) for Incubated for 30 min at room temperature (RT). Subsequently the wells were washed three times with wash buffer and 50 μl 1/100 in dilution buffer (10 mM Na phosphate, 150 mM NaCl, 1% BSA) diluted human serum samples for Incubated for 60 min at RT per well. After washing three times Dilution buffer was added to the wells with 50 μl Conjugate solution (anti-human IgG peroxidase, sheep, 1 μg / ml, Dilution buffer) and for 30 min incubated at RT. Subsequently, the cavities were washed again three times and 50 μl of the substrate solution (Tetramethylbenzidine) dispensed per cavity. After 10 After incubation at RT, the substrate reaction was stopped by adding 50 μl stop solution (0.3 M sulfuric acid). The optical density of the solution in the individual cavities was bichromatisch at 450 by means of microtiter plate photometer nm and 620 nm measured and computer supported with the software program EIAstar evaluated.

Indirect immunofluorescence (IIF) for determination of pancreatic antigen antibodies

Commercial monkey pancreas sections (Euroimmun, Lübeck) were used for the determination of pancreatic antigen antibodies using IIF. The serum samples were diluted with dilution buffer 1/40, 1/80 and Diluted 1/160. For each reaction field of the reagent carrier, 25 μl of diluted serum were pipetted and the slides were incubated with the tissue sections at RT for 30 min. Subsequently, the slides were washed with a phosphate buffer for 1 min. Then 20 μl of labeled antiserum (anti-human IgG FITC) were pipetted into each field of the purified reagent carrier and incubated with the tissue sections on the slides for 30 min at RT. After washing again with phosphate buffer for 1 min, the slides were fitted with a coverslip using a mounting medium. The evaluation of the fluorescence was carried out with the aid of a fluorescence microscope.

literature

• Bossuyt X. Serologic markers in inflammatory bowel disease. Clin Chem 2006, 52 (2): 171-181.
• Fricke H, Birkhofer A, Folwaczny C, Master W, Scriba PC. Characterization of antigens from the human exocrine pancreatic tissue (Pag) relevant as target antigens for autoantibodies in Crohn's disease. Eur J Clin Invest, 1999, 29: 41-45.
• Main J, McKenzie H, Yeaman GR, Kerr MA, Robson D, Pennington CR, Parratt D. Antibody to saccharomyces cerevisiae (bakers' yeast) in Crohn's disease. BMJ, 297: 1105-1106.
• Mayet WJ, Press AG, Hermann E, Minor R, Manns M, Ewe K, Meyer to Büchenfelde KH. Antibodies to cytoskeletal proteins in patients with Crohn's disease. Eur J Clin Invest, 1990, 20: 516-524.
• Seibold F, Weber P, Jens H, Wiedmann K H. Antibodies to a trypsin sensitive pancreatic antigen in chronic inflammatory bowel disease: specific markers for a subgroup of patients with Crohn's disease. Good 1991, 32: 1192-1197.
• Stöcker W, Otte M, Ulrich S, Normann D, Finkbeiner H, Stöcker K, Jantschek G, Scriba PC. Autoimmunity to pancreatic juice in Crohn's disease. Results of an autoantibody screening in patients with chronic inflammatory bowel disease. Scand J Gastroenterol, 1987, 22 (suppl 139), 41-52.

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA become.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• WO 99/6293 [0067]
• WO 02/38592 [0067]
• WO 99/56126 [0073]
• WO 02/26292 [0073]

Cited non-patent literature

• Mayet et al., 1990 [0021]
• Stoecker et al., 1987; Main et al., 1988 [0022]
• Fricke et al., 1999 [0028]
• Seibold et al., 1991 [0029]
• Fricke et al., 1999, Seibold et al., 1991 [0030]
• Bossuyt, 2006 [0030]
• Bossuyt X. Serologic markers in inflammatory bowel disease. Clin Chem 2006, 52 (2): 171-181. [0108]
• - Fricke H, Birkhofer A, Folwaczny C, Master W, Scriba PC. Characterization of antigens from the human exocrine pancreatic tissue (Pag) relevant as target antigens for autoantibodies in Crohn's disease. Eur J Clin Invest, 1999, 29: 41-45. [0108]
• - Main J, McKenzie H, Yeaman GR, Kerr MA, Robson D, Pennington CR, Parratt D. Antibody to saccharomyces cerevisiae (bakers' yeast) in Crohn's disease. BMJ, 297: 1105-1106. [0108]
• - Mayet WJ, Press AG, Hermann E, Minor R, Mann's M, Ewe K, Meyer to Büchenfelde KH. Antibodies to cytoskeletal proteins in patients with Crohn's disease. Eur J Clin Invest, 1990, 20: 516-524. [0108]
• - Seibold F, Weber P, Jenss H, Wiedmann K H. Antibodies to a trypsin sensitive pancreatic antigen in chronic inflammatory bowel disease: specific markers for a subgroup of patients with Crohn's disease. Good 1991, 32: 1192-1197. [0108]
• - Stöcker W, Otte M, Ulrich S, Normann D, Finkbeiner H, Stöcker K, Jantschek G, Scriba PC. Autoimmunity to pancreatic juice in Crohn's disease. Results of an autoantibody screening in patients with chronic inflammatory bowel disease. Scand J Gastroenterol, 1987, 22 (suppl 139), 41-52. [0108]

Claims (29)

One molecule GP2 according to the Sequence SEQ ID NO: 1 for use as a drug. Use of a molecule GP2 according to SEQ ID No. 1 for the prophylaxis, diagnosis, therapy and / or aftercare of autoimmune diseases. Use according to claim 1, characterized that the autoimmune disease is selected from the group inflammatory bowel disease (EDE) and / or autoimmune Liver diseases. Use according to claim 2 or 3, characterized that the inflammatory bowel disease Crohn's disease and / or is a chronic pancreatitis. Use according to one of the preceding claims, characterized in that the liver diseases are primary Sclerosing cholangitis and / or autoimmune enteritis are. Use according to one of the preceding claims, characterized in that the molecule GP2 is a linker and / or spacer selected from the group α-aminocarboxylic acids as well as their homo- and hetero-oligomers; , Ω-aminocarboxylic acids α as well as their branched homo- or hetero-oligomers; other amino acids and the linear and branched homo- or hetero-oligomers; Amino-oligoalkoxy-alkylamines; Maleimidocarboxylic acid derivatives; Oligomers of alkylamines; 4-alkylphenyl derivatives; 4-oligoalkoxyphenyl or 4-oligoalkoxyphenoxy derivatives; 4-oligoalkylmercaptophenyl or 4-oligoalkylmercaptophenoxy derivatives; 4-oligoalkylaminophenyl or 4-oligoalkylaminophenoxy derivatives; (Oligoalkylbenzyl) phenyl or 4- (oligo-alkylbenzyl) phenoxy derivatives and 4- (oligoalkoxybenzyl) phenyl or 4- (oligoalkoxybenzyl) phenoxy derivatives; Trityl derivatives; Benzyloxyaryl or benzyloxyalkyl derivatives; Xanthene-3-yl-oxyalkyl derivatives; (4-alkylphenyl) - or ω- (4-alkylphenoxy) -alkanoic acid derivatives; Oligoalkyl-phenoxylalkyl or oligoalkoxy-phonoalkyl derivatives; Carbamate derivatives; amines; Trialkylsilyl or dialkylakoxysilyl derivatives; Alkyl or aryl derivatives and / or combinations thereof. Use according to one of the preceding claims, characterized in that the molecule GP2 for the production a drug used to treat autoimmune diseases becomes. Use according to one of the preceding claims, characterized in that the molecule GP2 is soluble or solid phase bound for direct or indirect autoantibody detection in body fluids, especially blood and / or Serum is used. Use according to one of the preceding claims, characterized in that in addition to the soluble or solid-phase GP2 molecule and nonspecific Adsorber molecules are used, selected from the group comprising protein A, protein G, anti-human immunoglobulins and / or L-tryptophan. Use of the molecule GP2 for immunization of mammals for obtaining poly, monoclonal and / or anti-idiotypic autoantibodies. Use according to one of the preceding claims, thereby marked that the molecule GP2 selected is from the group comprising a) having a molecule an amino acid sequence that has sufficient homology to the molecule GP2, to this function analog to be, b) a molecule according to a), which by deletion, Addition, substitution, translocation, inversion and / or insertions modified and added to the molecule according to a) is functionally analogous. Use according to one of the preceding claims, characterized in that the molecule indicated under b) at least 40% homologous to the molecule indicated under a) is. Use according to the preceding claim, characterized in that the molecule indicated under b) is at least 60%, preferably 70%, preferably 80%, most preferably 90% homologous to the molecule indicated under a). Use according to one of the preceding claims, characterized in that the molecule GP2 is linear or in cyclic form, with peptide cyclization occurring in the presence of two cysteines by disulfide bond formation, or by amide cyclization, optionally via the side chains, via the terminal C and N, or a combination of these. Use according to one of the preceding claims, characterized in that the solid phase bound molecule GP2 to organic, inorganic, synthetic and / or mixed polymers is bound, preferably agarose, cellulose, silica gel, polyamides and / or polyvinyl alcohols. Pharmaceutical composition comprising at least a molecule GP2 optionally together with a pharmaceutical acceptable carrier. Pharmaceutical agent after the previous one Claim, characterized in that the pharmaceutical carrier is selected from the group comprising fillers, Disintegrants, binders, humectants, extenders, dissolution retardants, Absorption accelerator, wetting agents, absorbents and / or Lubricant. Use of a specific ligand for human immunoglobulins for the production of a ligand coupled to this ligand Column for the treatment of inflammatory bowel disease, this treatment involves passing a patient's plasma over includes the pillar, whereby conditions are chosen which is an effective binding of the specific ligand to the immunoglobulins allow in the patient's plasma, causing a significant amount the immunoglobulins are removed from the patient's plasma and returned the plasma thus obtained to the patient becomes. Method of treating inflammatory Intestinal diseases comprising the following steps: a) Provision a column to which specific ligands for human immunoglobulins are coupled, b) passing plasma the patient over the pillar under conditions, which is an effective binding of the specific ligand to the immunoglobulins allow in the patient's plasma, causing a significant amount the immunoglobulins are removed from the patient's plasma and c) recycling of the plasma thus obtained in the patient. Method of treating inflammatory Intestinal diseases according to the preceding claim, wherein the specific Ligand is selected from the group consisting of polyclonal anti-human immunoglobulin antibodies, monoclonal anti-human Immunoglobulin antibodies, fragments of these antibodies, recombinant molecules of the antibody idiotypes, synthesized peptides, protein A and / or protein B. Method of treating inflammatory Bowel disease according to claim 25, wherein the specific ligand recognizes autoantibodies directed against intestinal tissue. Method of treating inflammatory Intestinal diseases according to the preceding claim, wherein the specific Ligand is an antigen-imitating molecule selected from the group consisting of polyclonal and monoclonal anti-idiotypic Antibodies, fragments of these antibodies and / or synthesized peptides. Method of treating inflammatory Intestinal diseases according to the preceding claim, wherein the specific Ligand is a synthesized peptide which has a sequence of a Structure mimicked by GP2. Diagnostic test kit for the determination of autoimmune diseases optionally comprising the molecule GP2 with one instruction for combining the contents of the test kit and / or for providing a Formulation. Apparatus for chromatography, in particular for Apharese, comprising the molecule GP2. Device according to the preceding claim, characterized characterized in that the molecule GP2 to a solid phase is bound. Method of treating an autoimmune disease by the binding and / or removal of autoantibodies by means of molecules bound to a solid phase GP2. Test kit for the determination of autoimmune antibodies, in particular for the detection of inflammatory bowel disease, preferably Crohn's disease, chronic pancreatitis and / or colitis Colitis. Immunogenic agent, characterized in that it contains at least one molecule GP2.