EP0998492A1 - Composition et procedes de traitement d'affections inflammatoires - Google Patents

Composition et procedes de traitement d'affections inflammatoires

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Publication number
EP0998492A1
EP0998492A1 EP98928776A EP98928776A EP0998492A1 EP 0998492 A1 EP0998492 A1 EP 0998492A1 EP 98928776 A EP98928776 A EP 98928776A EP 98928776 A EP98928776 A EP 98928776A EP 0998492 A1 EP0998492 A1 EP 0998492A1
Authority
EP
European Patent Office
Prior art keywords
yopj
treatment
protein
microorganism
inflammatory diseases
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98928776A
Other languages
German (de)
English (en)
Inventor
Sven Centrum för Genomforskning PETTERSSON
Hans Wolf-Watz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Index Pharmaceuticals AB
Original Assignee
Karolinska Innovations AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Karolinska Innovations AB filed Critical Karolinska Innovations AB
Publication of EP0998492A1 publication Critical patent/EP0998492A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention concerns the treatment of inflammatory conditions in mammals and specifically an oral microbiological composition for the treatment of inflammatory diseases in the gastrointestinal tract.
  • Yops proteins that are present in several animal and plant pathogens. Up until now, the activity of only one of these cytoplasmically delivered Yops has been determined.
  • YopH which contains an eukaryotic-like phosphatase domain, likely dephosphorylates phosphotyrosine proteins involved in phagocytosis (Persson et al., 1997).
  • Yop proteins are presumed to interfere with cellular processes that normally occur in eukaryotic cells upon contact with bacteria.
  • Yersinia pestis and Y. pseudotuberculosis are able to colonize visceral organs and submucosal intestinal epithelial tissue, respectively, without eliciting infiltrating leukocytes to the site of infection (Une et al., 1986; Simonet et al., 1990).
  • this ability is dependent on the presence of the virulence plasmide and has been proposed to be due to the ability of Yersinia spp.
  • the treatment of inflammatory diseases is a source of problems and discomfort for patients and physicians alike.
  • the presently used pharmaceuticals, cortisone and derivatives thereof, such as the glycocorticoides, have severe drawbacks.
  • the systemic administration of steroids can cause changes in the metabolism, such as disorders of the fluid balance, which can lead to pulmonary oedema. Additionally, part of the patients develops resistance or fail to respond to the treatment.
  • the administration of salicylates another group of compounds used in the treatment of inflammatory diseases, is associated with similar problems. Part of the patients fail to respond to the treatment and others develop a hypersensitivity.
  • the salicylates themselves can also function as irritants of the gastric mucosa. Consequently, the objective of the present invention is to supply a new pharmaceutical composition and method for the treatment of inflammatory diseases, said composition and method being specific, effective and less harmful than the presently known approaches.
  • YopJ affects an eukaryotic signaling pathway involved in the early host immune response.
  • the gene encoding YopJ of Yersinia pseudo-tuberculosis has shown necessary and sufficient to suppress the expression of interleukin (IL)-8 in infected eukaryotic cells.
  • the present invention discloses a new composition and method for the treatment of inflammatory diseases and in particular inflammatory diseases in the gastro-intestinal tract. Further, the invention comprises an oral composition and a method for colonizing the intestinal tract with suitable microorganisms expressing YopJ.
  • the invention specifically discloses the incorporation of an organism expressing YopJ in an oral pharmaceutical composition, such as tablets or suspensions, or in a suitable food stuff, a so-called functional food.
  • Figure 1 shows the message levels of various cytokines in HeLa cells infected with different strains of Y. pseudotuberculosis.
  • Total RNA was isolated from either uninfected or infected cell cultures and analyzed by RT-PCR.
  • Figure 2 shows the NF- ⁇ B site-binding activity and NF- ⁇ B mRNA and protein levels in HeLa cell extracts following infection with various strains of Y. pseudotuberculosis.
  • YopJ the gene encoding YopJ of the enteropathogen Yersinia pseudotuberculosis is necessary and sufficient to downregulate DNA binding activity NF- ⁇ B transcription factors following infection.
  • the inventors have also shown that YopJ affects the levels of mRNAs encoding chemoattractant cytokines, whose inducibility have previously been shown to be dependent on NF- ⁇ B. Promoter activity of a NF- ⁇ B site-containing reporter gene is downregulated in eukaryotic cells infected with wild type Y.
  • YopJ which has been shown to be delivered into the cytoplasm of infected cells by a type III secretion system, inhibits expression of chemoattractant cytokines primarily through the NF- ⁇ B signaling pathway and that this activity accounts for the lack of an inflammatory host response observed in experimental yersiniosis.
  • HeLa cells infected with the yopJ mutant released significantly more IL-8 compared to cells infected with the isogenic wildtype strain (118 ng/ml and 18 ng/ml, respectively).
  • HeLa cells infected with the yopJ mutant containing a yopJ-encoding plasmide had IL-8 levels (20 ng/ml) similar to that observed in wildtype-infected cells.
  • NF- ⁇ B-mediated signaling either by targeted disruption of NF- ⁇ B-encoding genes (Sha et al., 1995) or by exposure to glucocorticoids (Scheinman et al., 1995) or NF- ⁇ B-specif ⁇ c antigens oligonucleotides (Neurath et al., 1996), result in a decreased inflammatory response.
  • YopJ apparently is able to block the various signals that feed into the NF- ⁇ B-mediated signaling pathway that are activated during a bacterial infection. In the absence of YopJ, it may be that the activities of the other cytoplasmically-delivered Yops activate NF- ⁇ B.
  • yopJ shares some degree of sequence similarity to genes found in two species of bacteria, one a plant pathogen and the other a plant symbioant (Whalen et al., 1993; Freiberg et al., 1997).
  • the nitrogen-fixing Rhizobium which from sequence analysis may contain a type in secretion system, possess an ORF predicted to encode a protein 43% similar to YopJ (Freiberg et al, 1997) suggesting that a plant symbioant and an animal pathogen may perform similar functions during their interactions with eukaryotic cells.
  • inflammatory diseases is, in this context, meant to include inflammatory diseases in general and inflammatory diseases in the gastrointestinal tract in particular.
  • the former comprises e.g. rheumatoid arthritis, localized neuritis and sepsis.
  • the latter comprises Crohn's disease (regional ileitis), colitis, ulcerative colitis and proctitis.
  • Further examples of gastrointestinal diseases with an inflammatory etiology or symptoms are e.g. celiac disease and colon irritabile.
  • the YopJ protein can, according to the present invention, be administered to the site of inflammation in many ways.
  • the mode of administration is governed by the nature of the inflammatory disease, its location in the mammal to be treated and by other factors, apparent to the treating physician.
  • die YopJ protein can be administered as such, incorporated in suitable carriers, generally approved for pharmaceutical use, such as liquids, gels and solid carriers.
  • suitable carriers include sterile water for injection or saline, oils and fats, oil-in- water emulsions and conventional tabletting aids.
  • the YopJ protein can be expressed by a suitable microorganism, colonizing or merely passing the site of inflammation.
  • a non-pathogenic microorganism expressing YopJ is administered to the site of inflammation.
  • This non- pathogenic microorganism can be an existing microorganism or a transgenic organism, developed using conventional technology of genetic engineering. An organism of choice is naturally one listed as GRAS (Generally Regarded As Safe). It is specially preferred that the yopJ gene is cloned into a microorganism, presently accepted for use in foodstuffs.
  • said non-pathogenic microorganism expressing YopJ is administered orally to a patient, in a composition so formulated, that it retains its viability in the passage through the ventriculus and intestines and accordingly can express and release YopJ protein in the gastrointestinal tract.
  • Said microorganism or culture of microorganisms can be incorporated in a pharmaceutical composition as such or after freezing, drying, lyophilization, or similar techniques, making the composition more easy to store awaiting use. Suitable techniques also include microencapsulation, following which the encapsulated microorganism can be incorporated in optional food stuff or pharmaceutical composition.
  • the yopJ gene is cloned into a microorganism, filling the GRAS-rements, the resulting transgenic micro-organism can be incorporated in or used for the production of a foodstuff without the destruction of the functionality of the yopJ gene or the YopJ protein, encoded by said gene.
  • suitable foodstuffs include, but are not limited to, fermented foodstuffs such as fermented dairy products, e.g. yogurt, sour milk, kefir and similar products, and fermented cereal products, such as fermented oatmeal gruel and similar products.
  • suitable host microorganisms include the Lactobacillus species, e.g. L. plantarum, L. lactis, L. cremoris, L. diacetylactis, L. bulgaricus, L. acidophilus, Leuconostoc cremoris, Streptococcus thermophilus, Candida kefir, the Saccaromyces species and related organisms.
  • other microorganisms can be used.
  • the chosen microorganism lacks the property of externalizing the YopJ protein, this property should be incorporated in the microorganism during the genetic engineering of the same.
  • the present invention further comprises a method of treating inflammatory diseases, in particular inflammatory diseases in the gastrointestinal tract in humans, characterized in that the intestinal tract is more or less colonized with microorganisms expressing a YopJ-type protein.
  • colonization in this context is meant that the microorganism passes through the gastrointestinal tract with unimpaired viability and with its YopJ expressing function intact, and that this passage is slow enough for the expressed YopJ to have die desired effect on the site of inflammation.
  • the colonization is achieved by administering to the patient a viable culture of microorganisms at intervals and in amounts, sufficient to ensure a fairly stabile level of YopJ at the site of inflammation. The exact dosage as to amounts and intervals can be determined by the treating physician.
  • the transgenic microorganism according to the invention is incorporated in a so-called functional foodstuff, a foodstuff exhibiting therapeutic properties to a certain degree, without being classified as a drug.
  • a so-called functional foodstuff a foodstuff exhibiting therapeutic properties to a certain degree, without being classified as a drug.
  • An example can be a fermented cereal product, e.g. on oatmeal basis, comprising a viable culture of microorganisms expressing YopJ.
  • Such a product could ttien be ingested by patients suffering from inflammatory diseases in the gastrointestinal tract.
  • the microorganism would then colonize the intestines of said patients and the YopJ protein expressed in me intestinal lumen could help to alleviate the symptoms of, or possibly cure the inflammatory disease without the systemic effects and side effects of presently used pharmaceutical regimens.
  • a viable culture of microorganisms can also be incorporated in tablets, suspensions and other forms, suitable for oral ingestion.
  • Microencapsulated microorganisms could, e.g. be provided in a dry, particulate composition or powder to be mixed with a suitable liquid before ingestion.
  • said powder further includes conventional additives, such as compounds increasing the storage time, anti caking agents, coloring and flavoring agents etc.
  • the present invention can naturally also find application in the local treatment of inflammatory diseases outside the intestinal tract.
  • a pharmaceutical composition comprising a YopJ protein can be applied locally, for example topically to inflammated areas.
  • a suitable vehicle is e.g. an oil-in- water emulsion or gel.
  • a protein of YopJ type can find utility in the systemic treatment of serious inflammatory diseases, such as sepsis.
  • a composition according to the present invention can also find utility in the treatment of other diseases that are accompanied by increased local cytokine expression levels, regardless of their cause, i.e. bacterial infections and macrophage triggered reactions.
  • Different bacteria is often very opportune to colonize inflammated sites, in particular ulcers in the gastrointestinal tract and skin lesions associated with psoriasis, mentioning only two, non-limiting examples.
  • the administration of a protein of YopJ type through an expression system, i.e. a GRAS classified organism expressing the desired protein will have the added positive effect of balancing or competing with and potentially eradicate the pathogen or unwanted organisms.
  • YopJ protein can inhibit phosphorylation of the kinase p38. This inhibition is highly specific. It is therefore suggested by the present inventors to use pure YopJ and/or coupled to a delivery substance or cassette, such as a DNA expression cassette or adeno virus vector, as a probe for discrimination or identification of different signal transduction pathways. YopJ would thus function as a specific instrument for mapping signal transduction pathways. Examples
  • Example 1 Mutant strain of Y. pseudotuberculosis containing deletions in vopM. vopE. vopK. vpkA. and vopj
  • YopJ affects IL-8 levels in the absence of the other known Y. Pseudotuberculosis ' Yop virulence genes
  • the present inventors utilized a mutant strain of Y. pseudotuberculosis containing deletions in yopM, yopE, yopK, ypkA, and yopJ, designated as MYM (Hakansson et al., 1996a).
  • the present inventors observed a more than four-fold decrease in the amount of IL-8 secreted by HeLa cells infected with the MYM strain containing a YopJ-expressing plasmide compared to HeLa cells infected with the parental MYM strain (34 ng/ml and 9 ng/ml, respectively).
  • HeLa cells infected with a MYM strain containing a plasmide encoding YopM (Leung et al, 1990) secreted levels of IL-8 (30 ng/ml) that were similar to the parental MYM strain.
  • the present inventors cannot account for the differences in the levels of IL-8 secreted between HeLa cells infected with the yopJ strain and the MEKA strain.
  • IL-8 mRNA levels were also observed in cells infected with the yopB strain (lane 5).
  • the yopB gene product has been shown to be required for d e delivery of Yop virulence factors into infected eukaryotic cells (Hakansson et al., 1996b).
  • Two other cytokines that have been reported to be chemoattractants for neutrophils, IL-1 (and MGSA (Dinarello et al., 1991; Richmond et al., 1985) had similar patterns of expression as IL-8 in HeLa cells infected widi the various Y. pseudotuberculosis strains (fig. lc and Id).
  • YopJ affected IL-8 promoter activity
  • HeLa cells were transiently transfected widi a reporter gene under the control of the IL-8 promoter and then infected with the various bacterial strains.
  • HeLa cells infected with wildtype Y. pseudotuberculosis had lower levels of luciferase activity compared to me uninfected control cells (table I).
  • HeLa cells infected wid the yopJ strain had higher levels of luciferase activity than cells infected wid the wildtype strain.
  • the YopJ-mediated effect on IL-8 promoter activity mimics the pattern observed in IL-8 secretion and mRNA levels and suggest mat YopJ affects IL-8 expression at the transcriptional level.
  • the IL-8 promoter region contains a number of binding sites specific for various families of transcription factors (Mukaida et al., 1989). Inducibility of IL-8 promoter activity, as well as the promoters of IL-1 (and MGSA, have been shown to be dependent on their respective NF- B binding site (Kunsch and Rosen, 1993; Matsusaka et al., 1993; IL-1 and MGSA refs). To determine whemer YopJ modulates me activity of NF- ⁇ B transcription factors, HeLa cells were transiently transfected with a reporter gene under die control of the TK promoter containing two NF- ⁇ B binding sites and were infected widi various strains of Y. pseudotuberculosis.
  • NF- ⁇ B site-binding transcription factors are retained in d e cytoplasm of uninduced cells by I ⁇ B proteins.
  • I ⁇ B is phosphorylated and degraded thereby unmasking NF- ⁇ B's nuclear localization signals and allowing NF- ⁇ B to translocate to the nucleus (Baeuerle and Baltimore, 1988).
  • the present inventors cotransfected the NF- ⁇ B-TKp-luciferase plasmide widi a plasmide encoding eid er a wildtype or a dominant-negative mutant (i.e.
  • NF- ⁇ B site-binding transcription factors are activated by various stress-related stimuli including exposure to bacterial padiogens such as Listeria monocytogenes and Shigella flexneri (Hauf et al, 1994; Dyer et al., 1993).
  • NF- ⁇ B site-binding activity was measured in nuclear extracts prepared from HeLa cells infected wid various Y. pseudotuberculosis strains in order to determine whether YopJ affects the activation of NF-
  • SUBST ⁇ UTE SHEET (RULE 26) KB site transcription factors.
  • pseudotuberculosis were similar to uninfected controls in contrast to the several-fold increase in binding activity observed in extracts prepared from cells infected wid the YopJ- strain. Wildtype-like binding activity could be restored to extracts prepared from cells infected with the trans-complemented YopJ- strain (compare lanes 3 and 6). Differences in NF- ⁇ B site-binding activity between extracts was not due to differences in levels of invasion among the various Yersinia strains since an identical pattern of binding activity was observed in the presence of cytochalasin D (not shown). A similar pattern of NF- ⁇ B site- binding activity was observed in die B cell line K46 infected widi either YopJ+ or YopJ- strains of Y.
  • the activity of the resulting fusion protein is activated by calmodulin, an intracellular eukaryotic protein.
  • calmodulin an intracellular eukaryotic protein.
  • the subcellular location of YopJ-Cya was compared to YopE-Cya and YopB-Cya; me former has been shown to be both exported from the bacterial cell and delivered into d e cytoplasm of infected eukaryotic cells (Sory et al., 1995) while YopB-Cya has been shown to be exported from die bacterial cell but not delivered into eukaryotic cells (Hikansson et al., 1996b).
  • YopE has been shown to cause a disruption of actin microfilaments resulting in an inability of die infected cell to undergo the cytoskeletal rearrangements necessary for phagocytosis (Rosqvist et al., 1991).
  • To determine whether the reporter protein was exported from the bacterial cell intact cells of Y. pseudotuberculosis containing the YopJ-Cya-encoding plasmide were assayed for calmodulin-dependent cyclase activity (Schesser et al, 1996). Low but significant levels of cyclase activity were detected in yopJ-cya-containing Y.
  • YopJ-Cya is similar to YopE-Cya in terms of its delivery into the cytoplasm of eukaryotic cells during infection.
  • Cells were transfected wid 26 (g of eitiier IL-8p-luc or NF- ⁇ B-TKp-luc with eittier none or
  • YopB protein of Yersinia pseudotuberculosis is essential for the translocation of Yop effector proteins across die target cell plasma membrane and displays a contact-dependent membrane-disrupting activity.
  • Characterization of a functional NF-kappa B site in the human interleukin 1 beta promoter evidence for a positive autoregulatory loop.
  • YopM inhibits platelet aggregation and is necessary for virulence of Yersinia pestis in mice. Infect. Immun. 58:3262-3271.
  • Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses.
  • HMGI(Y) and Spl in addition to NF-kappa B regulate transcription of the MGSA/GRO alpha gene.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pain & Pain Management (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Rheumatology (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'expression d'interleukines peut être régulée à la baisse par administration d'une protéine YopJ sur le site d'inflammation. La présente invention concerne des compositions pharmaceutiques et des procédés de traitement d'une affection inflammatoire chez les mamifères, notamment des affections inflammatoires de la région gastro-instestinale chez l'homme.
EP98928776A 1997-06-09 1998-06-09 Composition et procedes de traitement d'affections inflammatoires Withdrawn EP0998492A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE9702191 1997-06-09
SE9702191A SE9702191D0 (sv) 1997-06-09 1997-06-09 Coposition and methods for the treatment of inflammatory dideases
PCT/SE1998/001106 WO1998056817A1 (fr) 1997-06-09 1998-06-09 Composition et procedes de traitement d'affections inflammatoires

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EP0998492A1 true EP0998492A1 (fr) 2000-05-10

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EP (1) EP0998492A1 (fr)
AU (1) AU8049098A (fr)
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WO (1) WO1998056817A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0112687D0 (en) * 2001-05-24 2001-07-18 Microbiological Res Authority Pharmaceutical use of secreted bacterial effector proteins
CA2513584A1 (fr) * 2003-01-20 2004-08-05 Vib Vzw Utilisation de proteines yop ou d'inhibiteurs rho gtpase comme inhibiteurs de caspase-1
AU2006233929B2 (en) * 2005-04-11 2012-05-31 Yeda Research And Development Co.Ltd. Chimeric proteins comprising Yersinia Yop, their preparation and pharmaceutical compositions containing them
US8703152B2 (en) * 2008-06-03 2014-04-22 University Of Rochester Methods of treating inflammatory intestinal disease and managing symptoms thereof

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FI903414A (fi) * 1990-07-06 1992-01-07 Kansanterveyslaitos Produktion av proteiner i grampositiva bakterier.

Non-Patent Citations (1)

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See references of WO9856817A1 *

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WO1998056817A1 (fr) 1998-12-17
SE9702191D0 (sv) 1997-06-09
AU8049098A (en) 1998-12-30

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