EP1347776A2 - Gebrauch von polykationen als entzündungshemmende mittel - Google Patents

Gebrauch von polykationen als entzündungshemmende mittel

Info

Publication number
EP1347776A2
EP1347776A2 EP02702238A EP02702238A EP1347776A2 EP 1347776 A2 EP1347776 A2 EP 1347776A2 EP 02702238 A EP02702238 A EP 02702238A EP 02702238 A EP02702238 A EP 02702238A EP 1347776 A2 EP1347776 A2 EP 1347776A2
Authority
EP
European Patent Office
Prior art keywords
cpg
poly
arginine
odn
medicament
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Withdrawn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
EP02702238A
Other languages
English (en)
French (fr)
Inventor
Karen Lingnau
Alena Egyed
Walter Schmidt
Michael Buschle
Sonja Grill
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.)
Valneva Austria GmbH
Original Assignee
CISTEM Biotechnologies GmbH
Intercell Biomedizinische Forschungs und Entwicklungs AG
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
Priority claimed from PCT/EP2001/000087 external-priority patent/WO2001054720A1/en
Application filed by CISTEM Biotechnologies GmbH, Intercell Biomedizinische Forschungs und Entwicklungs AG filed Critical CISTEM Biotechnologies GmbH
Priority to EP02702238A priority Critical patent/EP1347776A2/de
Publication of EP1347776A2 publication Critical patent/EP1347776A2/de
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention relates to new uses for polycationic compounds.
  • polycationic compounds for example the polycationic amino acid polymers poly-L-arginine and poly-L-ly- sine, have been shown to allow very efficient charging of antigen presenting cells (APCs) with antigens in vitro and in vivo. This is thought to be the key event for triggering immune cascades, eventually leading to the induction of antigen specific immune effector cells that are able to destroy or neutralise targets. It has been shown previously that a number of polycationic compounds excert effects on immune cells (Buschle et al . , Gene Ther.Mol.Biol. 1 (1998), 309-321; Buschle et al . , Proc.Natl.Acad.Sci. USA, 94 (1997), 3256-3261).
  • Co-injection of a mixture of poly-L-arginine and poly-L-lysine together with an appropriate antigen as a vaccine protect animals from tumor growth in several animal models.
  • a vaccine consisting of polycationic compounds and antigens is accepted in the art as being a very effective form of treatment (WO 97/30721) .
  • polycationic compounds may be used to prevent inflammatory effects in an individual having or being at risk of inflammation or, alterna- tively, to reduce the inflammatory potential of a medicament or a medical treatment significantly which allows the administration of medicaments that are usually not administered or only rarely administered due to their inflammatory side-effects.
  • This anti-inflammatory effect of polycationic compounds used according to the present invention may be observed both locally and systemically.
  • the present invention is especially beneficial if the combined medicament is administered, e.g. subcutaneously, intravenously, intranasally, intramusculary, intradermally or transdermally.
  • other application forms such as parenteral or topical application, are also suitable for the present invention.
  • the depot effect seems to be mostly significant if the composition is injected or implanted.
  • the antigen to be used within the course of the present invention is not critical, it may preferably be selected from the group consisting of an antigen from a viral or a bacterial pathogen, an antigen from an eucaryotic pathogen, a tumor antigen, an autoimmune antigen or mixtures thereof. Especially preferred are negatively charged antigens or hydrophobic antigens . Further examples of antigens are whole-killed organisms, such as inactivated viruses or bacteria, fungi, protozoa or even cancer cells. Antigens may also consist of subfractions of these organisms/tissues, of proteins, or, in their most simple form, of peptides.
  • Antigens can also be recognised by the immune system in form of glycosy- lated proteins or peptides and may also be or contain polysaccha- rides or lipids.
  • Short peptides can be used, since e.g. cytotoxic T cells (CTL). recognise antigens in form of short usually 8-11 a ino acids long peptides in conjunction with major histocompati- bility complex (MHC) .
  • MHC major histocompati- bility complex
  • B cells recognise longer peptides starting at around 15 amino acids. By contrast to T cell epitopes, the three dimensional structure of B cell antigens may also be important for recognition by antibodies .
  • Preferred pathogens are selected from human immune deficiency virus (HIV) , hepatitis A and B viruses, hepatitis C virus (HCV) , Rous sarcoma virus (RSV) , Epstein Barr virus (EBV) , Influenza vi- rus, Rotavirus, Staphylococcus aureus, Chlamydia pneumoniae, Chlamydia trachomatis, Mycobacterium tuberculosis, Streptococcus pneumoniae, Bacillus anthracis, Vibrio cholerae, Plasmodium sp.
  • HCV human immune deficiency virus
  • HCV hepatitis A and B viruses
  • HCV hepatitis C virus
  • RSV Rous sarcoma virus
  • EBV Epstein Barr virus
  • Influenza vi- rus Influenza vi- rus
  • Rotavirus Staphylococcus aureus
  • Chlamydia pneumoniae Chlamydia
  • Antigens may also be molecules expressed by cancer cells
  • Antigens may also be derived antigens.
  • the derivation process may include the purification of a specific protein from the pathpgen/cancer cells, the inactivation of the pathogen as well as the proteolytic or chemical derivatisation or stabilisation of such a protein.
  • tumor antigens cancer vaccines
  • autoimmune antigens may be used together with a polycationic compound according to the present invention.
  • the polycationic compound (s) to be used according to the present invention may be any polycationic compound, which shows e.g. the characteristic effect according to the WO 97/30721, or others like cationic liposomes, polyethylene-a ine, chitosan, poly cations used for DNA transfer, etc..
  • Preferred polycationic compounds are selected from basic polypeptides, organic polycations, basic polyaminoacids or mixtures thereof. These polyaminoacids should have a chain length of at least 4 amino acid residues (see: Tuft- sin as described in Goldman et al (1983)) .
  • substances containing peptidic bounds like polylysine, polyarginine and polypeptides containing more than 20%, especially more than 50% of basic amino acid residues in a range of more than 8, especially more than 20, amino acid residues or mixtures thereof .
  • Other preferred polycations and their pharmaceutical compositons are described in WO 97/30721 (e.g. polyethyleneimine) and WO 99/38528.
  • these polypeptides contain between 5 and 500 amino acid residues, especially between 10 and 200 residues.
  • polycationic compounds may be produced chemically or reco - binantly or may be derived from natural sources .
  • Cationic (poly) peptides may also be polycationic anti-bacterial microbial peptides with properties as reviewed in (Ganz and Le- hrer, 1999; Hancock, 1999). These (poly)peptides may be of pro- karyotic or animal or plant origin or may be produced chemically or recombinantly (Andreu and Rivas , 1998; Ganz and Lehrer, 1999; Simmaco et al . , 1998) . Peptides may also belong to the class of defensins (Ganz, 1999; Ganz and Lehrer, 1999).
  • Such host defense peptides or defensines are also a preferred form of the polycationic polymer according to the present invention.
  • a compound 'allowing for activation (or down- regulation) of the adaptive immune system, preferably mediated by APCs (including dendritic cells) is used as polycationic polymer.
  • cathelicidin derived anti- icrobial peptides or derivatives thereof are especially preferred for use as polycationic substance in the present invention.
  • anti-microbial peptides derived from mammal cathelicidin preferably from human, bovine or mouse.
  • Polycationic compounds derived from natural sources include HIV- REV or HIV-TAT (derived cationic peptides, antennapedia peptides, chitosan or other derivatives of chitin) or other peptides derived from these peptides or proteins by biochemical or recombi- nant production.
  • Other preferred polycationic compounds are cathelin or related or derived substances from cathelin.
  • mouse cathelin is a peptide which has the amino acid sequence NH2-RLAGLLRKGGEKIGEKLKKIGOKIKNFFQKLVPQPE-COOH.
  • Related or derived cathelin substances contain the whole or parts of the cathelin sequence with at least 15-20 amino acid residues.
  • Derivations may include the substitution or modification of the natural amino acids by amino acids which are not among the 20 standard amino acids. Moreover, further cationic residues may be introduced into such cathelin molecules . These cathelin molecules are preferred to be combined with the antigen and the immunogenic ODNs according to the present invention. However, these cathelin molecules surprisingly have turned, out to be also effective as an adjuvant for an antigen without the addition of further adjuvants. It is therefore possible to use such cathelin molecules as efficient adjuvants in vaccine formulations with or without fur- ther immunostimulatory substances.
  • CpG-ODNs promotes the induction of a Thl-type response and the development of cytotoxic T cells.
  • immu- nogenic ODNs are used as vaccine adjuvants to enhance the antibody response to specific antigens (e.g. EP 0 468 520 A2 , WO 96/02555, WO 98/16247, etc.).
  • specific antigens e.g. EP 0 468 520 A2 , WO 96/02555, WO 98/16247, etc.
  • these CpG-ODNs exhibit strong inflammatory potential, an administration of CpG-ODNs is connected with severe local and systemic inflammatory events.
  • CpG-ODNs show local as well as systemic inflammatory reactions, but at the same time have also a potential to be used as a beneficial stimulatory medicament, this substance was applied to an animal to create and provide a model to show the advantages of the present invention. Indeed, it could be shown that both, the local and the systemic inflammatory events caused by administration of CpG-ODNs may be completely inhibited by administration of polycationic compounds .
  • a preferred embodiment of the present invention is characterised in that the medicament is to be applied together with the polycationic compound further comprises immunogenic oli- godesoxy nucleic acid molecules (ODNs) , especially ODNs containing CpG motifs (CpG-ODNs) , inosine containing ODNs (I-ODNs) or mixtures or combinations thereof.
  • ODNs immunogenic oli- godesoxy nucleic acid molecules
  • CpG-ODNs ODNs containing CpG motifs
  • I-ODNs inosine containing ODNs
  • this strong local immune response (day 4/draining lymph node cells) induced after one single injection of an anti ⁇ gen with a mixture of polycationic compounds (as an example polyarginine pR 60 is used) and CpG-ODNs converts to a systemic immune response which is very long lasting.
  • the complex formation ability of substances such as CpG-ODNs with polycationic compounds is used for preventing a systemic distribution and the subsequent fast resorption of such substances, thereby providing a strong prolongation of the properties of such substances, e.g. a prolongation of the immu- nostimulatory properties of CpG-ODNs.
  • preventing the systemic distribution avoids the induction of potential harmful systemic side effects of immunostimulatory agents.
  • This model using CpG-ODNs and polycationic peptides is f ⁇ rther described and analysed in the example section.
  • an Ovalbumin-derived peptide OVA257-26-
  • a model compound a model antigen
  • a second preferred compound with inflammatory potential to be used within the course of the present invention is lipopolysac- charide (LPS, endotoxin) , a cell wall component of Gram-negative bacteria.
  • LPS lipopolysac- charide
  • endotoxin a cell wall component of Gram-negative bacteria.
  • LPS plays a pivotal role in the induction of septic shock (endotoxic shock) (Karima et al (1999) .
  • septic shock endotoxic shock
  • leukocyte-induced tissue injury and activation of coagulation systems appear to be critical determinants in the development of sequential organ failure.
  • lethal shock can be associated with excessive secretion of pro-inflammatory cytokines like TNF-a and IL-6.
  • the present invention also relates to a method for treating or preventing inflammation, comprising administering to an individual (e.g. Patient or animal), suffering from inflammation or being at risk thereto, an effective amount of a polycationic compound.
  • the invention further relates to a method for reducing the inflammatory potential of a medicament when administering said medicament to a patient, comprising administering that medicament together with an effective amount of a polycationic compound.
  • polycationic compound to be administered is highly depending on the necessities of the individual composition and optionally on the drug to be administered together with the polycationic polymer.
  • preferred amounts of polycation are 0.001-1000 ⁇ g/administration unit, more preferred 0,1-10 mg/dose, especially around or beyond 0,1 mg/20g body weight (of mice) or the equivalent dose for humans .
  • Fig. 1 shows that the combined application of poly-L-arginine, CpG-ODN and antigen induces strong antigen-specific immune responses which are systemic and very long lasting.
  • the figure shows peripheral blood lymphocytes stimulated ex vivo with OVA257- 264-peptide;
  • Fig. 2a shows that poly-L-arginine induces the formation of a depot, at the injection site.
  • This figure shows photos from the injection sites at the indicated time points after vaccination.
  • White lines surround the area where the fluorescence labelled compounds of the vaccine can be detected;
  • Fig. 2b shows that the co-application of poly-L-arginine inhibits the spreading of CpG-0DN-Cy5 throughout the body.
  • This figure shows FACS analyses of lymphoid and non-lymphoid tissues at day 1 after injection of CpG-ODN-Cy5 (B) or CpG-ODN-Cy5 and pR 60-FITC (C) . Untreated mice were used as a control (A) ;
  • Fig. 3 shows that poly-L-arginine induces the formation of a depot at the injection site when co-injected at least with one more molecule. This figure shows photos from the injection sites at day 4 after vaccination;
  • Fig. 4 shows that co-injected poly-L-arginine prevents the CpG- ODN-induced systemic production of TNF- ⁇ and IL-6 in vivo.
  • Mice were injected into the hind footpads and one hour later serum was prepared. The amount of TNF- ⁇ and IL-6 in the sera was determined by ELISA;
  • Fig. 5 shows that poly-L-arginine abrogates CpG-ODN-induced production of TNF- ⁇ and IL-6 by mouse BM-DC in vitro.
  • CDllc + sorted BM-DC were incubated either with pR60, CpG-ODN 1668 or pR60 and CpG-ODN 1668 or, for control purposes, with.medium or LPS. After the incubation for 24 h, the amount of TNF- ⁇ and IL-6 were determined in the supernatant by ELISA;
  • Fig. 6 shows that poly-L-arginine abrogates poly I:C-induced production of pro-inflammatory cytokines by human DC in vitro.
  • Day 5-cultured human monocyte-derived DC were incubated (lxlOVwell) either with poly I:C, pR60, poly I:C and pR60 or, for control purposes, with LPS and medium alone in 24-well culture plates for 24 h. Thereafter, supernatants were collected and stored at -2p°C until use. The amount of TNF- ⁇ and IL-6 in the supernatants was determined by ELISA.
  • Fig.7 shows that KLK abrogates CpG-ODN-induced production of TNF- ⁇ and IL-6 by BM-DC in vitro: CD llc + sorted BM-DC were incubated either with KLK, CpG-ODN 1668 or KLK and CpG-ODN 1668 or, for control purposes, with medium or LPS. After the incubation for 24 h, the amount of TNF- ⁇ and IL-6 in the supernatants were determined by ELISA.
  • Fig.8 shows that co-injected poly-L-arginine prevents the BCG/CpG-ODN-induced systemic production of TNF- ⁇ and IL-6 in vivo.
  • Mice were injected s.c. Into the flank with 5xl0 3 cells of BCG and one hour later serum was prepared. The amount of TNF- ⁇ and IL-6 in the sera was determined by ELISA.
  • Fig.9 shows that poly-L-arginine decrease LPS-induced production of pro-inflammatory cytokines by murine bone-marrow-derived dendritic cells in vitro.
  • Fig.10 shows that poly-L-arginine, KLK and poly-D-lysine decrease LPS-induced production of pro-inflammatory cytokines by human dendritic cells in vitro.
  • fluorescence-labeled compounds were injected together subcutaneously into the flank of mice. At different time points after this treatment, injection sites were inspected for the presence of labeled compounds.
  • OVA 25 7-264-peptide unlabeled
  • poly-L-arginine- FITC yellow
  • CpG-ODN-Cy5 blue
  • CpG-ODN-Cy5 is also detectable in secondary lymphoid organs (draining lymph node, spleen) and non-lymphoid tissues (lung, liver, kidney, heart) .
  • TRP-2i8i-i88-peptide-FITC yellow
  • poly-L ⁇ arginine-TRITC red-violet
  • CpG-ODN-Cy5 blue
  • TRP-2 ⁇ s ⁇ - i88-peptide-FlTC was injected either alone or in combination with CpG-ODN-Cy5
  • the peptide was not detectable at the injection site at day 4.
  • CpG-ODNs are described to have potentially harmful side effects in that they induce the systemic release of high amounts of pro-inflammatory cytokines such as TNF- ⁇ and IL-6, which could induce a shock syndrome (Sparwasser 1997, Lipford 1997).
  • cytokines such as TNF- ⁇ and IL-6
  • Sparwasser 1997, Lipford 1997 a shock syndrome
  • CpG-ODNs are not systemically present when injected in combination with poly-L-arginine. Therefore, it was investigated whether the co-administration of poly-L-arginine affects the CpG-ODN-induced systemic production of TNF- ⁇ and IL-6. Serum levels of both cytokines were determined by ELISA one hour after injection.
  • Example 4 demonstrates that neither the injection of OVA 2 57-264-peptide alone nor in combination with poly-L-arginine led to the induction of significant amounts of TNF- ⁇ and IL-6 in the serum, whereas the injection of OVA 2S 7-264-peptide in combination with CpG-ODN induces high concentrations of both cytokines. However, upon co-administration of OVA 2 57-264-peptide with poly-L-arginine and CpG-ODN, this systemic production of TNF- ⁇ and IL-6 was totally abolished.
  • CDllc + dendritic cells were incubated either with poly-L-arginine, CpG-ODN or the combination of poly-L- arginine and CpG-ODN (example 5) .
  • the levels of TNF- ⁇ and IL-6 were determined in the supernatants derived from these cultures.
  • After incubation with poly-L-arginine neither TNF- ⁇ nor IL-6 were detectable, whereas after incubation with CpG-ODN significant amounts of both cytokines are produced.
  • the presence of poly-L-arginine inhibited the CpG-ODN-induced production of TNF- ⁇ and IL-6 by these cells.
  • poly-L-arginine also inhibits the polyinosinic-polycytidylic acid-induced production of pro-inflammatory cytokines by human dendritic cells (Example 6) .
  • Ovalbumin-peptide/poly-L-arginine (pR 60)/ CpG-ODN leads to the induction of strong antigen-specific immune responses which are systemic and very long lasting.
  • Peptide OVA 25 7-264-Peptide (SIINFEKL) , a MHC class I (H-2Kb)- restricted epitope of chicken Ovalbumin (Rotz- schke, 0. et al . , Eur. J. Immunol. 1991 21 (11): 2891-4) , synthesised by standard solid phase F- moc synthesis, HPLC purified and ana lysed by mass spectroscopy for purity. Dose: 300 ⁇ g/mouse
  • mice mice per group.
  • OVA 2 57-264-Pe tide CpG-ODN + pR 60
  • OVA 25 7-264-Peptide CpG-ODN 3 "
  • mice were injected into each hind footpad with a total volume of lOO ⁇ l (50 ⁇ l per footpad) containing the above mentioned compounds .
  • Blood was taken via the tail vein at the indicated time points and peripheral blood lymphocytes (PBLs) were isolated using a Ficoll gradient. PBLs were stimulated ex vivo with the antigen used for vaccination, with medium (background) and Conca- navalin A (positive control) .
  • IFN- ⁇ -ELISPOTs were carried out as described (Miyahira et al . , 1995). This method is a widely used procedure allowing the quantification of antigen-specific T cells. Spots representing single IFN- ⁇ producing T cells were counted and the number of background spots was substracted from all samples. There were many spots detected after the stimulation with Con A (data not shown) indicating a good condition of the used lymphocytes. For each experimental group of mice the number of spots/lxlO 6 PBLs are illustrated in Figure 1.
  • Example 2a Example 2a
  • Poly-L-arginine induces the formation of a depot at the injection site
  • Peptide OVA 25 7-264-Peptide (SIINFEKL) , a MHC class I (H- 2Kb) -restricted epitope of chicken Ovalbumin (Rotzschke, O.et al . , Eur. J. Immunol. 1991 21(11): 2891-4), synthesised by standard solid phase F-moc synthesis, HPLC purified and analysed by mass spectroscopy for purity. Dose: 300 ⁇ g/mouse
  • FITC fluorescein
  • mice On day 0 mice were injected subcutaneously into the right flank with a total volume of lOO ⁇ l containing the above mentioned compounds. Animals were sacrificed at the indicated time points after injection and photos were taken from the injection sites (Fig. 2a) .
  • FITC fluorescein
  • mice were injected subcutaneously into the right flank with a total volume of lOO ⁇ l containing the above mentioned compounds.
  • mice were sacrificed and FACS-analyses were performed from secondary lymphoid organs (draining lymph node, spleen) as well as non-lymphoid tissues (lung, liver, kidney, heart) (Fig. 2b) .
  • Poly-L-arginine induces the formation of a depot at the injection site when co-injected at least with one more molecule
  • TRP-2-Peptide VYDFFVWL
  • H-2Kb MHC class I
  • HPLC HPLC purified and analysed by mass spectroscopy for purity.
  • fluorescein (FITC) labeling the TRP-2 ⁇ 8 ⁇ -i88-peptide was dissolved in 1M sodium borate, pH 7,9. An 8-fold molar excess of FITC (Molecular Probes, Eugene, OR) in an equal volume of DMF was added to the peptide solution.
  • SIGMA chemi- cals For TRITC-labeling ' of poly-L-arginine, the poly-L-arginine was dissolved in 50mM HEPES pH 7,9 (10mg/500 ⁇ l) . A 5-fold molar excess of FITC (Molecular Probes, Eugene, OR) in an equal volume of DMSO was added to the poly-L-arginine solution. The solution was kept at room . temperature in the dark for.2 , 5 hours .
  • mice On day 0 mice were injected subcutaneously into the right flank with a total volume of lOO ⁇ l containing the above mentioned compounds. Animals were sacrificed at day 4 after injection and photos were taken from the injection sites (Fig. 3) .
  • Peptide OVA257-264 (SIINFEKL) , an MHC class I (H-2Kb) -restricted epitope of chicken ovalbumin (Rotzschke et al . , 1991), was synthesised using standard solid phase F-moc synthesis, HPLC-purified and analysed by mass spectroscopy for purity Dose: 300 ⁇ g/mouse
  • CpG-ODN 1668 phosphothioate-modified oligodeoxynucleotides containing a CpG motif TCC ATG ACG TTC CTG ATG CT, synthesised by NAPS GmbH, G ⁇ ttingen. Dose: 5 nmol/mouse
  • mice were injected into each hind footpad with a total volume of lOO ⁇ l (50 ⁇ l per footpad) , containing the above mentioned compounds.
  • lOO ⁇ l 50 ⁇ l per footpad
  • Poly-L-arginine abrogates CpG-ODN-induced production of pro-inflammatory cytokines by murine bone marrow-derived dendritic cells in vitro
  • Murine dendritic cells were generated from bone-marrow precursor cells of C57B1/6 mice as previously described (Inaba 1992) . Briefly, bone marrow cells were obtained by flushing the femurs and tibias with medium. Cells were depleted of lymphocytes, granulocytes and MHC class II + cells by Ab-complement-me- diated lysis. Dead cells were removed by Ficoll gradient (Ficoll Separating Solution, density l,077g/mol; Biochrom KG, Germany).
  • the remaining cells (1 x lOVml) were cultured in 24-well culture plates in high glucose DMEM medium (PAA Laboratories GmbH, Linz, Austria; E 15-009) supplemented with 10% heat-inactivated FCS, 10 mM sodium pyruvate, 2mM L-glutamine, 50mg/ml gentamicin, 0,5mM 2-mercaptoethanol (all supplements from PAA Laboratories GmbH, Linz, Austria), 20ng/ml GM-CSF and 250 U/ml IL-4. On day 3 of culture, cells were fed with fresh medium.
  • CDllc + sorted BM-DC were incubated either with poly-L-arginine (lO ⁇ g/ml) , CpG-ODN 1668 (lnmol/ml) or the combination of poly-L- arginine and CpG-ODN 1668, and, for control purposes, with medium or LPS.
  • Supernatants were harvested after 24h and analysed for the production of TNF- ⁇ and IL-6 using specific ELISAs .
  • CpG-ODN 1668 induces the production of TNF- ⁇ and IL-6 by murine BM-DC ( Figure 5) .
  • murine BM-DC were stimulated with poly-L-arginine alone, we could not detect any of the mentioned pro-inflammatory cytokines in DC-derived supernatants .
  • the CpG-ODN 1668-induced production of TNF- ⁇ and IL-6 was inhibited when BM-DCs were stimulated with a mixture of " CpG- ODN 1668 and poly-L-arginine.
  • Poly-L-arginine abrogates poly I:C-induced production of pro-inflammatory cytokines by human DC.
  • PBLs peripheral blood leukocytes
  • CD14-coated magnetic beads Miltenyi Biotec Inc., Germany
  • Example 7 KLK abrogates Cpg-ODN-induced production of pro-inflammatory cytokines by murine bone marrow-derived dendritic cells in vitro.
  • LPS Lipopolysaccharide
  • Escherichia coli Lipopolysaccharide from Escherichia coli; serotype 0111 :B4 (SIGMA Chemicals)
  • Murine dendritic cells were generated from bone-marrow precursor cells of C57B1/6 mice as described in example 5.
  • CDllc + sorted BM-DC were incubated either with KLK (16,8 ⁇ g/ml), CpG-ODN 1668 (0,5nmol/ml) or the combination of poly-L-arginine and CpG-ODN 1668, and, for control purposes, with medium or LPS.
  • Supernatants were harvested after 24h and analysed for the production of TNF-a and IL-6 using specific ELISAs.
  • CpG-ODN 1668 induces the production of TNF-a and IL-6 by murine BM-DC ( Figure 7) .
  • murine BM-DC were stimulated with KLK alone, we could not detect any of the mentioned pro-inflammatory cytokines in DC-derived supernatants.
  • the CpG-ODN 1668-induced production of TNF-a and IL-6 was inhib- ited when BM-DCs were stimulated with a mixture of CpG-ODN 166S and KLK.
  • Example 8 The co-injection of poly-L-arginine prevents the BCG/CpG-ODN-induced systemic production of TNF-a and IL-6 in vivo.
  • mice C57B1/6 (Harlan/Olac) Vaccine BCG (Bacille Calmette Guerin) - Vaccine "Pasteur Merieux” ; -live vaccine, containing an attenuated strain of Mycobacterium bovis; (Pasteur Merieux Connaught Austria)
  • Poly-L-arginine 60 (pR60) Poly-L-arginine with an average degree of polymerization of 60 arginine residues; SIGMA Chemicals Dose: lOO ⁇ g/mouse
  • CpG-ODN 1668 phosphothioate-modified oligode- oxynucleotides containing a CpG motif TCC ATG ACG TTC CTG ATG CT, synthesized by NAPS GmbH, G ⁇ ttingen. Dose: 5 nmol/mouse
  • mice were injected subcutaneously into the flank with a total volume of lOO ⁇ l, containing the above mentioned compounds.
  • the amount of the pro-inflammatory cytokines TNF-a and IL-6 in the sera was determined by cytokine-specific ELISAs according to the manufacturer's instructions (R&D Systems, Inc., Minneapolis, MN) .
  • This experiment shows that injection of BCG alone or in combination with poly-L-arginine does not induce the production of significant amounts of TNF-a or IL-6 ( Figure 8) .
  • the injection of CpG-ODN 1668 alone or in combination with BCG induces the systemic production of TNF-a and IL-6.
  • the BCG vaccine' and CpG-ODN were co-injected with poly-L-arginine, the CpG-ODN-induced production of pro-inflammatory cytokines was inhibited.
  • Example 9 Poly-L-arginine decrease LPS-induced production of pro-inflammatory cytokines by murine bone-marrow-derived dendritic cells in vitro.
  • LPS Lipopolysaccharide
  • Poly-L-arginine 60 (pR60) Poly-L-arginine with an average degree of polymerization of 60 arginine residues; SIGMA Chemicals,
  • Murine dendritic cells were generated from bone-marrow precursor cells of C57B1/6 mice as described in example 5.
  • the LPS-induced production of TNF-a and IL-6 was inhibited when BM-DCs were stimulated with a mixture of LPS/poly-L- arginine or when the cells were pre-incubated with poly- Larginine .
  • Example 10 Poly-L-arginine, KLK and poly-D-lysine decrease LPS- induced production of pro-inflammatory cytokines by human dendritic cells in vitro.
  • LPS Lipopolysaccharide
  • Poly-L-arginine 60 (pR60] Poly-L-arginine with an average degree of polymerizaTion of 60 arginine residues; SIGMA Chemicals,
EP02702238A 2001-01-05 2002-01-07 Gebrauch von polykationen als entzündungshemmende mittel Withdrawn EP1347776A2 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP02702238A EP1347776A2 (de) 2001-01-05 2002-01-07 Gebrauch von polykationen als entzündungshemmende mittel

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
WOPCT/EP01/00087 2001-01-05
PCT/EP2001/000087 WO2001054720A1 (en) 2000-01-28 2001-01-05 Pharmaceutical composition for immunomodulation and preparation of vaccines comprising an antigen and an immunogenic oligodeoxynucleotide and a polycationic polymer as adjuvants
AT6702001 2001-04-25
AT6702001 2001-04-25
EP02702238A EP1347776A2 (de) 2001-01-05 2002-01-07 Gebrauch von polykationen als entzündungshemmende mittel
PCT/EP2002/000071 WO2002053185A2 (en) 2001-01-05 2002-01-07 Anti-inflammatory use of polycationic compounds

Publications (1)

Publication Number Publication Date
EP1347776A2 true EP1347776A2 (de) 2003-10-01

Family

ID=25608381

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02702238A Withdrawn EP1347776A2 (de) 2001-01-05 2002-01-07 Gebrauch von polykationen als entzündungshemmende mittel

Country Status (3)

Country Link
EP (1) EP1347776A2 (de)
JP (1) JP2004519453A (de)
WO (1) WO2002053185A2 (de)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005533855A (ja) 2002-07-24 2005-11-10 インターツェル・アクチェンゲゼルシャフト 病原性ウイルスからの別のリーディングフレームによりコードされる抗原
EP2402026A3 (de) 2002-09-13 2012-04-18 Intercell AG Verfahren zum Isolieren von Hepatitis-C-Virus-Peptiden
JP2006521321A (ja) * 2003-03-24 2006-09-21 インターツェル・アクチェンゲゼルシャフト 免疫応答を促進するためのミョウバンおよびＴｈ１免疫応答誘起アジュバントの使用
ATE485056T1 (de) * 2003-03-24 2010-11-15 Intercell Ag Verbesserte impfstoffe
CA2617219A1 (en) 2005-07-29 2007-02-01 Suntory Limited Compositions comprising fucoidan or a fucoidan hydrolysate and an immuno-stimulating material
US9468650B2 (en) 2009-09-16 2016-10-18 Duke University Inhibition of endosomal toll-like receptor activation
WO2011101465A1 (en) 2010-02-19 2011-08-25 Intercell Ag Ic31 nanoparticles

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1290141A (de) * 1968-05-31 1972-09-20
JPH09176038A (ja) * 1995-09-18 1997-07-08 Hisamitsu Pharmaceut Co Inc 抗炎症性アンチセンス薬物
CO4600681A1 (es) * 1996-02-24 1998-05-08 Boehringer Ingelheim Pharma Composicion farmaceutica para la modulacion inmunitaria
DE69914911T2 (de) * 1998-06-26 2004-12-09 Naamloze Vennootschap Nutricia Pharmazeutische präparate zur bekämpfung oder prophylaxe von mikroorganismen infizierten oberflächen
JP2000256196A (ja) * 1999-03-05 2000-09-19 Pias Arise Kk 抗炎症，抗アレルギー剤
CZ303515B6 (cs) * 1999-04-19 2012-11-07 Smithkline Beecham Biologicals S. A. Adjuvantní prostredek
JP2001199903A (ja) * 1999-11-09 2001-07-24 Eizo Mori 核酸含有複合体
DE60100814T2 (de) * 2000-06-08 2004-07-01 Intercell Biomedizinische Forschungs- Und Entwicklungs Gmbh Immunstimulierende oligodeoxynukleotide
AT410173B (de) * 2000-06-08 2003-02-25 Cistem Biotechnologies Gmbh Antigene zusammensetzung

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO02053185A2 *

Also Published As

Publication number Publication date
WO2002053185A2 (en) 2002-07-11
JP2004519453A (ja) 2004-07-02
WO2002053185A3 (en) 2002-10-31

Similar Documents

Publication Publication Date Title
US20080025996A1 (en) Methods and Compositions Comprising Polycationic Compounds
AU784403B2 (en) Pharmaceutical composition for immunomodulation and preparation of vaccines comprising an antigen and an immunogenic oligodeoxynucleotide and a polycationic polymer as adjuvants
EP1326634B1 (de) Ein antigen und ein adjuvans-peptid enthaltende impfstoffzusammensetzung
EP1286695B1 (de) Antigene zusammensetzung die ein polykationisches peptid und inosin und cytosin enthält
EP1296713B1 (de) Immunstimulierende oligodeoxynukleotide
AU2002212326A1 (en) Vaccine composition comprising an antigen and a peptide having adjuvant properties
US20040248831A1 (en) Immunostimulatory oligodeoxynucleic molecules
JP2008222721A6 (ja) ワクチン組成物
WO2002053185A2 (en) Anti-inflammatory use of polycationic compounds
EP1347775B1 (de) Gebrauch von polykationischen substanzen als adjuvantien für impfstoffe
US20040081655A1 (en) Methods and compositions comprising polycationic compounds
AU2002235820A1 (en) Anti-inflammatory use of polycationic compounds
AU2002233302A1 (en) Uses for polycationic compounds as vaccine adjuvants

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030723

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: CISTEM BIOTECHNOLOGIES GMBH

Owner name: INTERCELL AG

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: INTERCELL BIOMEDIZINISCHE FORSCHUNGS- UND ENTWICKL

Owner name: INTERCELL AG

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: INTERCELL AG

17Q First examination report despatched

Effective date: 20070312

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20090815