EP1392268A2 - Arzneimittel enthaltend ein polyamin als wirksubstanz - Google Patents

Arzneimittel enthaltend ein polyamin als wirksubstanz

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Publication number
EP1392268A2
EP1392268A2 EP01985905A EP01985905A EP1392268A2 EP 1392268 A2 EP1392268 A2 EP 1392268A2 EP 01985905 A EP01985905 A EP 01985905A EP 01985905 A EP01985905 A EP 01985905A EP 1392268 A2 EP1392268 A2 EP 1392268A2
Authority
EP
European Patent Office
Prior art keywords
polyamine
medicament
dependent
medicament according
polyethyleneimine
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.)
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Application number
EP01985905A
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German (de)
English (en)
French (fr)
Inventor
Tobias Schlapp
Sonja Maria Friederichs
Martin Vollmer
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.)
Covestro Deutschland AG
Original Assignee
Bayer Healthcare AG
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Filing date
Publication date
Application filed by Bayer Healthcare AG filed Critical Bayer Healthcare AG
Publication of EP1392268A2 publication Critical patent/EP1392268A2/de
Withdrawn legal-status Critical Current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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the present invention relates to medicaments containing a polyamine as active substance, and to the use of a polyamine for the production of immunostimulating medicaments or of medicaments for the treatment and / or prophylaxis of various diseases in humans and animals.
  • Immune stimulators exist e.g. B. from chemically inactivated Parapoxvirus ovis, strain D 1701 (DE-A 35 04 940). A product made on the basis of this virus is BAYPAMU ⁇ ® .
  • the inactivated parapoxvirus induces non-specific protection against infections with a wide variety of pathogens in animals. It is believed that this
  • immune stimulators such as unmethylated, CpG-containing oligonucleotides (WO 98/18810) can be used to activate the non-adaptive immune system and to strengthen the organism against the occurrence of pathogens.
  • CpG-containing oligonucleotides WO 98/18810
  • a single dose can activate the initial immune response and prevent infection with various pathogens.
  • the object of the present invention was to provide pharmaceuticals which are new
  • Contain immunostimulators which have a similar efficacy to Parapox ovis, but can be synthesized chemically and are therefore cheaper to produce and easier to combine with chemotherapeutic agents.
  • the task was solved by providing medicinal products that a
  • the polyamines classified as active substance contain at least 10 monomer units or at least 10 nitrogen atoms, preferably at least 45 monomer units or at least 45 nitrogen atoms.
  • the polyamine can have a linear or branched structure.
  • the polyamine is preferably soluble or dispersible in water; in aqueous media, partial protonation depends on the pH value.
  • Degree of protonation can be determined by physico-chemical measurement methods such as zeta potential measurements.
  • the polyamine further preferably has hydrophobic substituents.
  • the hydrophobic substituents can be arranged as side chains or at the end of the polymer.
  • the degree of substitution is preferably between 0.01 and 10 percent.
  • hydrophobic substituents are alkyl chains, acyl chains or steroid-like substituents.
  • Acyl chains are particularly suitable as hydrophobic substituents.
  • hydrophobic substituents which can be introduced by adding the nitrogen functions of the main polymer chain to isocyanates or to ⁇ , ⁇ -unsaturated carbonyl compounds.
  • the polyamine is particularly preferably a polyethyleneimine.
  • a polyethyleneimine which can preferably be used for pharmaceutical production has the following general formula:
  • R represents hydrogen, methyl or ethyl
  • R 2 is alkyl having 1 to 23 carbon atoms, preferably alkyl having 12 to 23 carbon atoms, particularly preferably alkyl having 17 carbon atoms,
  • R 3 and R 4 (end groups) independently of one another are hydrogen and alkyl having 1 to 24 carbon atoms, preferably alkyl having 13 to 24 carbon atoms, particularly preferably alkyl with 18 carbon atoms, or have a structure dependent on the initiator,
  • R 5 is a substituent dependent on the termination reaction, for example hydroxyl, NH 2 , NHR or NR, the radicals R being the end groups
  • R 3 and R 4 can correspond,
  • the units m and n are not block structures, but statistically distributed in the polymer.
  • Another polyethyleneimine which can preferably be used for pharmaceutical production has the following general formula:
  • R 1 is hydrogen, methyl or ethyl
  • R> 2 denotes alkyl having 1 to 22 carbon atoms, preferably alkyl having 11 to 22 carbon atoms, particularly preferably alkyl having 16 carbon atoms, and in what
  • R 3 and R 4 (end groups) independently of one another are hydrogen or acyl with 1 to 24 carbon atoms, preferably acyl with 13 to 24 carbon atoms, particularly preferably acyl with 18 carbon atoms, or have a structure dependent on the initiator,
  • R 5 is a substituent which is dependent on the termination reaction, for example hydroxyl, NH 2 , NHR or NR 2 , where the radicals R can correspond to the end groups R 3 and R 4 ,
  • the units m and n are not block structures, but statistically distributed in the polymer.
  • Another one that can preferably be used for drug production is Another one that can preferably be used for drug production
  • Polyethyleneimine has the following general formula:
  • R, R and R are hydrogen or hydroxy
  • R 4 and R 5 (end groups) independently of one another denote hydrogen or steroid base bodies such as, for example, bile acids, or have a structure which is dependent on the initiator,
  • R 6 (end group) is a substituent dependent on the termination reaction, for example hydroxyl, NH 2 , NHR or NR 2 , where the radicals R can correspond to the end groups R 4 and R 5 ,
  • the substituents R 1 , R 2 and R 3 can be arranged both in the ⁇ and in the ⁇ configuration.
  • the substituent can be in the 5-position in the ⁇ as well as the ⁇ configuration (nomenclature according to Römpp chemistry lexicon, 9th edition, Georg Thieme Verlag, 1992).
  • the units m and n are not block structures, but statistically distributed in the polymer.
  • Another polyethyleneimine which can preferably be used for pharmaceutical production has the following general formula:
  • R 1 means OR 4 or NR 4 R 5 ,
  • R 4 and R 5 independently of one another are hydrogen or alkyl having 1 to 24 carbon atoms, preferably alkyl having 13 to 24 carbon atoms, particularly preferably alkyl having 18 carbon atoms,
  • R 2 and R 3 (end groups) independently of one another correspond to the substituents of the nitrogen atoms of the main polymer chain or have a structure which is dependent on the initiator,
  • R 6 is a substituent dependent on the termination reaction, for example hydroxyl, NH 2 , NHR or NR 2 , the radicals R being the end groups
  • R 2 and R 3 can correspond to
  • the units m and n are not block structures, but statistically distributed in the polymer.
  • Another polyethyleneimine which can preferably be used for pharmaceutical production has the following general formula:
  • R 1 is alkyl with 1 to 24 carbon atoms, preferably alkyl with 13 to 24 carbon atoms, particularly preferably alkyl with 18 carbon atoms,
  • R 4 is a substituent which is dependent on the termination reaction, for example hydroxyl, NH 2 , NHR or NR 2 , where the radicals R can correspond to the end groups R 2 and R 3 ,
  • the units m and n are not block structures, but statistically distributed in the polymer.
  • Another polyethyleneimine which can preferably be used for pharmaceutical production has the following general formula:
  • radical R is either hydrogen or a radical of the formula
  • R x is either hydrogen or again a residue of
  • Type R can be
  • polyethyleneimines have a branched or crosslinked structure.
  • the polymer preferably has an average molecular weight below 220,000 g / mol, particularly preferably a molecular weight between 2000 and 100,000 g / mol, very particularly preferably a molecular weight between 20,000 and 100,000 g / mol.
  • hydrophobic groups are introduced in polymer-analogous reactions, for example by alkylation with haloalkanes, acylation with carboxylic acid chlorides, acylation with reactive esters, Michael addition to ⁇ , ⁇ -unsaturated carbonyl compounds (carboxylic acids, carboxamides, carboxylic acid esters) or by addition to isocyanates. These are reaction types known from the literature (March, 1992).
  • the linear polyethyleneimines are prepared, for example, by cationic ring-opening polymerization of 2-ethyloxazoline with cationic initiators, preferably according to a regulation by B.L. Rivas and S.I. Ananias (1992).
  • the poly (ethyloxazolines) thus obtained are quantitated by treatment with a mixture of concentrated hydrochloric acid and water, preferably a 1: 1
  • the reaction temperature is preferably between 80 and 100 ° C. particularly preferred at 100 ° C.
  • the reaction time is preferably between 12 and 30 hours, particularly preferably 24 hours.
  • the product is preferably purified by repeated recrystallization from ethanol.
  • the linear polyethyleneimines can be produced in the desired molecular weight range from 2000 to 220,000 g / mol.
  • the alkyl groups such as, for example, C18 alkyl groups, are introduced, for example, by reacting a 5% strength solution of the corresponding linear polyethyleneimine in absolute ethanol at a reaction temperature of 40 to 75 ° C. preferably 60 ° C, with octadecyl chloride.
  • the dosing amount of the alkyl chloride is based exactly on the desired degree of substitution (0.1 to 10%).
  • the reaction time is preferably between 10 and 24 hours, particularly preferably 17 hours.
  • acyl groups e.g. C18 acyl groups
  • reaction temperature 40 to 60 ° C, preferably 50 ° C
  • the dosing amount of the acid chloride is based exactly on the desired degree of substitution (0.1 to 10%).
  • the reaction time is preferably between 10 and 24 hours, particularly preferably 20 hours.
  • Acyl groups can also be introduced using a reactive ester method with activation of a carboxylic acid derivative with N-hydroxysuccinimide.
  • This method is preferably used in the case of polyethyleneimine functionalization with bile acids.
  • the bile acid derivative chenodeoxycholic acid (3 ⁇ , 7 ⁇ -dihydroxy-5ß-cholanic acid), hereinafter abbreviated as a substituent with CDC is reacted with N-hydroxysuccinimide in dimethoxyethane as solvent in the presence of dicyclohexylcarbodiimide.
  • the reaction takes place at room temperature, the reaction time is 16 hours.
  • the reactive ester thus produced is reacted with a 5% solution of the corresponding linear polyethyleneimine in absolute ethanol.
  • the dosing amount of the reactive ester is based exactly on the desired degree of substitution (0.1 to 10%).
  • the reaction temperature is between 20 and 60 ° C, preferably 50 ° C.
  • the reaction time is preferably between 10 and 24 hours, particularly preferably 20 hours.
  • chenodeoxycholic acid into oligoamines such as, for example, spermine or pentaethylene hexamine via the reactive ester method has been described in the literature (Walker et al., 1998).
  • the bile acid-substituted polymers according to the invention have hydrophobic substituents, the degree of hydrophobicity being able to be controlled by the number of hydroxyl groups, analogously to that described by S. Walker et al. described "cationic facial amphiphiles".
  • Highly purified samples are prepared by dissolving the polyamines and especially the hydrophobic polyethyleneimines in water at pH 7 in a concentration of 0.1 to 1 mg / ml, preferably 0.5 mg / ml, and purifying them by column chromatography on Sephadex and subsequent freeze-drying , The polymers are then again in water or preferably more physiological
  • the concentration of the polyamine or polyethyleneimine stock solutions is preferably between 0.1 and 1 mg / ml, particularly preferably 0.5 mg / ml.
  • the stock solutions are stable at room temperature, preferably at 4 ° C.
  • Standard methods such as 1H NMR spectroscopy, FT-IR spectroscopy and zeta potential measurements can be used to characterize the cationic polymers.
  • the polyamines that can be used for drug production can also be coupled to cell-specific ligands.
  • cell-specific ligands can, for example, be such that they bind to the outer membrane of a target cell, preferably an animal or human target cell.
  • the target cell can be, for example, an endothelial cell, a muscle cell, a macrophage
  • Lymphocyte a glial cell, a blood-forming cell, a tumor cell, for example a leukemia cell, a virus-infected cell, a bronchial epithelial cell or a liver cell, for example a sinusoidal cell of the liver.
  • a ligand that binds specifically to endothelial cells can, for example, be selected from the group consisting of monoclonal antibodies or their fragments which are specific for endothelial cells, glycoproteins carrying terminal mannose, glycolipids or polysaccharides, cytokines, growth factors, adhesion molecules or, in a particularly preferred one Embodiment, from glycoproteins from the envelope of viruses that have a tropism for endothelial cells.
  • a ligand that binds specifically to smooth muscle cells can, for example, be selected from the group comprising monoclonal antibodies or their fragments that are specific for Actin, cell membrane receptors and growth factors or, in a particularly preferred embodiment, glycoproteins from the envelope of viruses that have a tropism for smooth muscle cells.
  • a ligand that binds specifically to macrophages and / or lymphocytes can, for example, be selected from the group comprising monoclonal antibodies that are specific for
  • Membrane antigens on macrophages and / or lymphocytes intact immunoglobulins or Fc fragments of polyclonal or monoclonal antibodies which are specific for membrane antigens on macrophages and / or lymphocytes, cytokines, growth factors, peptides, proteins, lipids or polysaccharides carrying terminal mannose or, in one particularly preferred
  • Embodiment from glycoproteins from the envelope of viruses, in particular the HEF protein from the influenza C virus with mutation in the nucleotide position 872 or HEF cleavage products of the influenza C virus containing the catalytic triad serine-71, histidine-368 or -369 and aspartic acid 261st
  • a ligand that binds specifically to glial cells can, for example, be selected from the group comprising
  • Antibodies and antibody fragments that bind specifically to membrane structures of glial cells, adhesion molecules, peptides bearing terminal mannose, proteins, lipids or polysaccharides, growth factors or, in a particularly preferred embodiment, from glycoproteins from the envelope of viruses that have a tropism for glial cells.
  • Cells binds can be selected, for example, from the group comprising antibodies or antibody fragments which are specific for a receptor of the stem cell factors, IL-1 (in particular receptor type I or II), IL-3 (in particular receptor type ⁇ or ⁇ ), LL- 6 or GM-CSF, as well as intact immunoglobulins or Fc fragments which have this specificity and growth factors such as SCF, IL-1,
  • a ligand that binds specifically to leukemia cells can, for example, be selected from the group comprising antibodies, antibody fragments, immunoglobulins or Fc fragments that bind specifically to membrane structures on leukemia cells, such as CD13, CD14, CD15, CD33, CAMAL, sialosyl Le, CD5,
  • a ligand that binds specifically to virus-infected cells can, for example, be selected from the group comprising antibodies, antibody fragments, intact immunoglobulins or Fc fragments which are specific for a virus antigen which, after infection by the virus, is expressed on the cell membrane of the infected cell ,
  • a ligand that can bind specifically to bronchial epithelial cells, sinusoidal cells of the liver or liver cells can, for example, be selected from the group comprising transferrin, asialoglycoproteins, such as asialoorosomucoid, neoglycoprotein or galactose, insulin, peptides carrying terminal mannose, proteins, lipids or polysaccharides, intact Immunoglobulins or
  • Fc fragments that bind specifically to the target cells and, in a particularly preferred embodiment, from glycoproteins from the envelope of viruses that bind specifically to the target cells.
  • ligands are e.g. in EP-A 0 790 312 and EP-A 0 846 772.
  • the medicaments according to the invention generally contain between 0.5 and 500 mg of polyamine per dose as active substance, preferably between 20 and 100 mg per dose.
  • the medicaments according to the invention can contain further pharmaceutical active substances, such as e.g. Parapox ovis (for example in the form of BAYPAMUN®), fragments of Parapox ovis, CpG-containing oligonucleotides, antibiotics, cytostatics.
  • active substances such as e.g. Parapox ovis (for example in the form of BAYPAMUN®), fragments of Parapox ovis, CpG-containing oligonucleotides, antibiotics, cytostatics.
  • Polyamines or the medicaments according to the invention themselves are preferably in solid form after cleaning and lyophilization of the polyamines and can then be dissolved in a suitable aqueous medium, preferably physiological saline, immediately before application or applied directly in a suitable formulation with the addition of additives, if necessary after Dispersion in aqueous media, preferably in physiological saline.
  • a suitable aqueous medium preferably physiological saline
  • formulation auxiliaries include biocompatible and biodegradable polymers such as polylactide, polylactide co-glycolide, polyacrylates.
  • Polyorthoesters, polyanhydrides, polyamides, polyamino acids, cellulose derivatives, starch derivatives or chitosan derivatives come into question.
  • the drug based on polyamines is administered systemically (e.g. orally, intramuscularly, subcutaneously, intraperitoneally, intravenously) or locally (e.g. into the organ in question).
  • polyamines can be used to treat the following diseases / lesions or for prophylaxis / metaphylaxis before the following
  • HSV He ⁇ es simples virus
  • HPV human papilloma virus
  • HIN human immunodeficiency virus
  • HCMN human cytomegalovirus
  • Organ fibrosis especially liver fibrosis or cirrhosis as a result of viral hepatitis or ethanol-induced liver diseases, and cystic
  • the polyamines can also be used as adjuvants.
  • Linear polyethyleneimines were synthesized by cationic ring-opening polymerization of 2-ethyloxazoline to poly (ethyloxazoline) (analogous to Rivas & Ananias, 1992) and subsequent acidic hydrolysis by splitting off propanoic acid.
  • Certain precursor polymers are also commercially available (Sigma-Aldrich Chemie GmbH, Germany). The precursor polymers were characterized by gel permeation chromatography, 1H-NMR and FT-IR.
  • High-purity batches (milligram amounts) were obtained by column chromatography over Sephadex G25 (Pharmacia disposable PD-10 desalting column) of saturated aqueous solutions (pH 7) of the polyethyleneimine with Millipore water as eluent and subsequent
  • H-LPEI hydrophobically functionalized linear polyethyleneimines
  • Quantities were obtained by column chromatography on Sephadex G25 (Pharmacia disposable PD-10 desalting column) of saturated aqueous solutions (pH 7) of the polyethyleneimine with Millipore water as eluent and subsequent freeze drying.
  • the alkylated linear polyethyleneimines were characterized by 1H-NMR and FT-IR, which confirmed the desired degree of alkylation.
  • H-LPEI hydrophobically functionalized linear polyethyleneimines
  • acylated linear polyethyleneimines were characterized by 1H-NMR and FT-IR, whereby the desired degree of acylation could be confirmed.
  • linear polyethyleneimines acyl-functionalized using the reactive ester method were characterized by 1H-NMR and FT-IR, which confirmed the desired degree of acylation.
  • polyethyleneimines LPEI, H-LPEI
  • stock solutions were prepared in physiological saline at pH 7 with a concentration of 0.5 mg / ml.
  • 25 mg of the LPEI or the H-LPEI were dissolved in 30 ml of water or physiological saline solution with heating and brief ultrasound treatment, adjusted to pH 7 with 0.1 N HC1 and made up to an volume of 50 ml.
  • the stock solutions were sterile filtered (0.2 ⁇ m) and can be stored at 20 ° C for a long time.
  • the interferon- ⁇ stimulating effect of the polyamines and subsequently the proof of effectiveness in vivo was demonstrated.
  • the NMRI mice (breeding strain, female, weight 18-20 g) were obtained 8 days before the start of the experiments from Charles River (Sulzfeld, Germany). The animals had free access to food and water and were kept in an artificial day-night rhythm (lighting from 7 a.m. to 7 p.m., darkness from 7 p.m. to 7 a.m.).
  • the animals were sacrificed by cervical dislocation and then the spleens were removed.
  • the spleens were freed from attached connective tissue and processed according to the following rule:
  • the spleen is placed on a metal sieve (mesh size approx. 70 ⁇ m) (petri dish) and crushed with scissors. Then 5 ml of PBS are added and the tissue pieces are pressed through the sieve with a glass pestle. The sieve is rinsed several times with PBS, the cells are transferred to a Falcon tube in a total of 50 ml PBS and then centrifuged for 10 minutes at 300 x g.
  • a metal sieve mesh size approx. 70 ⁇ m
  • petri dish petri dish
  • the stimulations were carried out in a volume of 1 ml in 24-well plates at 37 ° C. and 5% CO 2 for 72 hours. In a total volume of 1 ml (0.8 ml medium: RPMI, 10% FCS, 1% penicillin / streptomycin), 2 x 10 6 spleen cells were stimulated. Depending on the number of stimulants, the following was combined per approach:
  • the supernatants were transferred to 1.5 ml reaction vessels, and the remaining cells were separated by centrifugation at 300 ⁇ g for 10 minutes. The cell-free supernatants were removed and stored at -20 ° C. until the measurement of IFN- ⁇ by ELISA.
  • the determination of the iFN- ⁇ concentrations in the stimulation supernatants was carried out using the "Mouse IFN- ⁇ OptEIA®-ELISA Set” (Pharmingen, Heidelberg, Germany) according to the manufacturer's instructions.
  • the tested polyethyleneimines show a significant IFN- ⁇ induction in the mouse
  • NMRI mice (outbred strain HdsWimNMRI, female, weight 18-20 g, obtained from Harlan / Winkelmann, Borchen, Germany) were placed in autoclavable polycarbonate boxes lined with sawdust, in the S2 isolation stall at 20-22 ° C (humidity 50-60% ) and held in an artificial day / night rhythm (lighting from 6:30 a.m. to 6:30 p.m., darkness from 3:1 p.m. to 3:1 a.m.). They had free access to food and water.
  • HdsWimNMRI female, weight 18-20 g, obtained from Harlan / Winkelmann, Borchen, Germany
  • the animals were randomized and divided into two groups of 6 animals each.
  • mice were initially kept in the animal stable for 3 days without further treatment.
  • the substances to be analyzed were administered intraperitoneally in a volume of 0.2 ml.
  • the following treatment regimen was used:
  • Group 2 polyethyleneimine, H-LPEI, M w : 87000, C18 acyl, 3 mol%, according to Example 3 (0.1 mg per mouse).
  • mice were sacrificed 9 hours after the treatment and the peritoneal cells obtained by abdominal irrigation with 5 ml of ice-cold PBS.
  • the cells were concentrated using a centrifugation step (30 seconds at 16,000 ⁇ g, room temperature), the supernatant decanted and the cellular Total RNA extracted using the NucleoSpin RNA II kit (Machery-Nagel, Düren, Germany).
  • the cell pellet was resuspended in 400 ⁇ l RA1 buffer (from the NucleoSpin RNA II kit) and frozen at -80 ° C. After thawing to 37 ° C the
  • DNase I Cleavage of the DNA by DNase I.
  • 90 ⁇ l DNase reaction buffer was mixed with 10 ⁇ l DNase I (both from the NucleoSpin RNA II Kit) and 95 ⁇ l of this solution were applied to the dry filter. After incubation for 15 minutes (room temperature), the filter was washed first with 500 ⁇ l RA2, then with 600 ⁇ l RA3 and then with 250 ⁇ l RA3 (from the NucleoSpin RNA II Kit).
  • the washing buffer was applied and the column was centrifuged for 30 seconds at 8,000 x g and after the last washing step for 2 minutes at 16,000 x g.
  • the RNA was then eluted in 60 ⁇ l RNase-free, distilled water (16,000 ⁇ g for 1 minute).
  • the quality of the RNA was checked by photometric measurement.
  • the cDNA was synthesized by reverse transcription of the RNA using "random hexamers" as primers for the polymerase reaction.
  • the TaqMan Reverse Transcription Reagents (Applied Biosystems,
  • the mixture was incubated in the GeneAmp 2400 thermal cycler (Applied Biosystems, Rothstadt, Germany) first for 10 minutes at 25 ° C., then for 30 minutes at 48 ° C. and then cooled to + 4 ° C.
  • the cDNA synthesized in this way was stored at -20 ° C.
  • the amounts of the cDNAs were determined using a “housekeeping” gene (18S RNA), using the PDAR kit "Endogenous Control Ribosomal RNA Control
  • a calibrator cDNA was used for standardization and induction calculation. This consisted of a mixture of cDNA 11 from different mice, which were treated in accordance with group 1.
  • the amplifications were each carried out in a volume of 50 ⁇ l and were composed as follows: Endogenous 18S RNA control:
  • the Aujeszky mouse model is an in vivo exercise model for the detection of the effects of various immune stimulators (e.g. BAYPAMUN®, CpG oligonucleotides).
  • various immune stimulators e.g. BAYPAMUN®, CpG oligonucleotides.
  • mice (outbred strain HdsWimNMRI, female, weight 18-20 g, obtained from Harlan / Winkelmann, Borchen, Germany) were placed in autoclavable polycarbonate boxes lined with sawdust, in an S2 isolation stall at 20-22 ° C (humidity 50-60 %) and kept in an artificial day / night rhythm (lighting from 06:30 to 18:30, darkness from 18:30 to 06:30). They had free access to food and water.
  • HdsWimNMRI female, weight 18-20 g, obtained from Harlan / Winkelmann, Borchen, Germany
  • mice Maleted mice were formed for the investigations. The animals in a group received the same test substance.
  • mice were kept in the animal stall for 2-3 days after delivery.
  • the polyethyleneimines starting concentration 0.5 mg / ml
  • the polyethyleneimines were then diluted 1:10 and 1: 100 with physiological NaCl solution (pH 7.6). 0.2 ml of these solutions was administered intraperitoneally per mouse.
  • mice 24 hours after the treatment, the mice were challenged by intraperitoneal application of pseudorabies virus, strain Hannover H2.
  • the virus was diluted in PBS to a loading titer of 10 ⁇ -10 4 ' 1 TC ⁇ D 50 / ml and 0.2 ml of this suspension was applied.
  • a negative control a group of mice was treated with physiological NaCl solution and then exposed.
  • mice in this group died 3-8 days after exercise.
  • the mice treated with polyethylene imines survived to a large extent the pseudorabies virus
  • the strength of the induced immune stimulation resulted from the comparison of the dead mice in the NaCl control group and the test groups and was quantified by the action index. This gives the percentage number of
  • b indicates the percentage of dead mice in the control group
  • a indicates the percentage of dead mice in the test group.
  • the NMRI mice (breeding strain, female, weight 18-20 g) were obtained 8 days before the start of the experiments from Charles River (Sulzfeld, Germany). The animals had free access to food and water and were kept in an artificial day-night rhythm (lighting from 7 a.m. to 7 p.m., darkness from 7 p.m. to 7 a.m.).
  • the animals were randomized and divided into three groups of 4 animals each.
  • the substances to be analyzed were administered intraperitoneally in a volume of 0.2 ml.
  • the following treatment regimen was used:
  • Group 1 Placebo: Physiological NaCl solution.
  • Group 2 polyethyleneimine, H-LPEI, M: 87000, C18 acyl, 3 mol% (0.1 mg per
  • mice were sacrificed 6 hours after the treatment and the peritoneal cells were obtained by abdominal irrigation with 10 ml of medium (DMEM, 5% FCS).
  • medium DMEM, 5% FCS
  • the cells were then concentrated by centrifugation (10 minutes at 300 x g, room temperature) and then either used immediately in the RNA extraction or first subjected to erythrocyte lysis.
  • the pelleted peritoneal cells were resuspended in 1 ml PBS, with 10 ml lysis buffer (10 ml 0.17 M Tris, pH 7.2 + 90 ml 0.16 M
  • Total RNA was prepared from the peritoneal cells using the RNeasy Mini-Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Two approaches for a reprocessing were mixed.
  • the yields of total RNA from the peritoneal cells of four animals were between 10 and 20 ⁇ g RNA.
  • RNA is amplified from the total RNA preparation.
  • the cDNA of immunologically relevant genes (interleukins, differentiation clusters (CDs), transcription factors, receptors etc.) was applied to a chip.
  • the amplified RNA was used for the hybridization.
  • 2 ⁇ g of the amplified RNA were transcribed into cDNA and the fluorescence-labeled nucleotides were incorporated.
  • the controls were labeled with Cy3 and the samples with Cy5.
  • IL-lr II type II interleukin 1 receptor
  • monocytes a comparison to interleukin 1 beta (TL-1 beta) and interleukin 1 receptor antagonist (IL-lra).
  • TL-1 beta interleukin 1 beta
  • IL-lra interleukin 1 receptor antagonist
  • T cell-mediated viral clearance is dependent upon interferon-gamma (IFN-gamma). Virology, 202, 76-88.
  • hnmunostimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishrnaniasis via IL- 12- and IFN-gamma-dependent mechanisms.

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EP01985905A 2000-12-29 2001-12-19 Arzneimittel enthaltend ein polyamin als wirksubstanz Withdrawn EP1392268A2 (de)

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DE10065710 2000-12-29
DE10065710A DE10065710A1 (de) 2000-12-29 2000-12-29 Arzneimittel enthaltend ein Polyamin als Wirksubstanz
PCT/EP2001/015005 WO2002053149A2 (de) 2000-12-29 2001-12-19 Arzneimittel enthaltend ein polyamin als wirksubstanz

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JP3990972B2 (ja) * 2001-11-20 2007-10-17 有限会社 キック 血管再狭窄防止薬及び該防止薬がコーティングされた血管内埋め込み器具
GB0221942D0 (en) * 2002-09-20 2002-10-30 Univ Strathclyde Drug delivery
GB0422877D0 (en) * 2004-10-14 2004-11-17 Univ Glasgow Bioactive polymers
KR100904207B1 (ko) * 2007-06-01 2009-06-25 (주) 태웅메디칼 스텐트용 약물 방출 코팅제, 그의 제조방법 및 이 코팅제로 코팅된 약물 방출 스텐트
US20110318298A1 (en) 2008-10-14 2011-12-29 Nippon Shokubai Co., Ltd. Viral infection therapeutic drug containing polyalkyleneimine
US20110236512A1 (en) * 2009-09-28 2011-09-29 Toyo Boseki Kabushiki Kaisha Stress-reducing agent including plant-derived polyamine-containing extract serving as active component
JPWO2011142484A1 (ja) * 2010-05-14 2013-07-22 株式会社日本触媒 ポリアルキレンイミンを含むウイルス感染症治療薬
KR101420267B1 (ko) * 2011-10-10 2014-07-17 대구대학교 산학협력단 폴리아민을 유효성분으로 함유하는 염증의 예방 및 치료용 약학 조성물
AU2015287661B2 (en) * 2014-07-11 2019-07-11 Genzyme Corporation Main chain polyamines
CN112812298B (zh) * 2020-12-31 2022-07-19 广东省微生物研究所（广东省微生物分析检测中心） 一种可增强光动力抗菌效果的新型功能高分子材料及其制备方法和应用
JP2024515107A (ja) * 2021-04-20 2024-04-04 パーデュー リサーチ ファウンデーション 抗腫瘍免疫療法としての免疫機能担体、使用方法及び組成物

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BR0116653A (pt) 2004-02-17
WO2002053149A3 (de) 2003-12-11
ZA200304928B (en) 2005-05-04
CN1507349A (zh) 2004-06-23
NZ526721A (en) 2007-02-23
KR20030070085A (ko) 2003-08-27
US20040077610A1 (en) 2004-04-22
JP2004520328A (ja) 2004-07-08
CA2433125A1 (en) 2002-07-11
DE10065710A1 (de) 2002-07-04

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