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EP2029125A2 - Pharmaceutical composition for the treatment of viral infections and/or tumor diseases by inhibiting protein folding and protein breakdown - Google Patents

Pharmaceutical composition for the treatment of viral infections and/or tumor diseases by inhibiting protein folding and protein breakdown

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Publication number
EP2029125A2
EP2029125A2 EP07765312A EP07765312A EP2029125A2 EP 2029125 A2 EP2029125 A2 EP 2029125A2 EP 07765312 A EP07765312 A EP 07765312A EP 07765312 A EP07765312 A EP 07765312A EP 2029125 A2 EP2029125 A2 EP 2029125A2
Authority
EP
European Patent Office
Prior art keywords
chaperones
pharmaceutical composition
composition according
inhibitors
substances
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Withdrawn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
EP07765312A
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German (de)
French (fr)
Inventor
Ulrich Schubert
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.)
Virologik GmbH
Original Assignee
Virologik GmbH
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Filing date
Publication date
Application filed by Virologik GmbH filed Critical Virologik GmbH
Publication of EP2029125A2 publication Critical patent/EP2029125A2/en
Withdrawn legal-status Critical Current

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    • 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
    • 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
    • A61K31/325—Carbamic acids; Thiocarbamic acids; Anhydrides or salts thereof
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • composition for the treatment of viral infections and / or tumors by inhibiting protein folding and protein degradation.
  • the invention relates to a pharmaceutical composition containing as active components at least one proteasome inhibitor and an inhibitor of protein folding enzymes. These agents are useful in the treatment of acute and chronic human and animal pathogenic virus infections. These include in particular pathogens of infectious diseases such as AIDS, hepatitis, hemorrhagic fever, SARS, smallpox, measles, polio or influenza.
  • the invention relates to agents which contain on the one hand as active ingredients inhibitors of protein folding. These include inhibitors of cellular folding enzymes (the enzymatic chaperones) as well as substances that interfere with the folding of proteins by chemical chaperones. On the other hand, these agents contain components that interfere with the ubiquitin-proteasome system, especially agents that inhibit the 26S proteasome.
  • Inhibitors of protein folding enzymes are known from WO 2005/063281 A2.
  • Proteasome inhibitors have been described both for the treatment of tumor diseases (for example US Pat. No. 6,083,903) and for the treatment of viral infections (WO 02/30455).
  • the invention had the object of providing novel pharmaceutical compositions for the treatment of viral infections and / or tumors.
  • a pharmaceutical composition which contains as active components at least one inhibitor of the ubiquitin-proteasome system and an inhibitor of protein folding enzymes or a method for influencing protein folding.
  • the inhibitor of protein folding enzymes is preferably at least one inhibitor of cellular chaperones or at least one chemical substance which directly affects protein folding (chemical anti-chaperone).
  • a method for influencing the protein folding is preferably the local hyperthermia.
  • a further preferred embodiment of the invention is that substances are used as inhibitors of cellular chaperones or chemical anti-chaperones, which
  • a) inhibit, regulate or otherwise affect the folding and proteolytic maturation of viral proteins and thereby inhibit the release and replication of viruses, especially pathogens such as AIDS, hepatitis, haemorrhagic fever, SARS, smallpox, measles, polio, herpesvirus infections or influenza , or b) interfere with the proliferation of degenerate cells, especially tumor cells, by driving them into programmed cell death by accumulation of misfolded proteins.
  • viruses especially pathogens such as AIDS, hepatitis, haemorrhagic fever, SARS, smallpox, measles, polio, herpesvirus infections or influenza
  • the pharmaceutical composition according to the invention is characterized in that as inhibitors of cellular chaperones or of chemical anti-chaperones substances are used which specifically influence the enzymatic activities of molecular folding enzymes of the host cells.
  • the cells of higher eukaryotes receive these inhibitors or substances and, after cell uptake, block the protein folding of virus structure peats and of proteins from tumor cells.
  • the inhibitors or substances may be administered in vivo in various forms, orally, intravenously, intramuscularly, subcutaneously or in encapsulated form with or without cell specificity-bearing alterations, due to the application of a particular application and / or dose regimen having low cytotoxicity , cause no or negligible side effects, have a relatively high metabolic half-life and a relatively low clearance rate in the organism.
  • composition according to the invention is further characterized in that are used as inhibitors of cellular chaperones or chemical anti-chaperones substances that are a) isolated in natural form from microorganisms or other natural sources, or b) by chemical modifications of natural substances or c) be prepared totally synthetically or d) be synthesized in vivo by gene therapy methods.
  • the inhibitors of cellular chaperones or the chemical anti-chaperones disrupt the highly organized processes of assembly and proteolytic maturation of virus structural proteins and thereby inhibit the release and production of infectious progeny viruses.
  • these substances regulate, disrupt or block the folding of viral proteins and / or tumor-specific proteins by disrupting the late processes of viral replication such as assembly, budding, proteolytic maturation and virus release.
  • the proteolytic processing of precursor proteins of the viral polyproteins is thereby disturbed.
  • the activity of viral proteases is blocked.
  • a further preferred embodiment of the invention is that are used as inhibitors of cellular chaperones or of chemical anti-chaperones substances that lase the activities of cellular proteases and / or enzymes, such as ligases, kinases, Hydro, glycosylation enzymes , Phosphatases, DNAses, RNAses, helicases and transferases that are involved in virus maturation.
  • the inhibitors of cellular chaperones according to the invention or the chemical anti-chaperones have a broad spectrum of activity and can therefore be used as novel broad-spectrum antivirals for the prevention and / or therapy of different viral infections.
  • the pharmaceutical composition is characterized in that are used as inhibitors of cellular chaperones or chemical anti-chaperones substances that block or inhibit cellular chaperones such as heat shock proteins (hsp) proteins, in particular the activities of the heat shock proteins Hsp27, Hsp30 , Hsp40, Hsp60, Hsp70, Hsp72, Hsp73, Hsp90, HsplO4 and Hsc70.
  • heat shock proteins Hsp27, Hsp30 , Hsp40, Hsp60, Hsp70, Hsp72, Hsp73, Hsp90, HsplO4 and Hsc70.
  • substances can be used which belong to the following substance classes and their derivatives: geldanamycin (inhibits Hsp90), radicicol (tyrosine kinase inhibitor, inhibits Hsp90), deoxyspergualin (inhibits Hsc70 and Hsp90), 4-PBA (4 Downregulation of protein and mRNA expression of Hsc70), herbimycin A (tyrosine kinase inhibitor with Hsp72 / 73 induction), epolactaene (inhibitor of Hsp60), scythe and reaper (inhibit Hsp70), artemisinin (inhibitor of Hsp90), CCT0180159 (as pyrazole inhibitor of Hsp90) and SNX-2112 (Hsp90 inhibitor), radanamycin (macrololidimer of radicicol and geldanamycin), novobiocin (Hsp90 inhibitor), quercetin (
  • Substances which regulate, disrupt or block the protein conformation and the folding of viral and / or tumor-specific proteins can be used as chemical anti-chaperones. These include, for example, substances such as glycerol, trimethylamine, betaine, trehalose or deuterated water (D 2 O).
  • substances can be used which are suitable for the treatment, therapy and inhibition of infections with different human pathogenic or animal-pathogenic viruses or substances which are used for the treatment, therapy and inhibition of infections with agents of chronic infectious diseases such as AIDS (HIV-I and HIV-2), hepatitis (HCV and HBV), the cause of the "Severe Acute Respiratory Syndrome” (SARS), the SARS-CoV (coronavirus), of poxviruses, of viral haemorrhagic fever (VHF) pathogens, as well as the Ebola virus as a member of the family Filoviridae; influenza-causing agents such as the influenza A virus.
  • agents of chronic infectious diseases such as AIDS (HIV-I and HIV-2), hepatitis (HCV and HBV), the cause of the "Severe Acute Respiratory Syndrome” (SARS), the SARS-CoV (coronavirus), of poxviruses, of viral haemorrhagic fever (VHF) pathogens, as well as the Ebol
  • the pharmaceutical composition according to the invention is further characterized in that the inhibitors of the UPS is at least one substance which a) especially in the form of proteasome inhibitors, the enzymatic activities of the complete 26S proteasome complex and the free, not with regulatory
  • Subunits assembled 2OS influenced catalytically active proteasome structure or b) specifically inhibits the action of ubiquitin ligases or c) specifically inhibits the action of ubiquitin hydrolases or d) specifically inhibits the action of ubiquitin-activating enzymes or e) specifically the mono- ubiquitinylation of proteins or f) specifically inhibits the poly-ubiquitinylation of proteins
  • proteasome inhibitors are taken up by higher eukaryotes and interact after cell uptake with the catalytic subunits of the proteasome and block all or some of the proteolytic activities of the proteasome - the trypsin, the chymotrypsin and / or the postglutamyl peptide hydrolyzing activities - irreversible or reversible within the 26S or the 2OS proteasome complex.
  • proteasome inhibitors used are substances which a) are isolated in natural form from microorganisms or other natural sources; or b) result from chemical modifications of natural substances; or c) produced totally synthetically; or d) be synthesized in vivo by gene therapy methods; or e) by genetic engineering in vitro or f) in microorganisms.
  • the proteasome inhibitors are compounds which belong to the following substance classes: a) naturally occurring proteasome inhibitors:
  • Aclacinomycin A also referred to as Aclarubicin
  • modified peptide aldehydes such as N-carbobenzoxy-L-leucinyl-L-leucinyl-L-leucinal (also referred to as MG 132 or zLLL), its boric acid derivative MG232; N-carbobenzoxy-Leu-Leu-Nva-H (termed MGl 15; N-acetyl-leucinyl-L-leucinyl-L-norleucinal (referred to as LLnL), N-carbobenzoxy-Ile-Glu (OBut) -la-Leu -H (also referred to as PSI);
  • modified peptide aldehydes such as N-carbobenzoxy-L-leucinyl-L-leucinyl-L-leucinal (also referred to as MG 132 or zLLL), its boric acid derivative MG232; N-carbobenzoxy-Leu-Leu-Nva-H (termed
  • peptides which carry an ⁇ , ⁇ -epoxyketone C-terminal structure, furthermore vinylsulfones such as carbobenzoxy-L-leucinyl-L-leucinyl-L-leucine-vinyl-sulfone or 4-hydroxy-5-iodo-3-nitrophenylactetyl -L-leucinyl-L-leucinyl-L-leucine-vinylsulfone (NLVS);
  • glyoxal or boric acid radicals such as pyrazyl-CONH (CHPhe) CONH (CHisobutyl) B (OH) 2 ) as well as dipeptidyl-boric acid derivatives or
  • Pinacol esters such as benzyloxycarbonyl (Cbz) -Leu-Leu-boroLeu-Pinacol esters.
  • proteasome inhibitors the epoxyketones epoxomicin (epoxomycin, molecular formula: C28H86N4O7) and / or eponemycin (Eponemicin, molecular formula: C 20 H 36 N 2 O 5) or proteasome inhibitors from the PS series the compounds: a ) PS-519 as ⁇ -lactone as well as lactacystin derivative the compound IR- [IS, 4R, 5S]] - 1- (1-hydroxy-2-methylpropyl) -4-propyl-6-oxa-2-azabicyclo [3.2.0] heptane-3,7-dione -
  • PS-314 as peptidyl-boric acid derivative the compound N-pyrazinecarbonyl-L-phenylalanine-L-leucine-boric acid - molecular formula C1 9 H25BN4O4 - and / or c) PS-273 (morpholine-CONH- (CH-naphthyl) -CONH- (CH-isobutyl) -B (OH) 2 ) and its enantiomer PS-293 and / or d) the compound PS-296 (8-quinolyl -sulfonyl-CONH- (CH-Napthyl) -CONH (-CH-isobutyl) -B (OH) 2 ) and / or e) PS-303 (NH 2 (CH-Naphthyl) -CONH- (CH-isobutyl) - B (OH) 2 ) and / or f) PS
  • PS-383 pyridyl-CONH- (CH /? F-phenylalanine) -CONH- (CH-isobutyl) -B (OH) 2 ).
  • compositions described are useful as drugs or for the preparation of agents for the treatment of viral infections and / or tumor diseases.
  • CA carcinoma
  • AML - - Leukemia (AML, ALL, CML, CLL) - acute myeloid, chronic acute lymphocytic, chronic
  • the effect of these inhibitors for the treatment of Plasmazytomzellen of patients with multiple myeloma is used.
  • These B-cell tumors are characterized by an extremely high synthesis rate of immunoglobulins.
  • these plasmacytoma cells are particularly sensitive to the treatment with proteasome inhibitors. Therefore, proteasome inhibitors, especially in the form of boric acid peptides (trade name Velcade) are used successfully for the treatment of multiple myeloma.
  • a very narrow therapeutic window must be taken into account because the boundary between the therapeutic dose and the tolerable toxic dose is very narrow.
  • a further preferred embodiment of the invention relates to the antiviral effect in the combination of both active ingredients. It is known that proteasome inhibitors the
  • HIV human immunodeficiency virus
  • This embodiment of the invention is generally valid for all viral infections in which an ordered assembly of newly synthesized viral structure ports takes place.
  • Example 1 Hsp90 inhibitor 17-AAG shows no cytotoxicity in CEM cells up to a concentration of 10 mM.
  • CD4 + T lymphoid cells were seeded in a 96-well plate at a density of IxIO 4 cells per 100 ⁇ l.
  • To the medium (see Example 4a) were previously added appropriate amounts of 17-AAG to final concentrations of l ⁇ M; 10OnM; 1OnM; InM; 0.InM and 0.0 InM 17-AAG reach. After 30 hours incubation at 37 ° C and 5% CO 2 was added to 10 ⁇ l AlamarBlue TM (Invitrogen) and all batches were incubated for a further 4 hours at 37 ° C.
  • the measure of the vitality of the CEM cells (indicated in MTT CEM) under 17-AAG influence could be determined by measuring the color change of the medium by means of fluorescence measurement at 530/590 nm. All approaches were in triplicate.
  • Example 2 pNLenvl-transfected HeLaSS ⁇ cells show reduced Gag processing and increased Hsp70 expression in the cell fraction under 17-AAG influence in the virus fraction.
  • Example 4a / b For the biochemical analysis of the influence of 17-AAG on the kinetics of gag processing and virus release time kinetics were performed. The experimental details of cultivation, transfection, media change and time kinetics are given in Example 4a / b.
  • cultures of HeLaSS ⁇ cells were used, which were transfected with pNLenvl (Schubert et al, 1995).
  • 17-AAG-containing medium (10OnM 17-AAG), or inhibitor-free medium
  • the kinetics was started after distinct washing steps and aliquoting of the batches. Aliquots of cells were collected at each time point and separated by centrifugation into cell, virus and cell culture supernatant fractions.
  • the HIV proteins were separated by SDS-PAGE, transferred to PVDF membrane, and then visualized by X-ray on antibody-mediated chemo-luminescence.
  • Example 3 17-AAG, as well as the combination with PS341, inhibits virus replication of X4-tropic HI viruses in the HLAC model.
  • CEM cells were cultured in RPMI 1640 with 10% (v / v) fetal calf serum, 2 mM L-glutamine, 100 U ml-1 penicillin and 100 ⁇ g ml-1 streptomycin.
  • Heia cells were cultured in Dulbecco's modified Eagle's Medium (DMEM) with 10% fetal calf serum, 2 mM L-glutamine, 100 U ml-1 penicillin, and 100 ⁇ g / ml streptomycin.
  • DMEM Dulbecco's modified Eagle's Medium
  • Tonsillar cells were transfected into RPMI 1640 with 15% (v / v) fetal calf serum, 2 mM L-glutamine, 100 U ml-1 penicillin, 100 ⁇ g ml-1 streptomycin, 2.5 ⁇ g / ml fungizone, ImM sodium pyrovate, 1 % MEM non-essential amino acid solution and 50 ⁇ g / ml gentanamycin cultured ("tonsil medium").
  • Example 4b Transfection, change of media and kinetics Heia cells (ATCC CCL2) were transfected into OPTI-MEM by means of pNL ⁇ env-Lipofectamine 2000 mixture. After 8 hours of incubation in 37 ° C and 5% CO 2 , a change of media was performed. In one of the two batches a final concentration of 10OnM 17-AAG was added to the medium and incubated for a further 16 hours. After distinct washes in PBS, the respective times were aliquoted. At the appropriate time points, the cells were separated from the supernatant by centrifugation (5min, 5000rpm) and later lysed by CHAPS / DOC lysis (3min on ice).
  • VLP's in the supernatant were pelleted on a 20% sucrose pad (90 min; 14000 rpm) and, like the cell pellet lysates, separated by 10% SDS-PAGE, transferred to PVDF membrane by wet blot and placed in 10 % milk powder (in PBS-Tween 0.1%) blocked.
  • Detection of the HIV or cell-specific proteins was carried out via specific antibodies (against Hsp70, Hsp90, p24, PR55, ⁇ -actin). Through the reaction with secondary antibodies and their coupled chemo luminescence, the signals could be detected on X-ray films.
  • plasmid DNA from HIV-1 molecular DNA was transfected into HeLa cells using the calcium-phosphate precipitation method. These were confluent cultures of Heia cells (5x10 6 cells) with 25 ⁇ g plasmid DNA in calcium phosphate crystals, prepared by a method of Graham and van der Eb (1973), and then a glycerol shock according to Gorman et al. (1982). For the recovery of concentrated virus preparations, the cell culture supernatants were harvested two days after transfection. Subsequently, the cells and their components were separated by centrifugation (1.000 xg, 5 min, 4oC) and filtration (0.45 micron pore size).
  • Virus particles were pelleted by ultra-centrifugation (Beckman SW55 rotor, 1.5 hr, 35,000 rpm, 10oC) and subsequently resuspended in 1 ml of DMEM medium.
  • the virus preparations were sterilized by filtration (0.45 .mu.m pore size) and portioned frozen (-80 0 C).
  • Single virus preparations were standardized by determination of reverse transcriptase activity using a previously described assay (Willey et al, 1988) using [32 P] -TTP incorporation into an oligo (dT) -poly (A) template.
  • Example 4d HLAC model (extraction, infection, kinetics)
  • the tonsil tissue was washed in PBS, cleaned by scalpels of blood clots and divided into 1-mm pieces. Single cells were obtained by mechanical squeezing through a filter mesh. After centrifugation of the isolated cells (5 min, 1200rpm), the cells were counted, seeded in 96-well plates and incubated overnight at 37 ° C and 5% CO 2 . The infection of the cells was carried out by addition of 10ng X4-tropher HIV stocks with simultaneous application of the corresponding inhibitor concentrations. The following day, 50 .mu.l of supernatant were taken ( "1 dpi") and stored at -80 0 C. The cells were then centrifuged (5 min, 1200 rpm)...
  • FIG. 1 is a diagrammatic representation of FIG. 1:
  • the Hsp90 inhibitor 17-AAG shows up to a concentration of 1OnM no cytotoxicity in CEM cells.
  • CD4 + T lymphoid cells CEM cells
  • AlamarBlue TM Invitrogen
  • FIG. 2 is a diagrammatic representation of FIG. 1
  • HeLaSS ⁇ cells transf ⁇ instance with subgenomic HIV-I expression vector pNLenvl show under 17-AAG influence in the virus fraction reduced Gag processing and increased Hsp70 expression in the cell fraction.
  • FIG. 3 is a diagrammatic representation of FIG. 3
  • Tonsil B (B) also showed no effect on X4-tropic HIV replication when incubated with InM PS341 or InM 17-AAG. In contrast to tonsil A, a significant reduction in virus replication could be detected in tonsil B even with the addition of 1OnM 17-AAG. In any combination of proteasome inhibitor PS341 with Hsp90 inhibitor 17-AAG, no viral replication could be detected.

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Abstract

The invention relates to a pharmaceutical composition containing at least one proteasome inhibitor and an inhibitor of protein folding enzymes as active components. Said substances are suitable for the treatment of acute and chronic infections with viruses that are pathogenic to humans and animals.

Description

Pharmazeutische Zusammensetzung zur Behandlung von Virusinfektionen und / oder Tumorerkrankungen durch Inhibition der Proteinfaltung und des Proteinabbaus.Pharmaceutical composition for the treatment of viral infections and / or tumors by inhibiting protein folding and protein degradation.
Beschreibungdescription
[0001] Die Erfindung betrifft eine pharmazeutische Zusammensetzung, die als wirksame Komponenten mindestens einen Proteasom-Inhibitor und einen Inhibitor von Proteinfaltungsenzymen enthält. Diese Mittel sind zur Behandlung von akuten und chronischen Infektionen mit für Mensch und Tier pathogenen Viren geeignet. Hierzu zählen insbesondere Erreger von Infektionserkrankungen wie AIDS, Hepatitis, hämorrhagische Fieber, SARS, Pocken, Masern, Polio oder Grippe. Gegenstand der Erfindung sind Mittel, die zum Einen als Wirkstoffe Inhibitoren der Proteinfaltung enthalten. Hierzu zählen Inhibitoren von zellulären Faltungsenzymen (den enzymatischen Chaperonen), wie auch Substanzen, welche die Faltung von Proteinen durch chemische Chaperone stören. Zum Anderen enthalten diese Mittel Komponenten, welche das Ubiquitin-Proteasom-System stören, insbesondere Mittel, welche das 26S Proteasom inhibieren. In Kombination dieser therapeutischen Mittel soll getrennt voneinander, oder gleichzeitig, die Effizienz der Proteinbiosynthese und der Abbau von falsch gefalteten Proteinen gestört werden. In der Summe dieser Wirkungen sollen gezielt entartete Tumorzellen und/oder akut und/oder chronisch Virus- infizierte Zellen in ihrer Vitalität beeinträchtigt, und somit in den programmierten Zelltod (Apoptose) dirigiert werden. Anwendungsgebiete sind die Behandlung von Virusinfektionen und / oder Tumorerkrankungen.The invention relates to a pharmaceutical composition containing as active components at least one proteasome inhibitor and an inhibitor of protein folding enzymes. These agents are useful in the treatment of acute and chronic human and animal pathogenic virus infections. These include in particular pathogens of infectious diseases such as AIDS, hepatitis, hemorrhagic fever, SARS, smallpox, measles, polio or influenza. The invention relates to agents which contain on the one hand as active ingredients inhibitors of protein folding. These include inhibitors of cellular folding enzymes (the enzymatic chaperones) as well as substances that interfere with the folding of proteins by chemical chaperones. On the other hand, these agents contain components that interfere with the ubiquitin-proteasome system, especially agents that inhibit the 26S proteasome. In combination of these therapeutic agents, separately or simultaneously, the efficiency of protein biosynthesis and degradation of misfolded proteins should be disturbed. In the sum of these effects, specifically degenerate tumor cells and / or acutely and / or chronically virus-infected cells should be impaired in their vitality, and thus be directed into programmed cell death (apoptosis). Areas of application are the treatment of viral infections and / or tumor diseases.
Stand der TechnikState of the art
[0002] Inhibitoren von Proteinfaltungsenzymen sind aus der Druckschrift WO 2005/063281 A2 bekannt.Inhibitors of protein folding enzymes are known from WO 2005/063281 A2.
[0003] Proteasom-Inhibitoren sind sowohl zur Behandlung von Tumorerkrankungen (z.B. US 6,083,903) als auch zur Behandlung von Virus-Infektionen beschrieben worden (WO 02/30455).Proteasome inhibitors have been described both for the treatment of tumor diseases (for example US Pat. No. 6,083,903) and for the treatment of viral infections (WO 02/30455).
[0004] Eine Kombination aus Inhibitoren von Proteinfaltungsenzymen und Proteasom- Inhibitoren ist bisher nicht beschrieben worden. Lediglich die Kombination nicht Proteasom- selektiver Protease-Inhibitoren mit Inhibitoren von Proteinfaltungsenzymen wurde in der Druckschrift WO 2005/063281 A2 erwähnt.[0004] A combination of inhibitors of protein folding enzymes and proteasome inhibitors has not previously been described. Only the combination of proteasome Selective protease inhibitors with inhibitors of protein folding enzymes was mentioned in WO 2005/063281 A2.
Aufgabe der ErfindungObject of the invention
[0005] Der Erfindung lag die Aufgabe zugrunde, neue pharmazeutische Zusammensetzungen zur Behandlung von Virusinfektionen und / oder Tumorerkrankungen bereitzustellen.The invention had the object of providing novel pharmaceutical compositions for the treatment of viral infections and / or tumors.
Lösung der AufgabeSolution of the task
[0006] Die Aufgabe wurde gemäß den Merkmalen der Patentansprüche gelöst. Die erfindungsgemäße Kombination aus Inhibitoren von Proteinfaltungsenzymen und Proteasom- Inhibitoren ist dem Stand der Technik überlegen. Erfindungsgemäß wurde eine pharmazeutische Zusammensetzung, die als wirksame Komponenten mindestens einen Inhibitor des Ubiquitin-Proteasom-Systems und einen Inhibitor von Proteinfaltungsenzymen oder eine Methode zur Beeinflussung der Proteinfaltung enthält, bereitgestellt.The problem has been solved according to the features of the claims. The combination of inhibitors of protein folding enzymes and proteasome inhibitors according to the invention is superior to the prior art. According to the invention, a pharmaceutical composition has been provided which contains as active components at least one inhibitor of the ubiquitin-proteasome system and an inhibitor of protein folding enzymes or a method for influencing protein folding.
[0007] Bei dem Inhibitor von Proteinfaltungsenzymen handelt es sich vorzugsweise um mindestens einen Inhibitor zellulärer Chaperone oder um mindestens eine chemische Substanz, welche die Proteinfaltung direkt beeinfiusst (chemisches Anti-Chaperon).The inhibitor of protein folding enzymes is preferably at least one inhibitor of cellular chaperones or at least one chemical substance which directly affects protein folding (chemical anti-chaperone).
[0008] Als Methode zur Beeinflussung der Proteinfaltung dient vorzugsweise die lokale Hyperthemie.As a method for influencing the protein folding is preferably the local hyperthermia.
[0009] Eine weitere bevorzugte Ausführungsform der Erfindung besteht darin, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti-Chaperonen Substanzen eingesetzt werden, welcheA further preferred embodiment of the invention is that substances are used as inhibitors of cellular chaperones or chemical anti-chaperones, which
a) die Faltung und proteolytische Reifung von Virusproteinen hemmen, regulieren oder anderweitig beeinflussen und dadurch die Freisetzung und die Replikation von Viren hemmen, speziell von Erregern von Infektionserkrankungen wie AIDS, Hepatitis, hämorrhagischem Fieber, SARS, Pocken, Masern, Polio, Herpesvirusinfektionen oder Grippe, oder b) die Vermehrung von entarten Zellen, speziell Tumorzellen, stören, indem diese durch Akkumulation von falsch gefalteten Proteinen in den programmierten Zelltod getrieben werden.a) inhibit, regulate or otherwise affect the folding and proteolytic maturation of viral proteins and thereby inhibit the release and replication of viruses, especially pathogens such as AIDS, hepatitis, haemorrhagic fever, SARS, smallpox, measles, polio, herpesvirus infections or influenza , or b) interfere with the proliferation of degenerate cells, especially tumor cells, by driving them into programmed cell death by accumulation of misfolded proteins.
[0010] Die erfindungsgemäße pharmazeutische Zusammensetzung ist dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti-Chaperonen Substanzen eingesetzt werden, die speziell die enzymatischen Aktivitäten von molekularen Faltungsenzymen der Wirtszellen beeinflussen. Die Zellen höherer Eukaryonten nehmen diese Inhibitoren oder Substanzen auf und blockieren nach Zellaufnahme die Proteinfaltung von Virusstrukturpoteinen und von Proteinen aus Tumorzellen. Die Inhibitoren oder Substanzen können in verschiedenen Formen in vivo oral, intravenös, intramuskulär, subkutan oder in verkapselter Form mit oder ohne Zell-Spezifität-tragenden Veränderungen verabreicht werden, aufgrund der Anwendung eines bestimmten Applikations- und/oder Dosis-Regimes eine geringe Zytotoxizität aufweisen, keine oder unbedeutende Nebenwirkungen auslösen, eine relative hohe metabolische Halbwertszeit und eine relative geringe Clearence-Rate im Organismus aufweisen.The pharmaceutical composition according to the invention is characterized in that as inhibitors of cellular chaperones or of chemical anti-chaperones substances are used which specifically influence the enzymatic activities of molecular folding enzymes of the host cells. The cells of higher eukaryotes receive these inhibitors or substances and, after cell uptake, block the protein folding of virus structure peats and of proteins from tumor cells. The inhibitors or substances may be administered in vivo in various forms, orally, intravenously, intramuscularly, subcutaneously or in encapsulated form with or without cell specificity-bearing alterations, due to the application of a particular application and / or dose regimen having low cytotoxicity , cause no or negligible side effects, have a relatively high metabolic half-life and a relatively low clearance rate in the organism.
[0011] Die erfindungsgemäße pharmazeutische Zusammensetzung ist weiterhin dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti- Chaperonen Substanzen eingesetzt werden, die a) in natürlicher Form aus Mikroorganismen oder anderen natürlichen Quellen isoliert werden oder b) durch chemische Modifikationen aus natürlichen Substanzen hervorgehen oder c) total-synthetisch hergestellt werden oder d) durch gentherapeutische Verfahren in vivo synthetisiert werden.The pharmaceutical composition according to the invention is further characterized in that are used as inhibitors of cellular chaperones or chemical anti-chaperones substances that are a) isolated in natural form from microorganisms or other natural sources, or b) by chemical modifications of natural substances or c) be prepared totally synthetically or d) be synthesized in vivo by gene therapy methods.
[0012] Die Inhibitoren zellulärer Chaperone oder die chemischen Anti-Chaperone stören die hoch organisierten Prozesse der Assemblierung und der proteolytischen Reifung von Virusstrukturproteinen und unterbinden dadurch die Freisetzung und die Produktion von infektiösen Nachkommenviren. Außerdem regulieren, stören oder blockieren diese Substanzen die Faltung von viralen Proteinen und/oder von Tumor-spezifischen Proteinen dadurch, dass die späten Prozesse der Virusreplikation wie Assemblierung, Knospung, proteolytische Reifung und Virusfreisetzung, gestört werden. Die proteolytische Prozessierung von Vorläuferproteinen der viralen Polyproteine wird dadurch gestört. Außerdem wird die Aktivität von viralen Proteasen blockiert. [0013] Eine weitere bevorzugte Ausführungsform der Erfindung besteht darin, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti-Chaperonen Substanzen eingesetzt werden, welche die Aktivitäten von zellulären Proteasen und/oder von Enzymen, wie zum Beispiel Ligasen, Kinasen, Hydro lasen, Glykosylierungsenzymen, Phosphatasen, DNAsen, RNAsen, Helikasen und Transferasen stören, die an der Virusreifung beteiligt sind. Die erfindungsgemäßen Inhibitoren zellulärer Chaperone bzw. die chemischen Anti-Chaperone besitzen ein breites Wirkspektrum und können daher als neuartige Breitbandvirostatika zur Vorbeugung und/oder zur Therapie von unterschiedlichen Virusinfektionen eingesetzt werden.The inhibitors of cellular chaperones or the chemical anti-chaperones disrupt the highly organized processes of assembly and proteolytic maturation of virus structural proteins and thereby inhibit the release and production of infectious progeny viruses. In addition, these substances regulate, disrupt or block the folding of viral proteins and / or tumor-specific proteins by disrupting the late processes of viral replication such as assembly, budding, proteolytic maturation and virus release. The proteolytic processing of precursor proteins of the viral polyproteins is thereby disturbed. In addition, the activity of viral proteases is blocked. A further preferred embodiment of the invention is that are used as inhibitors of cellular chaperones or of chemical anti-chaperones substances that lase the activities of cellular proteases and / or enzymes, such as ligases, kinases, Hydro, glycosylation enzymes , Phosphatases, DNAses, RNAses, helicases and transferases that are involved in virus maturation. The inhibitors of cellular chaperones according to the invention or the chemical anti-chaperones have a broad spectrum of activity and can therefore be used as novel broad-spectrum antivirals for the prevention and / or therapy of different viral infections.
[0014] Die pharmazeutische Zusammensetzung ist dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti-Chaperonen Substanzen eingesetzt werden, die zelluläre Chaperone wie Hitzeschockproteine (heat shock proteins (hsp)) blockieren oder hemmen, insbesondere die Aktivitäten der Hitzeschockproteine Hsp27, Hsp30, Hsp40, Hsp60, Hsp70, Hsp72, Hsp73, Hsp90, HsplO4 und Hsc70.The pharmaceutical composition is characterized in that are used as inhibitors of cellular chaperones or chemical anti-chaperones substances that block or inhibit cellular chaperones such as heat shock proteins (hsp) proteins, in particular the activities of the heat shock proteins Hsp27, Hsp30 , Hsp40, Hsp60, Hsp70, Hsp72, Hsp73, Hsp90, HsplO4 and Hsc70.
[0015] Als Inhibitoren zellulärer Chaperone können Substanzen eingesetzt werden, die folgenden Substanzklassen und deren Derivaten angehören: Geldanamycin (inhibiert Hsp90), Radicicol (Tyrosinkinase-Inhibitor; inhibiert Hsp90), Deoxyspergualin (inhibiert an Hsc70 und Hsp90), 4-PBA (4-Phenylbutyrat; Downregulation der Protein- und mRNA-Expression der Hsc70), Herbimycin A (Tyrosinkinase-Inhibitor mit Hsp72/73 Induktion), Epolactaene (Inhibitor der Hsp60), Scythe und Reaper (inhibieren Hsp70), Artemisinin (Inhibitor der Hsp90), CCT0180159 (als Pyrazole-Inhibitor von Hsp90) und SNX-2112 (Hsp90 Inhibitor), Radanamycin (Macrolidchimera von Radicicol und Geldanamycin), Novobiocin (Hsp90- Inhibitor), Quercetin (Inhibitor der Hsp70-Expression).As inhibitors of cellular chaperones, substances can be used which belong to the following substance classes and their derivatives: geldanamycin (inhibits Hsp90), radicicol (tyrosine kinase inhibitor, inhibits Hsp90), deoxyspergualin (inhibits Hsc70 and Hsp90), 4-PBA (4 Downregulation of protein and mRNA expression of Hsc70), herbimycin A (tyrosine kinase inhibitor with Hsp72 / 73 induction), epolactaene (inhibitor of Hsp60), scythe and reaper (inhibit Hsp70), artemisinin (inhibitor of Hsp90), CCT0180159 (as pyrazole inhibitor of Hsp90) and SNX-2112 (Hsp90 inhibitor), radanamycin (macrololidimer of radicicol and geldanamycin), novobiocin (Hsp90 inhibitor), quercetin (inhibitor of Hsp70 expression).
[0016] Als chemische Anti-Chaperone können Substanzen eingesetzt werden, welche die Proteinkonformation und die Faltung von viralen und/ oder Tumor-spezifischen Proteinen regulieren, stören oder blockieren. Dazu zählen z.B. Substanzen wie Glycerol, Trimethylamine, Betain, Trehalose oder deuteriertes Wasser (D2O). Weiterhin können Substanzen eingesetzt werden, welche für die Behandlung, Therapie und Hemmung von Infektionen mit unterschiedlichen humanpathogenen oder auch tierpathogenen Viren geeignet sind oder Substanzen, die für die Behandlung, Therapie und Hemmung von Infektionen mit Erregern von chronischen Infektionserkrankungen wie AIDS (HIV-I und HIV-2), von Hepatitis (HCV und HBV), von dem Erreger des "Severe Acute Respiratory Syndroms" (SARS), dem SARS-CoV (Coronavirus), von Pockenviren, von Erregern des viralen hämorrhagischen Fiebers (VHF) wie den Ebola- Viren als Vertreter der Familie der Filoviridae; von Grippe-Erregern wie dem Influenza- A-Virus, geeignet sind. Dazu zählen z.B. Cyclosporin A und / oder Tacrolimus.Substances which regulate, disrupt or block the protein conformation and the folding of viral and / or tumor-specific proteins can be used as chemical anti-chaperones. These include, for example, substances such as glycerol, trimethylamine, betaine, trehalose or deuterated water (D 2 O). Furthermore, substances can be used which are suitable for the treatment, therapy and inhibition of infections with different human pathogenic or animal-pathogenic viruses or substances which are used for the treatment, therapy and inhibition of infections with agents of chronic infectious diseases such as AIDS (HIV-I and HIV-2), hepatitis (HCV and HBV), the cause of the "Severe Acute Respiratory Syndrome" (SARS), the SARS-CoV (coronavirus), of poxviruses, of viral haemorrhagic fever (VHF) pathogens, as well as the Ebola virus as a member of the family Filoviridae; influenza-causing agents such as the influenza A virus. These include, for example, cyclosporin A and / or tacrolimus.
[0017] Die erfindungsgemäße pharmazeutische Zusammensetzung ist weiterhin dadurch gekennzeichnet, dass es sich bei den Hemmern des UPS um mindestens eine Substanz handelt, die a) speziell in Form von Proteasom-Inhibitoren die enzymatischen Aktivitäten des kompletten 26S Proteasom-Komplexes und der freien, nicht mit regulatorischenThe pharmaceutical composition according to the invention is further characterized in that the inhibitors of the UPS is at least one substance which a) especially in the form of proteasome inhibitors, the enzymatic activities of the complete 26S proteasome complex and the free, not with regulatory
Untereinheiten assemblierten 2OS katalytisch aktiven Proteasom-Struktur beeinflusst oder b) speziell die Wirkung von Ubiquitin-Ligasen hemmt oder c) speziell die Wirkung von Ubiquitin-Hydrolasen hemmt oder d) speziell die Wirkung von Ubiquitin-aktivierenden Enzymen hemmt oder e) speziell die Mono-ubiquitinylierung von Proteinen hemmt oder f) speziell die Poly-ubiquitinylierung von Proteine hemmtSubunits assembled 2OS influenced catalytically active proteasome structure or b) specifically inhibits the action of ubiquitin ligases or c) specifically inhibits the action of ubiquitin hydrolases or d) specifically inhibits the action of ubiquitin-activating enzymes or e) specifically the mono- ubiquitinylation of proteins or f) specifically inhibits the poly-ubiquitinylation of proteins
[0018] Die Proteasom-Inhibitoren werden von höheren Eukaryoten aufgenommen und treten nach Zellaufnahme mit den katalytischen Untereinheiten des Proteasoms in Wechselwirkung und blockieren dabei alle oder einzelne der proteolytischen Aktivitäten des Proteasoms - die Trypsin-, die Chymotrypsin- und/oder die Postglutamyl-Peptid hydrolysierenden Aktivitäten - innerhalb des 26S oder auch des 2OS Proteasom-Komplexes irreversibel oder reversibel.The proteasome inhibitors are taken up by higher eukaryotes and interact after cell uptake with the catalytic subunits of the proteasome and block all or some of the proteolytic activities of the proteasome - the trypsin, the chymotrypsin and / or the postglutamyl peptide hydrolyzing activities - irreversible or reversible within the 26S or the 2OS proteasome complex.
[0019] Als Proteasom-Inhibitoren werden Substanzen eingesetzt, die a) in natürlicher Form aus Mikroorganismen oder anderen natürlichen Quellen isoliert werden; oder b) durch chemische Modifikationen aus natürlichen Substanzen hervorgehen; oder c) total-synthetisch hergestellt werden; oder d) durch gentherapeutische Verfahren in vivo synthetisiert werden; oder e) durch gentechnische Verfahren in vitro oder f) in Mikroorganismen hergestellt werden. [0020] Bei den Proteasom-Inhibitoren handelt es sich um Verbindungen, die folgenden Substanzklassen angehören: a) natürlich vorkommende Proteasom-Inhibitoren:Proteasome inhibitors used are substances which a) are isolated in natural form from microorganisms or other natural sources; or b) result from chemical modifications of natural substances; or c) produced totally synthetically; or d) be synthesized in vivo by gene therapy methods; or e) by genetic engineering in vitro or f) in microorganisms. The proteasome inhibitors are compounds which belong to the following substance classes: a) naturally occurring proteasome inhibitors:
- Peptid-Derivate, welche C-terminal Epoxyketon-Strukturen enthalten; oder - ß-Lacton-Derivate; oderPeptide derivatives containing C-terminal epoxy ketone structures; or β-lactone derivatives; or
- Aclacinomycin A (auch bezeichnet als Aclarubicin); oderAclacinomycin A (also referred to as Aclarubicin); or
- Lactacystin und dessen chemische modifizierte Varianten, wie der Zellmembran- penetrierenden Variante "Clasto lactacystein ß-Lacton"- Lactacystin and its chemically modified variants, such as the cell membrane-penetrating variant "Clasto lactacysteine β-lactone"
b) synthetisch hergestellte Proteasom-Inhibitoren: modifizierte Peptidaldehyde wie N-carbobenzoxy-L-leucinyl-L-leucinyl-L-leucinal (auch bezeichnet als MG 132 oder zLLL), dessen Borsäure-Derivat MG232; N- Carbobenzoxy-Leu-Leu-Nva-H (bezeichnet als MGl 15; N-AcetylL-Leuzinyl-L- Leuzinyl-L-Norleuzinal (bezeichnet als LLnL), N-Carbobenzoxy-Ile-Glu(OBut)-Ala- Leu-H (auch bezeichnet als PSI);b) synthetically prepared proteasome inhibitors: modified peptide aldehydes such as N-carbobenzoxy-L-leucinyl-L-leucinyl-L-leucinal (also referred to as MG 132 or zLLL), its boric acid derivative MG232; N-carbobenzoxy-Leu-Leu-Nva-H (termed MGl 15; N-acetyl-leucinyl-L-leucinyl-L-norleucinal (referred to as LLnL), N-carbobenzoxy-Ile-Glu (OBut) -la-Leu -H (also referred to as PSI);
c) Peptide, welche C-terminal eine α, ß -Epoxyketon-Struktur tragen, ferner Vinylsulfone wie Carbobenzoxy-L-Leucinyl-L-Leucinyl-L-Leucin-vinyl-sulfon oder 4-Hydroxy-5- iodo-3-nitrophenylactetyl-L-Leucinyl-L-Leucinyl-L-Leucin-vinyl-sulfon (NLVS);c) peptides which carry an α, β-epoxyketone C-terminal structure, furthermore vinylsulfones such as carbobenzoxy-L-leucinyl-L-leucinyl-L-leucine-vinyl-sulfone or 4-hydroxy-5-iodo-3-nitrophenylactetyl -L-leucinyl-L-leucinyl-L-leucine-vinylsulfone (NLVS);
d) Glyoxal- oder Borsäure-Reste wie Pyrazyl-CONH(CHPhe)CONH(CHisobutyl)B(OH)2) sowie Dipeptidyl-Borsäure-Derivate oderd) glyoxal or boric acid radicals such as pyrazyl-CONH (CHPhe) CONH (CHisobutyl) B (OH) 2 ) as well as dipeptidyl-boric acid derivatives or
e) Pinacol-Ester - wie Benzyloxycarbonyl(Cbz)-Leu-Leu-boroLeu-Pinacol-Ester.e) Pinacol esters - such as benzyloxycarbonyl (Cbz) -Leu-Leu-boroLeu-Pinacol esters.
[0021] Besonders geeignete Proteasom-Inhibitoren sind die Epoxyketone Epoxomicin (Epoxomycin, Molekülformel: C28H86N4O7) und/oder Eponemycin (Eponemicin, Molekülformel: C20H36N2O5) oder Proteasom-Inhibitoren aus der PS-Serie die Verbindungen: a) PS-519 als ß-Lacton- sowie als Lactacystin-Derivat die Verbindung IR-[IS, 4R, 5S]]- l-(l-Hydroxy-2-methylpropyl)-4-propyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione -[0021] Particularly suitable proteasome inhibitors the epoxyketones epoxomicin (epoxomycin, molecular formula: C28H86N4O7) and / or eponemycin (Eponemicin, molecular formula: C 20 H 36 N 2 O 5) or proteasome inhibitors from the PS series the compounds: a ) PS-519 as β-lactone as well as lactacystin derivative the compound IR- [IS, 4R, 5S]] - 1- (1-hydroxy-2-methylpropyl) -4-propyl-6-oxa-2-azabicyclo [3.2.0] heptane-3,7-dione -
Molekülformel C12H19NO4- und/oder b) PS-314 als Peptidyl-Borsäure-Derivat die Verbindung N-Pyrazinecarbonyl-L- Phenylalanin-L-Leuzin-Borsäure - Molekülformel C19H25BN4O4 - und/oder c) PS-273 (Morpholin-CONH-(CH-Naphthyl)-CONH-(CH-isobutyl)-B(OH)2) und dessen Enantiomer PS-293 und/oder d) die Verbindung PS-296 (8-Quinolyl-sulfonyl-CONH-(CH-Napthyl)-CONH(-CH- isobutyl)-B(OH)2) und/oder e) PS-303 (NH2(CH-Naphthyl)-CONH-(CH-isobutyl)-B(OH)2) und/oder f) PS-321 als (Morpholin-CONH-(CH-Napthyl)-CONH-(CH-Phenylalanin)-B(OH)2); - und/oder g) PS-334 (CH3-NH-(CH-Naphthyl-CONH-(CH-Isobutyl)-B(OH)2) und/oder h) die Verbindung PS-325 (2-Quinol-CONH-(CH-Aomo-Phenylalanin)-CONH-(CH- isobutyl)-B(OH)2) und/oder i) PS-352 (Phenyalanin-CH2-CH2-CONH-(CH-Phenylalanin)-CONH-(CH-isobutyl)l-Molecular Formula C12H1 9 NO4- and / or b) PS-314 as peptidyl-boric acid derivative the compound N-pyrazinecarbonyl-L-phenylalanine-L-leucine-boric acid - molecular formula C1 9 H25BN4O4 - and / or c) PS-273 (morpholine-CONH- (CH-naphthyl) -CONH- (CH-isobutyl) -B (OH) 2 ) and its enantiomer PS-293 and / or d) the compound PS-296 (8-quinolyl -sulfonyl-CONH- (CH-Napthyl) -CONH (-CH-isobutyl) -B (OH) 2 ) and / or e) PS-303 (NH 2 (CH-Naphthyl) -CONH- (CH-isobutyl) - B (OH) 2 ) and / or f) PS-321 as (morpholine-CONH- (CH-Napthyl) -CONH- (CH-phenylalanine) -B (OH) 2 ); - and / or g) PS-334 (CH 3 -NH- (CH-naphthyl-CONH- (CH-isobutyl) -B (OH) 2 ) and / or h) the compound PS-325 (2-quinol-CONH - (CH-Aomo-phenylalanine) -CONH- (CH-isobutyl) -B (OH) 2 ) and / or i) PS-352 (phenylalanine-CH 2 -CH 2 -CONH- (CH-phenylalanine) -CONH- (CH-isobutyl) l-
B(OH)2) und/oder j) PS-383 (Pyridyl-CONH-(CH/?F-Phenylalanin)-CONH-(CH-isobutyl)-B(OH)2) eingesetzt werden.B (OH) 2 ) and / or j) PS-383 (pyridyl-CONH- (CH /? F-phenylalanine) -CONH- (CH-isobutyl) -B (OH) 2 ).
[0022] Die beschriebenen pharmazeutischen Zusammensetzungen eignen sich als Arzneimittel oder zur Herstellung von Mitteln zur Behandlung von Virusinfektionen und / oder Tumorerkrankungen. Eine Kombination mit anderen Mitteln, die zur Behandlung von Virusinfektionen und / oder Tumorerkrankungen ist ebenfalls möglich.The pharmaceutical compositions described are useful as drugs or for the preparation of agents for the treatment of viral infections and / or tumor diseases. A combination with other agents that is also available to treat viral infections and / or tumors.
[0023] Die erfindungsgemäße Verwendung dieser Mittel erfolgt in Form vonThe inventive use of this agent is in the form of
Inhalateninhalants
Depotformendepot forms
- Pflaster - in mikroelektronischen Systemen („intelligente Pille")- plasters - in microelectronic systems ("intelligent pill")
[0024] Eine Verwendung in der Onkologie und / oder Onkologie und Virologie zur Behandlung von[0024] Use in oncology and / or oncology and virology for the treatment of
- Glioblastom (Maligne Hirntumore) - - Mamma-CA (CA = Carcinom)- glioblastoma (malignant brain tumors) - - mamma CA (CA = carcinoma)
- - Kopf-, HaIs-CA- - Head, HaIs-CA
- - Plattenepithel-CA- - squamous epithelium CA.
- - Ovarial-CA- - ovarian CA.
- Bronchial-CA (kleinzellig, großzellig) - - Schilddrüsen-CA - Lungen-CA- Bronchial CA (small cell, large cell) - - Thyroid CA. - Lung CA.
- - Kolon-CA- - Colon CA.
- Pankreas-CA- pancreatic CA.
- - Leukemie (AML, ALL, CML, CLL) - akute myeloische, chronische acute lymphatische, chronische- - Leukemia (AML, ALL, CML, CLL) - acute myeloid, chronic acute lymphocytic, chronic
- Lymphom (Non-Hodgkin)- Lymphoma (Non-Hodgkin)
- Zervix-Ca- Cervical Ca
- Neuroblastom - - Haut-CA (Melanom)- neuroblastoma - - skin CA (melanoma)
- - Prostata-CA- - Prostate CA.
- - Blasen-CA- - Bladder CA
- Sarkome (Knochen u. Weichteile)- Sarcomas (bones and soft tissues)
- Zwerchfell-CA - - Gastrointestinale-CA (z. B. Magen, Oesophagus)- diaphragmatic CA - - gastrointestinal CA (eg, stomach, esophagus)
- Hoden-Ca- Testicle Ca
- Metastasen (z. B. Knochenmark)- metastases (eg bone marrow)
- Lymphoma- Viren- Lymphoma viruses
- Herpes Simplex - - Zytomegalie- Herpes Simplex - - Cytomegalovirus
- Varizellen- Varicella
- Varicella Zoster- Varicella zoster
- Masern- Measles
- Lassa-Fieber - - Aids- Lassa fever - - AIDS
- Mumps (- Meningitis, - Orchitis)- mumps (- meningitis, - orchitis)
- Enteritis ; Grippe (alle Formen)- enteritis; Flu (all forms)
- Enzephalitis- Encephalitis
- - Hepatitis (A, B, C, D, E, G) - - Röteln- - Hepatitis (A, B, C, D, E, G) - - Rubella
- Coxsackie B- Coxsackie B
- Polio (-Myelitis)- polio (myelitis)
- Enzephalomyelitis- Encephalomyelitis
- Pankreatitits - - Pneumonie- Pancreatitis - - Pneumonia
- Myokarditis - Tropenkrankheiten (Virale)- Myocarditis - tropical diseases (viral)
- alle doppel- und einsträngigen DNS und RNS humanpathogenen Viren ist möglich.- All double- and single-stranded DNA and RNA human-pathogenic viruses is possible.
[0025] Überraschenderweise hat sich herausgestellt, dass durch die Störung der Mechanismen der Proteinfaltung verstärkt fehlgefaltete Proteine gebildet werden. Diese Fehlprodukte der Proteinbiosynthese werden normalerweise durch das Ubiquitin-Proteasom-System (UPS) abgebaut und somit aus dem Zellstoffwechsel entfernt. Bei Inhibition des UPS, zum Beispiel durch Proteasom-Inhibitoren und/oder durch Inhibitoren von Ubiquitin-Ligasen, werden diese, in der Regel poly-ubiquitinylierten und falsch gefalteten Fehlprodukte der Proteinbiosynthese in der Zelle akkumulieren und dadurch vielfältige Störungen des Zellstoffwechsel auslösen, welche in der Summe der Wirkungen die betreffende Zelle bevorzugt in den programmierten Zelltod (Apoptose) treiben wird. Da sowohl in Virus- infizierten, als auch in sich rasch teilenden Tumorzellen eine besonders hohe Rate der Proteinbiosynthese vorliegt, werden insbesondere solche Zellen verstärkt auf die Wirkung von Inhibitoren des UPS und der Proteinfaltung reagieren, während normale und gesunde Zellen von diesen Inhibitoren weitestgehend unbeeinfiusst bleiben. Darauf beruht der grundsätzliche Wirkmechanismus des erfindungsgemäß vorgeschlagenen neuen Therapieverfahrens.Surprisingly, it has been found that increased misfolded proteins are formed by the disruption of the mechanisms of protein folding. These protein biosynthesis defects are normally degraded by the ubiquitin-proteasome (UPS) system and thus removed from the cell metabolism. Upon inhibition of the UPS, for example by proteasome inhibitors and / or by inhibitors of ubiquitin ligases, these, usually poly-ubiquitinated and misfolded protein biosynthesis defective products, will accumulate in the cell, thereby triggering a variety of cellular metabolism disorders the sum of the effects will drive the cell in question preferably into programmed cell death (apoptosis). Since a particularly high rate of protein biosynthesis is present both in virus-infected and in rapidly dividing tumor cells, in particular such cells will increasingly respond to the effect of inhibitors of UPS and protein folding, while normal and healthy cells of these inhibitors remain largely unaffected , This is the basis of the fundamental mechanism of action of the new therapeutic method proposed according to the invention.
[0026] In einer besonderen Ausführungsform der Erfindung wird die Wirkung dieser Inhibitoren zur Behandlung von Plasmazytomzellen von Patienten mit Multiplen Myelom benutzt. Diese B-Zelltumore zeichnen sich durch eine extrem hohe Synthese-Rate an Immunglobulinen aus. Bekannter Maßen sind diese Plasmazytomzellen besonders sensitiv gegenüber der Behandlung mit Proteasom-Inhibitoren. Daher werden Proteasom-Inhibitoren, insbesondere in Form von Borsäure-Peptiden (Handelsname Velcade) erfolgreich für die Behandlung von Multiplen Myelom eingesetzt. Allerdings muss bei der Behandlung mit Proteasom-Inhibitoren ein sehr enges therapeutisches Fenster berücksichtigt werden, da die Grenze zwischen der therapeutischen Dosis und der tolerierbaren toxischen Dosis sehr eng ist. Durch die Behandlung mit Inhibitoren der Proteinfaltung werden solche Plasmazytomzellen für die Wirkung auf Proteasom-Inhibitoren sensibilisiert. Durch die Kombination von Proteasom-Inhibitoren und Inhibitoren der Proteinfaltung wird die Wirkung beider Wirkstoffe in synergistischer Weise verstärkt. Gleichzeitig können beide Medikamente in sub-toxischen Dosen mit höherer Wirksamkeit eingesetzt werden, was in der Summe die Erfolgsaussichten der Therapie wesentlich erhöht. [0027] Die erfindungsgemäße Lösung bietet gegenüber dem Stand der Technik folgende Vorteile:In a particular embodiment of the invention, the effect of these inhibitors for the treatment of Plasmazytomzellen of patients with multiple myeloma is used. These B-cell tumors are characterized by an extremely high synthesis rate of immunoglobulins. As is known, these plasmacytoma cells are particularly sensitive to the treatment with proteasome inhibitors. Therefore, proteasome inhibitors, especially in the form of boric acid peptides (trade name Velcade) are used successfully for the treatment of multiple myeloma. However, when treating with proteasome inhibitors, a very narrow therapeutic window must be taken into account because the boundary between the therapeutic dose and the tolerable toxic dose is very narrow. By treatment with inhibitors of protein folding, such plasmacytoma cells are sensitized for the action on proteasome inhibitors. The combination of proteasome inhibitors and protein folding inhibitors synergistically enhances the action of both agents. At the same time, both drugs can be used in sub-toxic doses with greater efficacy, which in total significantly increases the chances of success of the therapy. The solution according to the invention offers the following advantages over the prior art:
- Vermeidung von Resistenzen - Heilung bestimmter Krankheiten- Avoidance of drug resistance - cure certain diseases
- Höhere Responderrate- higher responder rate
- Behandlung mehrerer Tumorformen (Leichte, mittlere, schwere Fälle)- treatment of several types of tumors (mild, moderate, severe cases)
[0028] Eine weitere bevorzugte Ausführungsform der Erfindung betrifft die anti- virale Wirkung in der Kombination beider Wirkstoffe. Es ist bekannt, dass Proteasom-Inhibitoren dieA further preferred embodiment of the invention relates to the antiviral effect in the combination of both active ingredients. It is known that proteasome inhibitors the
Replikation von humanen Immundefiziensviren (HIV) und anderen Viren stört, indem es dieReplication of human immunodeficiency virus (HIV) and other viruses by interfering with the
Akkumulation von falsch gefalteten Gag-Proteinen induziert, welche mit den geordnetenAccumulation of misfolded gag proteins induced with the ordered
Prozessen der Assemblierung und Freisetzung von Nachkommenviren interferiert. Diese therapeutische Wirkung von Proteasom-Inhibitoren wird wesentlich verstärkt, wenn die virus infizierte Zelle gleichzeitig mit Inhibitoren der Proteinfaltung behandelt wird. Dadurch erhöht sich die Zahl an falsch gefalteten Strukturproteinen des Virus, welche in einem trans- negativen Mechanismus, sozusagen in einer Prion-ähnlichen Wirkungsweise, verstärkt dieProcesses of assembly and release of progeny viruses. This therapeutic effect of proteasome inhibitors is significantly enhanced if the virus-infected cell is treated simultaneously with inhibitors of protein folding. This increases the number of misfolded structural proteins of the virus, which in a trans-negative mechanism, so to speak in a prion-like mode of action, increases the
Assemblierung von Virusproteinen und dadurch die Formierung von Nachkommenviren stören wird. Diese Ausführungsform der Erfindung ist für alle Virusinfektionen, bei denen ein geordneter Zusammenbau von neu synthetisierten Virusstrukturporteinen stattfindet, allgemein gültig.Assembly of viral proteins and thereby disrupt the formation of progeny viruses. This embodiment of the invention is generally valid for all viral infections in which an ordered assembly of newly synthesized viral structure ports takes place.
[0029] Die Erfindung soll anhand von Ausführungsbeispielen näher erläutert werden, ohne sie auf diese Beispiele zu beschränken.The invention will be explained in more detail by means of embodiments, without limiting them to these examples.
Ausführungsbeispieleembodiments
Beispiel 1: Der Hsp90-Inhibitor 17- AAG zeigt bis zu einer Konzentration von 1OnM keine Zyto-Toxizität in CEM-Zellen.Example 1: Hsp90 inhibitor 17-AAG shows no cytotoxicity in CEM cells up to a concentration of 10 mM.
[0030] CD4+-T-lymphatische Zellen (CEM-Zellen) wurden in einer 96-well-Platte in einer Dichte von IxIO4 Zellen je lOOμl ausgesät. Zum Medium (siehe Beispiel 4a) wurden zuvor entsprechende Mengen 17-AAG gegeben, um Endkonzentrationen von lμM; 10OnM; 1OnM; InM; 0.InM und 0.0 InM 17-AAG zu erreichen. Nach 30-stündiger Inkubation bei 37°C und 5% CO2 wurden 10μl AlamarBlue™ (Invitrogen) zugegeben und alle Ansätze für weitere 4 Stunden bei 37°C inkubiert. Das Maß für die Vitalität der CEM-Zellen (angegeben in MTT CEM) unter 17-AAG Einfluss konnte durch die Messung des Farbumschlages des Mediums mittels Fluoreszenzmessung bei 530/590nm bestimmt werden. Alle Ansätze erfolgten in Triplikat.CD4 + T lymphoid cells (CEM cells) were seeded in a 96-well plate at a density of IxIO 4 cells per 100μl. To the medium (see Example 4a) were previously added appropriate amounts of 17-AAG to final concentrations of lμM; 10OnM; 1OnM; InM; 0.InM and 0.0 InM 17-AAG reach. After 30 hours incubation at 37 ° C and 5% CO 2 was added to 10 μl AlamarBlue ™ (Invitrogen) and all batches were incubated for a further 4 hours at 37 ° C. The measure of the vitality of the CEM cells (indicated in MTT CEM) under 17-AAG influence could be determined by measuring the color change of the medium by means of fluorescence measurement at 530/590 nm. All approaches were in triplicate.
Beispiel 2: pNLenvl-transfizierte HeLaSSό-Zellen zeigen unter 17-AAG-Einfluß in der Virusfraktion eine verminderte Gag-Prozessierung und eine verstärkte Hsp70-Expression in der Zellfraktion.Example 2: pNLenvl-transfected HeLaSSό cells show reduced Gag processing and increased Hsp70 expression in the cell fraction under 17-AAG influence in the virus fraction.
[0031] Zur biochemischen Analyse des Einflusses von 17-AAG auf die Kinetik der Gag- Prozessierung und Virusfreisetzung wurden Zeitkinetiken durchgeführt. Die experimentellen Details der Kultivierung, Transfektion, Medienwechsel und der Zeitkinetik sind in Beispiel 4a/b angegeben. Hierzu wurden Kulturen von HeLaSSό-Zellen eingesetzt, welche mit pNLenvl (Schubert et al, 1995) transfiziert wurden. Nach Inkubation in 17-AAG-haltigem Medium (10OnM 17- AAG), bzw. Inhibitor- freiem Medium, wurde die Kinetik nach distinkten Waschschritten und Aliquotierung der Ansätze begonnen. Aliquote Zellkulturen wurden zu jedem Zeitpunkt gewonnen und durch Zentrifugation in Zell-, Virus- und Zellkulturüberstand- Fraktionen aufgetrennt. Die HIV-Proteine wurden im SDS-PAGE aufgetrennt, auf PVDF- Membran transferiert und anschließend durch Antikörper- vermittelte Chemo luminiszenz auf Röntgenfilmen sichtbar gemacht.For the biochemical analysis of the influence of 17-AAG on the kinetics of gag processing and virus release time kinetics were performed. The experimental details of cultivation, transfection, media change and time kinetics are given in Example 4a / b. For this purpose, cultures of HeLaSSό cells were used, which were transfected with pNLenvl (Schubert et al, 1995). After incubation in 17-AAG-containing medium (10OnM 17-AAG), or inhibitor-free medium, the kinetics was started after distinct washing steps and aliquoting of the batches. Aliquots of cells were collected at each time point and separated by centrifugation into cell, virus and cell culture supernatant fractions. The HIV proteins were separated by SDS-PAGE, transferred to PVDF membrane, and then visualized by X-ray on antibody-mediated chemo-luminescence.
Beispiel 3: 17-AAG, als auch die Kombination mit PS341, hemmt die Virusreplikation von X4-trophen HI-Viren im HLAC-Modell.Example 3: 17-AAG, as well as the combination with PS341, inhibits virus replication of X4-tropic HI viruses in the HLAC model.
[0032] Humane Tonsillen wurden mazeriert und in 96-well Platten überführt. Nach eintägiger Inkubation wurden die Zellen mit X4-trophen HI-Viren infiziert, mit den entsprechenden Inhibitoren versetzt und tags darauf gewaschen. Diese Schritte, wie auch die Folgenden, sind unter Beispiel 4c-d im Detail beschrieben. Zu jedem Kinetikpunkt wurden 150μl des Mediums entfernt und bei -8O0C zur RT-Messung aufbewahrt. Das wieder zugegeben Medium enthielt die für den speziellen Ansatz nötigen Konzentrationen an Inhibitoren.Human tonsils were macerated and transferred to 96-well plates. After one day of incubation, the cells were infected with X4-tropic HI viruses, added with the appropriate inhibitors and washed the next day. These steps, as well as the following, are described in detail under Example 4c-d. For each kinetic point of the medium was removed and stored at -8O 150μl 0 C to RT measurement. The re-added medium contained the concentrations of inhibitors necessary for the particular approach.
[0033] Nach 15 Tagen wurde der Anteil funktioneller gebildeter HI -Viren mittels RT-Assays (siehe Beispiel 4e) aus den aufbewahrten Überständen ermittelt.After 15 days, the proportion of functional HI virus formed by RT assays (see Example 4e) was determined from the stored supernatants.
11 Beispiel 4: Material und Methoden11 Example 4: Material and Methods
Beispiel 4a: ZellkulturExample 4a: cell culture
[0034] CEM-Zellen wurden in RPMI 1640 mit 10% (V /V) fötalem Kälberserum, 2 mM L- Glutamin, 100 U ml-1 Penicillin und 100 μg ml-1 Streptomycin kultiviert.CEM cells were cultured in RPMI 1640 with 10% (v / v) fetal calf serum, 2 mM L-glutamine, 100 U ml-1 penicillin and 100 μg ml-1 streptomycin.
[0035] Heia-Zellen (ATCC CCL2) wurden in Dulbeccos' modifizierten Eagle's Medium (DMEM) mit 10% fötalem Kälberserum, 2 mM L-Glutamin, 100 U ml-1 Penicillin, und 100 μg/ml Streptomycin kultiviert.Heia cells (ATCC CCL2) were cultured in Dulbecco's modified Eagle's Medium (DMEM) with 10% fetal calf serum, 2 mM L-glutamine, 100 U ml-1 penicillin, and 100 μg / ml streptomycin.
[0036] Tonsilläre Zellen wurden in RPMI 1640 mit 15% (V /V) fötalem Kälberserum, 2 mM L- Glutamin, 100 U ml-1 Penicillin, 100 μg ml-1 Streptomycin, 2.5 μg/ml Fungizone, ImM Sodiumpyrovate, 1% MEM Non-Essential Aminosäure-Lösung und 50μg/ml Gentanamycin kultiviert („Tonsillenmedium").Tonsillar cells were transfected into RPMI 1640 with 15% (v / v) fetal calf serum, 2 mM L-glutamine, 100 U ml-1 penicillin, 100 μg ml-1 streptomycin, 2.5 μg / ml fungizone, ImM sodium pyrovate, 1 % MEM non-essential amino acid solution and 50μg / ml gentanamycin cultured ("tonsil medium").
Beispiel 4b: Transfektion, Medienwechsel und Kinetik [0037] Heia-Zellen (ATCC CCL2) wurden mittels pNLΔenv-Lipofectamine2000 Gemisch in OPTI-MEM transfiziert. Nach 8-stündiger Inkubation in 37°C und 5% CO2 wurde ein Medienwechsel vollzogen. Bei einem der beiden Ansätze wurde dem Medium eine Endkonzentration von 10OnM 17-AAG zugegeben und für weitere 16 Stunden inkubiert. Nach distinkten Waschschritten in PBS wurden die entsprechenden Zeitpunkte aliquotiert. Zu den entsprechenden Zeitpunkten wurden die Zellen durch Zentrifugation (5min; 5000rpm) vom Überstand getrennt und später mittels CHAPS/DOC-Lyse (3min auf Eis) lysiert. Die VLP's im Überstand wurden über ein 20%-iges Sucrose-Kissen pelletiert (90min; 14000rpm), und wie auch die Lysate der Zellpellets mittels 10%-iger SDS-PAGE aufgetrennt, per Nass-Blot auf PVDF-Membran überführt und in 10%-igem Milchpulver (in PBS-Tween 0.1%) geblockt. Die Detektion der HIV- bzw. Zeil-spezifischen Proteine erfolgte über spezifische Antikörper (gegen Hsp70; Hsp90; p24; PR55; ß-Aktin). Durch die Reaktion mit Sekundärantikörpern und deren gekoppelter Chemo luminiszenz konnten die Signale auf Röntgenfilmen detektiert werden.Example 4b: Transfection, change of media and kinetics Heia cells (ATCC CCL2) were transfected into OPTI-MEM by means of pNLΔenv-Lipofectamine 2000 mixture. After 8 hours of incubation in 37 ° C and 5% CO 2 , a change of media was performed. In one of the two batches a final concentration of 10OnM 17-AAG was added to the medium and incubated for a further 16 hours. After distinct washes in PBS, the respective times were aliquoted. At the appropriate time points, the cells were separated from the supernatant by centrifugation (5min, 5000rpm) and later lysed by CHAPS / DOC lysis (3min on ice). The VLP's in the supernatant were pelleted on a 20% sucrose pad (90 min; 14000 rpm) and, like the cell pellet lysates, separated by 10% SDS-PAGE, transferred to PVDF membrane by wet blot and placed in 10 % milk powder (in PBS-Tween 0.1%) blocked. Detection of the HIV or cell-specific proteins was carried out via specific antibodies (against Hsp70, Hsp90, p24, PR55, β-actin). Through the reaction with secondary antibodies and their coupled chemo luminescence, the signals could be detected on X-ray films.
Beispiel 4c: Transfektion und Gewinnung von VirusstocksExample 4c: Transfection and Recovery of Virus Stocks
[0038] Für die Herstellung von Virus-Präparaten wurde Plasmid-DNA von molekularer HIV- 1-DNA unter Anwendung der Kalzium-Phosphat-Präzipitationsmethode in HeLa-Zellen transfiziert. Dazu wurden konfluente Kulturen von Heia-Zellen (5xlO6 Zellen) mit 25-μg Plasmid DNA in Kalziumphosphat-Kristallen, hergestellt nach einer Methode von Graham und van der Eb (1973), inkubiert und anschließend einem Glycerol-Schock nach Gorman et al. (1982) unterzogen. Für die Gewinnung von konzentrierten Viruspräparaten wurden zwei Tage nach Transfektion die Zellkulturüberstände geerntet. Anschließend wurden die Zellen sowie deren Bestandteile durch Zentrifugation (1,000 x g, 5 min, 4oC) und Filtration (0.45 μm Porengröße) abgetrennt. Viruspartikel wurden durch Ultra-Zentrifugation (Beckman SW55 Rotor, 1.5 hr, 35,000 rpm, 10oC) pelletiert und nachfolgend in 1 ml of DMEM Medium resuspendiert. Die Viruspräparate wurden durch Filtration sterilisiert (0.45 μm Porengröße) und portioniert eingefroren (-800C). Einzelne Viruspräparate wurden durch Bestimmung der Reverse Transkriptase- Aktivität, und zwar anhand eines bereits beschriebenen Tests (Willey et al, 1988), unter Verwendung von [32P]-TTP Einbau in ein oligo(dT)-poly(A) Template standardisiert.For the preparation of virus preparations, plasmid DNA from HIV-1 molecular DNA was transfected into HeLa cells using the calcium-phosphate precipitation method. These were confluent cultures of Heia cells (5x10 6 cells) with 25 μg plasmid DNA in calcium phosphate crystals, prepared by a method of Graham and van der Eb (1973), and then a glycerol shock according to Gorman et al. (1982). For the recovery of concentrated virus preparations, the cell culture supernatants were harvested two days after transfection. Subsequently, the cells and their components were separated by centrifugation (1.000 xg, 5 min, 4oC) and filtration (0.45 micron pore size). Virus particles were pelleted by ultra-centrifugation (Beckman SW55 rotor, 1.5 hr, 35,000 rpm, 10oC) and subsequently resuspended in 1 ml of DMEM medium. The virus preparations were sterilized by filtration (0.45 .mu.m pore size) and portioned frozen (-80 0 C). Single virus preparations were standardized by determination of reverse transcriptase activity using a previously described assay (Willey et al, 1988) using [32 P] -TTP incorporation into an oligo (dT) -poly (A) template.
Beispiel 4d: HLAC-Modell (Gewinnung; Infektion; Kinetik)Example 4d: HLAC model (extraction, infection, kinetics)
[0039] Das Tonsillengewebe wurde in PBS gewaschen, mittels Skalpells von Blutgerinseln gesäubert und in l-2mm große Stücke zerteilt. Einzelne Zellen wurden durch mechanische Pressung durch ein Filternetz erzielt. Nach erfolgter Zentrifugation der vereinzelten Zellen (5min, 1200rpm) wurden die Zellen gezählt, in 96-well-Platten ausgesät und über Nacht bei 37°C und 5% CO2 inkubiert. Die Infektion der Zellen erfolgte durch Zugabe von 10ng X4- tropher HIV-Stocks bei gleichzeitiger Applikation der entsprechenden Inhibitorkonzentrationen. Am folgenden Tag wurden 50μl Überstand entnommen („1dpi") und bei -800C aufbewahrt. Die Zellen wurden daraufhin zentrifugiert (5min; 1200rpm) und weitere 50μl Überstand entfernt. Nach Resuspension der Zellen in lOOμl Tonsillenmediums wurde dieser Waschschritt zweifach wiederholt. Nach Zugabe von Tonsillen-Medium mit den entsprechenden Inhibitorkonzentrationen wurden die Zellen erneut bei 37°C und 5%CO2 inkubiert. An den Tagen 3, 6, 9 und 12 wurden 150μl Medium entnommen, bei -800C aufbewahrt und 150μl Medium mit den entsprechenden Inhibitorkonzentrationen zugegeben. An Tag 15 wurden lediglich 150μl Überstand abgenommen, bei -800C aufbewahrt und die Zellen danach entsorgt.The tonsil tissue was washed in PBS, cleaned by scalpels of blood clots and divided into 1-mm pieces. Single cells were obtained by mechanical squeezing through a filter mesh. After centrifugation of the isolated cells (5 min, 1200rpm), the cells were counted, seeded in 96-well plates and incubated overnight at 37 ° C and 5% CO 2 . The infection of the cells was carried out by addition of 10ng X4-tropher HIV stocks with simultaneous application of the corresponding inhibitor concentrations. The following day, 50 .mu.l of supernatant were taken ( "1 dpi") and stored at -80 0 C. The cells were then centrifuged (5 min, 1200 rpm)... And other 50 .mu.l of supernatant removed after resuspension of the cells in lOOμl Tonsillenmediums this washing step was repeated twice after addition of tonsil medium with the appropriate inhibitor concentrations the cells were incubated again at 37 ° C and 5% CO 2. on days 3, 6, 9 and 12, 150μl of medium was removed, stored at -80 0 C and 150μl medium with the appropriate concentrations of inhibitor added. on day 15, only 150μl of supernatant was removed, stored at -80 0 C and the cells then disposed of.
Beispiel 4e: RT-AssayExample 4e: RT assay
[0040] Die bei -800C aufbewahrten Tonsillenüberstände wurden durch Bestimmung der Reverse Transkriptase-Aktivität, und zwar anhand eines bereits beschriebenen Tests (Willey et al, 1988), unter Verwendung von [32P]-TTP Einbau in ein oligo(dT)-poly(A) Template, ermittelt. Legende zu den Figuren[0040] The kept at -80 0 C were Tonsillenüberstände by determining the reverse transcriptase activity, namely using an assay described previously (Willey et al, 1988), using [32P] -TTP installation in an oligo (dT) -poly (A) template, determined. Legend to the figures
Figur 1:FIG. 1:
[0041] Der Hsp90-Inhibitor 17-AAG zeigt bis zu einer Konzentration von 1OnM keine Zyto- Toxizität in CEM-Zellen. CD4+-T-lymphatische Zellen (CEM-Zellen) wurden mit verschiedenen Konzentrationen 17-AAG inkubiert und der zeitabhängige Farbumschlag, gleichbedeutend mit der Anzahl lebender Zellen, nach AlamarBlue™ (Invitrogen)-Zugabe mittels Fluoreszenzmessung ermittelt.The Hsp90 inhibitor 17-AAG shows up to a concentration of 1OnM no cytotoxicity in CEM cells. CD4 + T lymphoid cells (CEM cells) were incubated with different concentrations of 17-AAG and the time-dependent color change, equivalent to the number of living cells, after AlamarBlue ™ (Invitrogen) addition was determined by fluorescence measurement.
Figur 2:FIG. 2:
[0042] HeLaSSό-Zellen transfϊziert mit subgenomischem HIV-I Expressionsvektor pNLenvl zeigen unter 17-AAG-Einfluß in der Virusfraktion eine verminderte Gag-Prozessierung und eine verstärkte Hsp70-Expression in der Zellfraktion.HeLaSSό cells transfϊziert with subgenomic HIV-I expression vector pNLenvl show under 17-AAG influence in the virus fraction reduced Gag processing and increased Hsp70 expression in the cell fraction.
Figur 3:FIG. 3:
[0043] Antiviraler Effekt von 17-AAG allein, als auch in Kombination mit dem Proteasom- Inhibitor PS341, gegen X4-trophe HI-Viren im HLAC-Modell, dargestellt anhand der RT- Daten der jeweiligen Kinetikpunkte zweier unterschiedlicher Tonsillen (A und B). [0044] Die Virusreplikation des X4-trophen HI-Viruses konnte in Tonsille A (A) weder durch Inkubation mit InM Proteasom-Inhibitor PS341, InM 17-AAG noch mittels 1OnM 17-AAG deutlich beeinflusst werden. Erst die Kombination beider Substanzen (5nM PS341 und InM 17-AAG) erreichte eine deutliche Abnahme der Virusreplikation. Hierbei zeigte sich, dass dieser additive Effekt bei Applikation beider Substanzen durch eine höhere Konzentration des Hsp90-Inhibitors 17-AAG (1OnM) noch verstärkt werden konnte. Die Tonsille B (B) zeigte ebenfalls keine Beeinflussung der X4-trophen HIV-Replikation bei Inkubation mit InM PS341 oder InM 17-AAG. Im Gegensatz zu Tonsille A konnte bei Tonsille B bereits bei Zugabe von 1OnM 17- AAG ein deutliche Reduktion der Virusreplikation nachgewiesen werden. Bei jeglicher Kombination zwischen Proteasom-Inhibitor PS341 mit Hsp90-Inhibitor 17-AAG konnte keinerlei Virusreplikation mehr nachgewiesen werden. Antiviral effect of 17-AAG alone, as well as in combination with the proteasome inhibitor PS341, against X4-tropic HI viruses in the HLAC model, shown on the basis of the RT data of the respective kinetic points of two different tonsils (A and B ). The virus replication of X4-tropic HI virus could not be significantly influenced in tonsil A (A) either by incubation with InM proteasome inhibitor PS341, InM 17-AAG or by 1OnM 17-AAG. Only the combination of both substances (5nM PS341 and InM 17-AAG) achieved a significant decrease in virus replication. It was shown that this additive effect could be enhanced by a higher concentration of the Hsp90 inhibitor 17-AAG (1OnM) when both substances were applied. Tonsil B (B) also showed no effect on X4-tropic HIV replication when incubated with InM PS341 or InM 17-AAG. In contrast to tonsil A, a significant reduction in virus replication could be detected in tonsil B even with the addition of 1OnM 17-AAG. In any combination of proteasome inhibitor PS341 with Hsp90 inhibitor 17-AAG, no viral replication could be detected.

Claims

Patentansprüche claims
1. Pharmazeutische Zusammensetzung, die als wirksame Komponenten mindestens einen Inhibitor des Ubiquitin-Proteasom-Systems und einen Inhibitor von Proteinfaltungsenzymen.1. A pharmaceutical composition containing as active components at least one inhibitor of the ubiquitin proteasome system and an inhibitor of protein folding enzymes.
2. Pharmazeutische Zusammensetzung nach Anspruch 1, dadurch gekennzeichnet, dass es sich bei dem Inhibitor von Proteinfaltungsenzymen mindestens um einen Inhibitor zellulärer Chaperone oder um mindestens eine chemische Substanz handelt, welche die Proteinfaltung direkt beeinflusst (chemisches Anti-Chaperon)handelt.2. Pharmaceutical composition according to claim 1, characterized in that the inhibitor of protein folding enzymes is at least an inhibitor of cellular chaperones or at least one chemical substance which directly affects protein folding (chemical anti-chaperone).
3. Pharmazeutische Zusammensetzung nach Anspruch 2, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti-Chaperonen Substanzen eingesetzt werden, welche a) die Faltung und proteolytische Reifung von Virusproteinen hemmen, regulieren oder anderweitig beeinflussen und dadurch die Freisetzung und die Replikation von Viren hemmen, speziell von Erregern von Infektionserkrankungen wie AIDS, Hepatitis, hämorrhagischem Fieber, SARS, Pocken, Masern, Polio, Herpesvirusinfektionen oder Grippe. oder b) die Vermehrung von entarten Zellen, speziell Tumorzellen, stören, indem diese durch Akkumulation von falsch gefalteten Proteinen in den programmierten Zelltod getrieben werden.3. A pharmaceutical composition according to claim 2, characterized in that are used as inhibitors of cellular chaperones or of chemical anti-chaperones substances which a) inhibit the folding and proteolytic maturation of viral proteins, regulate or otherwise influence and thereby the release and replication of Inhibiting viruses, especially pathogens of infectious diseases such as AIDS, hepatitis, haemorrhagic fever, SARS, smallpox, measles, polio, herpesvirus infections or influenza. or b) interfere with the proliferation of degenerate cells, especially tumor cells, by driving them into programmed cell death by accumulation of misfolded proteins.
4. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 oder 3, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti- Chaperonen Substanzen eingesetzt werden, die speziell die enzymatischen Aktivitäten von molekularen Faltungsenzymen der Wirtszellen beeinflussen.4. A pharmaceutical composition according to any one of claims 2 or 3, characterized in that are used as inhibitors of cellular chaperones or of chemical anti-chaperones substances that specifically affect the enzymatic activities of molecular folding enzymes of the host cells.
5. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 4, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti- Chaperonen Substanzen eingesetzt werden, die von Zellen höherer Eukaryonten aufgenommen werden und nach Zellaufnahme die Proteinfaltung von Virusstrukturpoteinen und von Proteinen aus Tumorzellen blockieren. 5. A pharmaceutical composition according to any one of claims 2 to 4, characterized in that are used as inhibitors of cellular chaperones or chemical anti-chaperones substances that are absorbed by cells of higher eukaryotes and after cell uptake block the protein folding of Virusstrukturpoteinen and proteins from tumor cells ,
6. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti- Chaperonen Substanzen eingesetzt werden, die in verschiedenen Formen in vivo oral, intravenös, intramuskulär, subkutan oder in verkapselter Form mit oder ohne Zell-Spezifität- tragenden Veränderungen verabreicht werden, aufgrund der Anwendung eines bestimmten Applikations- und/oder Dosis-Regimes eine geringe Zytotoxizität aufweisen, keine oder unbedeutende Nebenwirkungen auslösen, eine relative hohe metabolische Halbwertszeit und eine relative geringe Clearence-Rate im Organismus aufweisen.6. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that are used as inhibitors of cellular chaperones or of chemical anti-chaperones substances which in various forms in vivo orally, intravenously, intramuscularly, subcutaneously or in encapsulated form with or without Cell specificity-bearing changes administered, due to the application of a particular application and / or dose regimen have low cytotoxicity, trigger no or insignificant side effects, have a relatively high metabolic half-life and a relatively low clearance rate in the organism.
7. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti- Chaperonen Substanzen eingesetzt werden, die a) in natürlicher Form aus Mikroorganismen oder anderen natürlichen Quellen isoliert werden oder b) durch chemische Modifikationen aus natürlichen Substanzen hervorgehen oder c) total-synthetisch hergestellt werden oder d) durch gentherapeutische Verfahren in vivo synthetisiert werden.7. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that are used as inhibitors of cellular chaperones or of chemical anti-chaperones substances which are a) isolated in natural form from microorganisms or other natural sources, or b) by chemical modifications originate from natural substances or c) be prepared in a totally synthetic manner or d) be synthesized in vivo by gene therapy methods.
8. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti- Chaperonen Substanzen eingesetzt werden, welche die hoch organisierten Prozesse der Assemblierung und der proteolytischen Reifung von Virusstrukturproteinen stören und dadurch die Freisetzung und die Produktion von infektiösen Nachkommenviren unterbinden.8. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that are used as inhibitors of cellular chaperones or chemical anti-chaperones substances that interfere with the highly organized processes of assembly and proteolytic maturation of virus structural proteins and thereby the release and the Prevent production of infectious progeny viruses.
9. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti- Chaperonen Substanzen eingesetzt werden, welche die Faltung von viralen Proteinen und/oder von Tumor-spezifischen Proteinen regulieren, stören oder blockieren.9. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that are used as inhibitors of cellular chaperones or of chemical anti-chaperones substances that regulate the folding of viral proteins and / or tumor-specific proteins interfere or block.
10. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Chaperonen Substanzen eingesetzt werden, die späte Prozessen der Virusreplikation wie Assemblierung, Knospung, proteolytische Reifung und Virusfreisetzung stören. 10. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that are used as inhibitors of cellular chaperones or chemical chaperones substances that interfere with late processes of virus replication such as assembly, budding, proteolytic maturation and virus release.
11. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Chaperonen Substanzen eingesetzt werden, welche die proteolytische Prozessierung von Vorläuferproteinen der viralen Polyproteine stören.11. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that are used as inhibitors of cellular chaperones or chemical chaperones substances that interfere with the proteolytic processing of precursor proteins of the viral polyproteins.
12. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti- Chaperonen Substanzen eingesetzt werden, welche die Aktivität von viralen Proteasen blockieren.12. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that are used as inhibitors of cellular chaperones or of chemical anti-chaperones substances which block the activity of viral proteases.
13. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti- Chaperonen Substanzen eingesetzt werden, welche die Aktivitäten von zellulären Proteasen und/oder von Enzymen, wie zum Beispiel Ligasen, Kinasen, Hydrolasen,13. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that are used as inhibitors of cellular chaperones or of chemical anti-chaperones substances containing the activities of cellular proteases and / or of enzymes such as ligases, kinases, hydrolases .
Glykosylierungsenzymen, Phosphatasen, DNAsen, RNAsen, Helikasen und Transferasen stören, die an der Virusreifung beteiligt sind.Interfere with glycosylation enzymes, phosphatases, DNAses, RNAses, helicases, and transferases involved in viral maturation.
14. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti-14. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that as inhibitors of cellular chaperones or of chemical anti-
Chaperonen Substanzen eingesetzt werden, welche ein breites Wirkspektrum besitzen und daher als neuartige Breitbandvirostatika zur Vorbeugung und/oder zur Therapie von unterschiedlichen Virusinfektionen eingesetzt werden.Chaperones substances are used, which have a broad spectrum of action and are therefore used as a novel broad-spectrum antivirals for the prevention and / or treatment of different viral infections.
15. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Anti- Chaperonen Substanzen eingesetzt werden, die zelluläre Chaperone wie Hitzeschockproteine (heat shock proteins (hsp)) blockieren.15. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that are used as inhibitors of cellular chaperones or chemical anti-chaperones substances that block cellular chaperones such as heat shock proteins (hsp) proteins.
16. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone oder von chemischen Chaperonen Substanzen eingesetzt werden, welche die Aktivitäten der Hitzeschockproteine Hsp27, Hsp30, Hsp40, Hsp60, Hsp70, Hsp72, Hsp73, Hsp90, Hsp 104 und Hsc70 hemmen. 16. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that are used as inhibitors of cellular chaperones or chemical chaperones substances which the activities of the heat shock proteins Hsp27, Hsp30, Hsp40, Hsp60, Hsp70, Hsp72, Hsp73, Hsp90, Hsp 104 and Hsc70 inhibit.
17. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren zellulärer Chaperone Substanzen eingesetzt werden, die folgenden Substanzklassen und deren Derivaten angehören: Geldanamycin (inhibiert Hsp90), Radicicol (Tyrosinkinase-Inhibitor; inhibiert Hsp90), Deoxyspergualin (inhibiert an Hsc70 und Hsp90), 4-PBA (4-Phenylbutyrat; Downregulation der Protein- und mRNA- Expression der Hsc70), Herbimycin A (Tyrosinkinase-Inhibitor mit Hsp72/73 Induktion), Epolactaene (Inhibitor der Hsp60), Scythe und Reaper (inhibieren Hsp70), Artemisinin (Inhibitor der Hsp90), CCTO 180159 (als Pyrazole-Inhibitor von Hsp90) und SNX-2112 (Hsp90 Inhibitor), Radanamycin (Macrolidchimera von Radicicol und Geldanamycin),17. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that as inhibitors of cellular chaperones substances are used which belong to the following substance classes and their derivatives: geldanamycin (inhibits Hsp90), radicicol (tyrosine kinase inhibitor, inhibits Hsp90), deoxyspergualin ( inhibits Hsc70 and Hsp90), 4-PBA (4-phenylbutyrate, downregulation of protein and mRNA expression of Hsc70), herbimycin A (tyrosine kinase inhibitor with Hsp72 / 73 induction), Epolactaene (inhibitor of Hsp60), Scythe and Reaper (inhibit Hsp70), artemisinin (inhibitor of Hsp90), CCTO 180159 (as pyrazole inhibitor of Hsp90) and SNX-2112 (Hsp90 inhibitor), radanamycin (macrolidechimera of radicicol and geldanamycin),
Novobiocin (Hsp90-Inhibitor), Quercetin (Inhibitor der Hsp70-Expression).Novobiocin (Hsp90 inhibitor), quercetin (inhibitor of Hsp70 expression).
18. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als chemische Anti-Chaperone Substanzen eingesetzt werden, welche die Proteinkonformation und die Faltung von viralen und/oder Tumor-spezifischen18. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that substances are used as chemical anti-chaperones, the protein conformation and the folding of viral and / or tumor-specific
Proteinen regulieren, stören oder blockieren.Regulate, disrupt or block proteins.
19. Pharmazeutische Zusammensetzung Anspruch 18, dadurch gekennzeichnet, dass als chemische Anti-Chaperone Substanzen wie Glycerol, Trimethylamine, Betain, Trehalose oder deuteriertes Wasser (D2O) eingesetzt werden.19. Pharmaceutical composition according to claim 18, characterized in that substances such as glycerol, trimethylamine, betaine, trehalose or deuterated water (D 2 O) are used as chemical anti-chaperones.
20. Pharmazeutische Zusammensetzung nach Anspruch 18 und 19, dadurch gekennzeichnet, dass als chemische Anti-Chaperone Substanzen eingesetzt werden, welche für die Behandlung, Therapie und Hemmung von Infektionen mit unterschiedlichen humanpathogenen oder auch tierpathogenen Viren geeignet sind.20. A pharmaceutical composition according to claim 18 and 19, characterized in that substances are used as chemical anti-chaperones, which are suitable for the treatment, therapy and inhibition of infections with different human pathogenic or animal-pathogenic viruses.
21. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass als Inhibitoren von molekularen Chaperonen oder von chemischen Anti-Chaperonen Substanzen eingesetzt werden, welche für die Behandlung, Therapie und Hemmung von Infektionen mit Erregern von chronischen Infektionserkrankungen wie21. A pharmaceutical composition according to any one of claims 2 to 5, characterized in that are used as inhibitors of molecular chaperones or of chemical anti-chaperones substances which are used for the treatment, therapy and inhibition of infections with agents of chronic infectious diseases such
AIDS (HIV-I und HIV-2), von Hepatitis (HCV und HBV), von dem Erreger des "Severe Acute Respiratory Syndroms" (SARS), dem SARS-CoV (Coronavirus), von Pockenviren, von Erregern des viralen hämorrhagischen Fiebers (VHF) wie den Ebola- Viren als Vertreter der Familie der Filoviridae; von Grippe-Erregern wie dem Influenza-A-Virus, geeignet sind.AIDS (HIV-1 and HIV-2), hepatitis (HCV and HBV), Severe Acute Respiratory Syndrome (SARS), SARS-CoV (Coronavirus), poxviruses, viral haemorrhagic fever pathogens (VHF) as the Ebola virus as Representative of the family Filoviridae; flu infectious agents such as the influenza A virus.
22. Pharmazeutische Zusammensetzung nach einem der Ansprüche 2 bis 5, dadurch gekennzeichnet, dass Cyclosporin A und / oder Tacrolimus eingesetzt werden.22. Pharmaceutical composition according to one of claims 2 to 5, characterized in that cyclosporin A and / or tacrolimus are used.
23. Pharmazeutische Zusammensetzung nach Anspruch 1, dadurch gekennzeichnet, dass es sich bei den Hemmern des UPS um mindestens eine Substanz handelt, die a) speziell in Form von Proteasom-Inhibitoren die enzymatischen Aktivitäten des kompletten 26S Proteasom-Komplexes und der freien, nicht mit regulatorischen23. A pharmaceutical composition according to claim 1, characterized in that the inhibitors of the UPS is at least one substance which a) especially in the form of proteasome inhibitors, the enzymatic activities of the complete 26S proteasome complex and the free, not with regulatory
Untereinheiten assemblierten 2OS katalytisch aktiven Proteasom-Struktur beeinflusst oder b) speziell die Wirkung von Ubiquitin-Ligasen hemmt oder c) speziell die Wirkung von Ubiquitin-Hydrolasen hemmt oder d) speziell die Wirkung von Ubiquitin-aktivierenden Enzymen hemmt oder e) speziell die Mono-ubiquitinylierung von Proteinen hemmt oder f) speziell die Poly-ubiquitinylierung von Proteine hemmt.Subunits assembled 2OS influenced catalytically active proteasome structure or b) specifically inhibits the action of ubiquitin ligases or c) specifically inhibits the action of ubiquitin hydrolases or d) specifically inhibits the action of ubiquitin-activating enzymes or e) specifically the mono- ubiquitinylation of proteins or f) specifically inhibits the poly-ubiquitinylation of proteins.
24. Pharmazeutische Zusammensetzung nach Anspruch 23, dadurch gekennzeichnet, dass Substanzen eingesetzt werden, die als Proteasom-Inhibitoren von höheren Eukaryoten aufgenommen werden und nach Zellaufnahme mit den katalytischen Untereinheiten des Proteasoms in Wechselwirkung treten und dabei alle oder einzelne der proteolytischen Aktivitäten des Proteasoms - die Trypsin-, die Chymotrypsin- und/oder die Postglutamyl- Peptid hydrolysierenden Aktivitäten - innerhalb des 26S oder auch des 2OS Proteasom- Komplexes irreversibel oder reversibel blockieren.24. A pharmaceutical composition according to claim 23, characterized in that substances are used which are taken up as proteasome inhibitors of higher eukaryotes and after cell uptake interact with the catalytic subunits of the proteasome and thereby all or some of the proteolytic activities of the proteasome - the Trypsin, the chymotrypsin and / or the postglutamyl-peptide-hydrolyzing activities - irreversibly or reversibly block within the 26S or the 2OS proteasome complex.
25. Pharmazeutische Zusammensetzung nach Anspruch 23 oder 24, dadurch gekennzeichnet, dass als Proteasom-Inhibitoren Substanzen eingesetzt werden, die g) in natürlicher Form aus Mikroorganismen oder anderen natürlichen Quellen isoliert werden; oder h) durch chemische Modifikationen aus natürlichen Substanzen hervorgehen; oder i) total-synthetisch hergestellt werden; oder j) durch gentherapeutische Verfahren in vivo synthetisiert werden; oder k) durch gentechnische Verfahren in vitro oder 1) in Mikroorganismen hergestellt werden.25. A pharmaceutical composition according to claim 23 or 24, characterized in that as proteasome inhibitors substances are used, the g) are isolated in natural form from microorganisms or other natural sources; or h) result from chemical modifications of natural substances; or i) are produced totally synthetically; or j) are synthesized in vivo by gene therapy methods; or k) by genetic engineering in vitro or 1) are produced in microorganisms.
26. Pharmazeutische Zusammensetzung nach einem der Ansprüche 23 bis 25, dadurch gekennzeichnet, dass als Proteasom-Inhibitoren Substanzen eingesetzt werden, die folgenden Substanzklassen angehören: f) natürlich vorkommende Proteasom-Inhibitoren:26. A pharmaceutical composition according to any one of claims 23 to 25, characterized in that as proteasome inhibitors substances are used which belong to the following substance classes: f) naturally occurring proteasome inhibitors:
- Peptid-Derivate, welche C-terminal Epoxyketon-Strukturen enthalten; oderPeptide derivatives containing C-terminal epoxy ketone structures; or
- ß-Lacton-Derivate; oder- β-lactone derivatives; or
- Aclacinomycin A (auch bezeichnet als Aclarubicin); oder - Lactacystin und dessen chemische modifizierte Varianten, wie der Zellmembran- penetrierenden Variante "Clasto lactacystein ß-Lacton"Aclacinomycin A (also referred to as Aclarubicin); or - Lactacystin and its chemically modified variants, such as the cell membrane penetrating variant "Clasto lactacysteine β-lactone"
g) synthetisch hergestellte Proteasom-Inhibitoren: modifizierte Peptidaldehyde wie N-carbobenzoxy-L-leucinyl-L-leucinyl-L-leucinal (auch bezeichnet als MG 132 oder zLLL), dessen Borsäure-Derivat MG232; N-g) synthetically prepared proteasome inhibitors: modified peptide aldehydes such as N-carbobenzoxy-L-leucinyl-L-leucinyl-L-leucinal (also referred to as MG 132 or zLLL), its boric acid derivative MG232; N-
Carbobenzoxy-Leu-Leu-Nva-H (bezeichnet als MGl 15; N-AcetylL-Leuzinyl-L- Leuzinyl-L-Norleuzinal (bezeichnet als LLnL), N-Carbobenzoxy-Ile-Glu(OBut)-Ala- Leu-H (auch bezeichnet als PSI);Carbobenzoxy-Leu-Leu-Nva-H (referred to as MGl 15; N-acetyl-leucinyl-L-leucinyl-L-norleucinal (referred to as LLnL); N-carbobenzoxy-Ile-Glu (OBut) -Ala- Leu-H (also referred to as PSI);
h) Peptide, welche C-terminal eine α, ß -Epoxyketon-Struktur tragen, ferner Vinylsulfone wie Carbobenzoxy-L-Leucinyl-L-Leucinyl-L-Leucin-vinyl-sulfon oder 4-Hydroxy-5- iodo-3-nitrophenylactetyl-L-Leucinyl-L-Leucinyl-L-Leucin-vinyl-sulfon (NLVS);h) peptides which carry an α, β-epoxyketone C-terminal structure, furthermore vinylsulfones such as carbobenzoxy-L-leucinyl-L-leucinyl-L-leucine-vinylsulfone or 4-hydroxy-5-iodo-3-nitrophenylactetyl -L-leucinyl-L-leucinyl-L-leucine-vinylsulfone (NLVS);
i) Glyoxal- oder Borsäure-Reste wie Pyrazyl-CONH(CHPhe)CONH(CHisobutyl)B(OH)2) sowiei) glyoxal or boric acid radicals such as pyrazyl-CONH (CHPhe) CONH (CHisobutyl) B (OH) 2 ) as well as
Dipeptidyl-Borsäure-Derivate oderDipeptidyl-boric acid derivatives or
j) Pinacol-Ester - wie Benzyloxycarbonyl(Cbz)-Leu-Leu-boroLeu-Pinacol-Ester.j) Pinacol esters - such as benzyloxycarbonyl (Cbz) -Leu-Leu-boroLeu-Pinacol esters.
27. Pharmazeutische Zusammensetzung nach einem der Ansprüche 23 bis 25, dadurch gekennzeichnet, dass als besonders geeignete Proteasom-Inhibitoren die Epoxyketone Epoxomicin (Epoxomycin, Molekülformel: C28H86N4O7) und/oder Eponemycin (Eponemicin, Molekülformel: C20H36N2O5) eingesetzt werden. 27. A pharmaceutical composition according to any one of claims 23 to 25, characterized in that as particularly suitable proteasome inhibitors, the epoxyketones epoxomicin (epoxomycin, molecular formula: C 28 H 86 N 4 O 7 ) and / or eponemicin (eponemicin, molecular formula: C 20 H 36 N 2 O 5 ) can be used.
28. Pharmazeutische Zusammensetzung nach einem der Ansprüche 23 bis 25, dadurch gekennzeichnet, dass als besonders geeignete Proteasom-Inhibitoren aus der PS-Serie die Verbindungen k) PS-519 als ß-Lacton- sowie als Lactacystin-Derivat die Verbindung IR-[IS, 4R, 5S]]- 5 l-(l-Hydroxy-2-methylpropyl)-4-propyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione -28. A pharmaceutical composition according to any one of claims 23 to 25, characterized in that as particularly suitable proteasome inhibitors from the PS series, the compounds k) PS-519 as ß-lactone and lactacystin derivative as the compound IR- [IS , 4R, 5S]] - 5 l- (1-hydroxy-2-methylpropyl) -4-propyl-6-oxa-2-azabicyclo [3.2.0] heptane-3,7-dione
Molekülformel C12H19NO4- und/oder 1) PS-314 als Peptidyl-Borsäure-Derivat die Verbindung N-Pyrazinecarbonyl-L-Molecular formula C12H1 9 NO4- and / or 1) PS-314 as peptidyl-boric acid derivative, the compound N-pyrazinecarbonyl-L-
Phenylalanin-L-Leuzin-Borsäure - Molekülformel C19H25BN4O4 - und/oder m) PS-273 (Morpholin-CONH-(CH-Naphthyl)-CONH-(CH-isobutyl)-B(OH)2) und 0 dessen Enantiomer PS-293 und/oder n) die Verbindung PS-296 (8-Quinolyl-sulfonyl-CONH-(CH-Napthyl)-CONH(-CH- isobutyl)-B(OH)2) und/oder o) PS-303 (NH2(CH-Naphthyl)-CONH-(CH-isobutyl)-B(OH)2) und/oder p) PS-321 als (Morpholin-CONH-(CH-Napthyl)-CONH-(CH-Phenylalanin)-B(OH)2); - 5 und/oder q) PS-334 (CH3-NH-(CH-Naphthyl-CONH-(CH-Isobutyl)-B(OH)2) und/oder r) die Verbindung PS-325 (2-Quinol-CONH-(CH-Aomo-Phenylalanin)-CONH-(CH- isobutyl)-B(OH)2) und/oder s) PS-352 (Phenyalanin-CH2-CH2-CONH-(CH-Phenylalanin)-CONH-(CH-isobutyl)l- o B(OH)2) und/oder t) PS-383 (Pyridyl-CONH-(CH/?F-Phenylalanin)-CONH-(CH-isobutyl)-B(OH)2) eingesetzt werden.Phenylalanine-L-leucine boric acid - molecular formula C1 9 H25BN4O4 - and / or m) PS-273 (morpholine-CONH- (CH-naphthyl) -CONH- (CH-isobutyl) -B (OH) 2 ) and 0 its enantiomer PS-293 and / or n) the compound PS-296 (8-quinolyl-sulfonyl-CONH- (CH-naphthyl) -CONH (-CH-isobutyl) -B (OH) 2 ) and / or o) PS-303 (NH 2 (CH-naphthyl) -CONH- (CH-isobutyl) -B (OH) 2 ) and / or p) PS-321 as (morpholine-CONH- (CH-naphthyl) -CONH- (CH-phenylalanine) -B (OH) 2 ); - 5 and / or q) PS-334 (CH 3 -NH- (CH-naphthyl-CONH- (CH-isobutyl) -B (OH) 2 ) and / or r) the compound PS-325 (2-quinol CONH- (CH-aomo-phenylalanine) -CONH- (CH-isobutyl) -B (OH) 2 ) and / or s) PS-352 (phenylalanine-CH 2 -CH 2 -CONH- (CH-phenylalanine) -CONH - (CH-isobutyl) 1-0 B (OH) 2 ) and / or t) PS-383 (pyridyl-CONH- (CH /? F-phenylalanine) -CONH- (CH-isobutyl) -B (OH) 2 ) are used.
29. Mittel zur Behandlung von Virus infektionen und / oder Tumorerkrankungen die eine 5 Zusammensetzung gemäß einem der Ansprüche 1 bis 28 enthalten.29. Agents for the treatment of virus infections and / or tumor diseases containing a composition according to any one of claims 1 to 28.
30. Verwendung der Zusammensetzung gemäß einem der Ansprüche 1 bis 28 zur Behandlung von Virusinfektionen und / oder Tumorerkrankungen.30. Use of the composition according to any one of claims 1 to 28 for the treatment of viral infections and / or tumor diseases.
0 31. Verwendung der Zusammensetzung gemäß einem der Ansprüche 1 bis 28 zur Herstellung von Mitteln zur Behandlung von Virusinfektionen und / oder Tumorerkrankungen.0 31. Use of the composition according to any one of claims 1 to 28 for the preparation of agents for the treatment of viral infections and / or tumor diseases.
32. Verwendung nach Anspruch 30 oder 31 in Kombination mit anderen Mitteln, die zur Behandlung von Virusinfektionen und / oder Tumorerkrankungen eingesetzt werden. 32. Use according to claim 30 or 31 in combination with other agents which are used for the treatment of viral infections and / or tumor diseases.
33. Verwendung nach einem der Ansprüche 30 bis 32 in Form von33. Use according to one of claims 30 to 32 in the form of
Inhalaten Depotformen - Pflaster in mikroelektronischen Systemen („intelligente Pille")Inhaled depot forms - patches in microelectronic systems ("intelligent pill")
34. Verwendung nach einem der Ansprüche 30 bis 33 in der Onkologie und / oder Onkologie und Virologie34. Use according to any one of claims 30 to 33 in oncology and / or oncology and virology
35. Verwendung nach Anspruch 30 oder 31 zur Behandlung von:35. Use according to claim 30 or 31 for the treatment of:
- Glioblastom (Maligne Hirntumore)- glioblastoma (malignant brain tumors)
- Mamma-CA (CA = Carcinom)- mamma CA (CA = carcinoma)
- - Kopf-, HaIs-CA - - Plattenepithel-CA- - Head, HaIs-CA - - Squamous-epithelial-CA
- - Ovarial-CA- - ovarian CA.
- Bronchial-CA (kleinzellig, großzellig)- Bronchial CA (small cell, large cell)
- Schilddrüsen-CA- Thyroid CA
- Lungen-CA - - Kolon-CA- Lung CA - - Colon CA.
- Pankreas-CA- pancreatic CA.
- - Leukemie (AML, ALL, CML, CLL) akute myeloische, chronische acute lymphatische, chronische - - Lymphom (Non-Hodgkin)- - Leukemia (AML, ALL, CML, CLL) Acute myeloid, chronic acute lymphocytic, chronic - Lymphoma (Non-Hodgkin)
- Zervix-Ca- Cervical Ca
- Neuroblastom- Neuroblastoma
- Haut-CA (Melanom)- skin CA (melanoma)
- - Prostata-CA - - Blasen-CA- - Prostate CA - - Bladder CA.
- Sarkome (Knochen u. Weichteile)- Sarcomas (bones and soft tissues)
- - Zwerchfell-CA- - Diaphragm CA.
- Gastrointestinale-CA (z. B. Magen, Oesophagus)Gastrointestinal CA (eg stomach, esophagus)
- Hoden-Ca - - Metastasen (z. B. Knochenmark)- testes Ca - - metastases (eg bone marrow)
- Lymphoma- Viren - Herpes Simplex- Lymphoma viruses - herpes simplex
- Zytomegalie- cytomegalovirus
- Varizellen- Varicella
- Varicella Zoster - - Masern- Varicella Zoster - - Measles
- Lassa-Fieber- Lassa fever
- Aids- AIDS
- Mumps (- Meningitis, - Orchitis)- mumps (- meningitis, - orchitis)
- Enteritis ; Grippe (alle Formen) - - Enzephalitis- enteritis; Flu (all forms) - - Encephalitis
- - Hepatitis (A, B, C, D, E, G)- - Hepatitis (A, B, C, D, E, G)
- - Röteln- - rubella
- Coxsackie B- Coxsackie B
- Polio (-Myelitis) - - Enzephalomyelitis- polio (myelitis) - - encephalomyelitis
- Pankreatitits- pancreatitis
- Pneumonie- Pneumonia
- Myokarditis- Myocarditis
- Tropenkrankheiten (Virale) - - alle doppel- und einsträngigen DNS und RNS humanpathogenen Viren. - Tropical diseases (viral) - - all double- and single-stranded DNA and RNA human-pathogenic viruses.
EP07765312A 2006-06-01 2007-06-01 Pharmaceutical composition for the treatment of viral infections and/or tumor diseases by inhibiting protein folding and protein breakdown Withdrawn EP2029125A2 (en)

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