WO2000051578A2 - Use of ceramides for the treatment of cystic fibrosis - Google Patents

Use of ceramides for the treatment of cystic fibrosis Download PDF

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Publication number
WO2000051578A2
WO2000051578A2 PCT/EP2000/001682 EP0001682W WO0051578A2 WO 2000051578 A2 WO2000051578 A2 WO 2000051578A2 EP 0001682 W EP0001682 W EP 0001682W WO 0051578 A2 WO0051578 A2 WO 0051578A2
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ceramide
substance
building block
ceramides
treatment
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PCT/EP2000/001682
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German (de)
French (fr)
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WO2000051578A3 (en
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Florian Lang
Erich Gulbins
Lepple-Wienhues
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Florian Lang
Erich Gulbins
Lepple Wienhues
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Priority to CA002365290A priority Critical patent/CA2365290A1/en
Priority to AU31613/00A priority patent/AU3161300A/en
Priority to EP00909267A priority patent/EP1156854A2/en
Publication of WO2000051578A2 publication Critical patent/WO2000051578A2/en
Publication of WO2000051578A3 publication Critical patent/WO2000051578A3/en

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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

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  • the invention relates to the use of ceramides, derivatives of ceramides and / or precursors of ceramides.
  • the invention relates to the use of these substances in connection with the treatment of cystic fibrosis.
  • Cystic fibrosis is the most common congenital metabolic disease and the most common lethal genetic disorder with hundreds of thousands of deaths worldwide each year. The frequency of occurrence of this disease varies from region to region. In Europe, one in every 2000 newborns is affected.
  • Cystic fibrosis is an autosomal recessive metabolic disorder that is associated with a generalized malfunction of exocrine glands.
  • An increased electrolyte content in the secretions of sweat glands leads to fluid and electrolyte losses.
  • the viscosity of secretions is increased, so that there are serious complications in the area of the respiratory tract and digestive tract with secondary cyst formation. The disease causes a significant reduction in life expectancy.
  • CF cystic fibrosis
  • the cystic fibrosis is caused by a large number of different mutations in the CFTR gene, which codes for chloride channels and / or regulators of chloride channels, Na + channels and K + channels.
  • the mutations lead to a disturbed transepithelial transport with reduced Cl ⁇ and HC0 3 " secretion and increased Na + absorption.
  • the object of the invention is to provide substances and methods which have a positive influence on the processes described which are disturbed in CF patients.
  • the fluid secretion in the epithelia of CF patients is to be stimulated, thus at least alleviating the course of the disease.
  • ORCC channels outwardly rectifying chloride channels
  • the body's own substances are known to be ceramides, which occur particularly in the brain and in the myelin of the central nervous system. They are lipophilic amides.
  • the ceramides are found in the organism as choline phosphate ester or as glycosides.
  • the ceramides form the building blocks of the choline phosphate esters, also known as sphingomyelines, or of the glycosides which occur as so-called cerebrosides, gangliosides and sulfatides.
  • the two groups of substances mentioned with ceramides as building blocks are called sphingolipids. Chemically, these are lipids that contain, instead of glycerin (in fats and oils) as an alcohol component, sphingosine (4-sphingenine), which is not naturally available.
  • the invention includes the use of ceramides and / or the other substances mentioned for the treatment of cystic fibrosis. By administering an effective amount of such substances, fluid secretion in epithelia of patients can preferably be stimulated.
  • the lung, pancreas and intestinal epithelia of CF patients are particularly worth mentioning here.
  • the invention further includes the treatment of cystic fibrosis by administration of ceramides and / or the other substances mentioned and the use of all of these substances for the production of medicaments, in particular medicaments for the treatment of cystic fibrosis.
  • the ceramides or substances with a ceramide building block used according to the invention are longer-chain ceramides, such as C12 ceramide, or their derivatives, which can be isolated directly from cell material as frequently occurring substances.
  • the ceramides / substances used can also be obtained by enzymatic treatment of corresponding precursors which can be isolated from cell material.
  • ceramides / substances which are obtained with the aid of molecular biological methods known to the person skilled in the art are used.
  • corresponding, synthetically produced ceramides / substances are used according to the invention.
  • the C2 or C6 ceramides or substances with these ceramides as building blocks are used here.
  • ceramides which are chemically or biologically modified in their ceramide building blocks.
  • cystic fibrosis these derivatives have a similar, preferably better, effect than ceramides and / or have similar or better pharmaceutical properties. This can be, for example, slower breakdown rates of the active ingredient in the body or better uptake rates in the cells.
  • the substances used according to the invention are biological precursor molecules of ceramides or of substances with a ceramide building block, which are converted into the active ceramides in the cell, for example by an enzymatic cleavage by sphingomyelinas.
  • the substances used according to the invention act on metabolic pathways which lead to the formation of ceramides.
  • activators of sphingomyelinases which cause the cleavage of precursor molecules to the active ceramides, are used.
  • activators are cell components, preferably enzymes, which are connected downstream of the ceramides in the signal chain.
  • Kinases which are activated by ceramides are particularly suitable for this purpose.
  • the introduction of nucleic acids which code for these substances is also encompassed by the invention.
  • the invention further comprises pharmaceutical compositions which contain at least one ceramide and / or the other substances mentioned and preferably additionally a pharmaceutically acceptable carrier. Whether or not which pharmaceutical carrier is used depends on the form of administration of the drug.
  • the pharmaceutical composition is administered systemically.
  • the pharmaceutical composition is administered via the digestive tract.
  • Administration by inhalation is also particularly preferred, the active ingredient being introduced quickly and directly into the lungs. This form of administration is particularly useful in the treatment of cystic fibrosis, since this disease leads to serious complications in the area of the respiratory system.
  • Treatment with ceramides and / or the other substances mentioned can also achieve positive results in the case of other diseases which are associated with a disturbed regulation of molecular transport processes, preferably on membranes. Therefore, the use of all of these substances in the treatment of such diseases is included in the invention.
  • the transport processes should preferably take place on ion channels, in particular on ion channels, preferably chloride channels, directed outwards (out of a cell or cell organelle).
  • the pictures show:
  • FIG. 3 Influence of the chloride channels in normal lymphocytes (A, B) and CF lymphocytes (C, D) by the tyrosine kinase Lck 56 .
  • a membrane is sucked in with a micropipette and vacuum at the edge of the micropipette pette sealed.
  • the membrane fixed in this way as part of an intact cell or as an isolated membrane piece is immersed in a suitable electrolyte bath and the conductivity between it and the electrolyte solution inside the pipette (patch clamp electrode) is measured.
  • the opening state of the ion channels of the membrane can be influenced by adding various agents to the electrolyte solutions. Such an influence can be demonstrated by a sudden change in the conductivity.
  • T-lymphocytes were isolated from normal and from patients suffering from cystic fibrosis (CF), placed in a perfusion chamber and flooded with an electrolyte solution (145 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 2 M CaCl, 10 mM Glucose, 10mM Hepes / NaOH, pH 7.4).
  • an electrolyte solution 145 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 2 M CaCl, 10 mM Glucose, 10mM Hepes / NaOH, pH 7.4
  • Patch clamp electrodes (filled with 160 mM Cs-glutamate, 2 mM MgCl 2 , 0.1 mM CaCl 2 , 1.1 mM EGTA, 4 Na 2 ATP, 10 mM Hepes / NaOH, pH 7) were examined under the inverted microscope , 2) brought to the cell membrane and a connection to the intracellular space is established by sucking in the cell membrane (whole cell patch clamp) or the conductivity of the aspirated membrane spot is measured (cell attached patch clamp). This made a continuous registration of the cell membrane conductivity possible.
  • the cells were stimulated either extracellularly with cAMP (200 ⁇ M), extracellularly with C2-ceramide (50 ⁇ M) or intracellularly with the tyrosine kinase Lck 56 (10 U / ml) and the conductivity was measured continuously.
  • the electrodes were connected to a patch clamp amplifier (EPC-9) by means of suitable preamplifiers, with the aid of which the potential between the patch clamp electrode and a reference electrode in the bath was varied from -100 mV to +100 mV.
  • EPC-9 patch clamp amplifier
  • the respective currents across the cell membrane between patch The clamp electrode and reference electrode were recorded using the patch clamp amplifier.
  • the choice of solutions allowed an exclusive analysis of currents through chloride channels.
  • FIG. 1 the application of cAMP, as expected, led to an activation of the Cl ⁇ current in normal lymphocytes (FIG. 1A, B), but not in CF lymphocytes (FIG. 1 C, D).
  • FIGS. 1 A, C, D the measurement curves determined at intervals are each one above the other, but not in FIG. 1B.
  • ceramide in CF lymphocytes led to activation of the chloride channels.
  • tyrosine kinase Lck 56 which is known to be activated by ceramides, led to an activation of chloride channels in normal (FIG. 3A, B) and in CF lymphocytes (FIG. 3 C, D).
  • the curves lie one above the other in FIGS. 3A and 3C, but not in FIGS. 3A and 3D. This is in contrast to the results with cAMP.

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Abstract

The invention relates to the use of ceramides and/or substances which contain ceramides as a building block. Said ceramides and substances are used for the treatment of cystic fibrosis. The ceramides are especially C2 and/or C6 ceramides. The invention also relates to the use of the above-mentioned substances for the treatment of diseases which are linked to a disturbed regulation of transport processes at membranes.

Description

Beschreibung description
Verwendung von Ceramiden zur Behandlung cystischer FibröseUse of ceramides to treat cystic fibrosis
Die Erfindung betrifft die Verwendung von Ceramiden, Derivaten von Ceramiden und/oder Vorläufern von Ceramiden. Insbesondere betrifft die Erfindung die Verwendung dieser Stoffe im Zusammenhang mit der Behandlung von cystischer Fibröse.The invention relates to the use of ceramides, derivatives of ceramides and / or precursors of ceramides. In particular, the invention relates to the use of these substances in connection with the treatment of cystic fibrosis.
Die cystische Fibröse ist die häufigste angeborene Stoffwechselkrankheit und mit weltweit mehreren Hunderttausend Todesfällen pro Jahr die häufigste letale genetische Erkrankung. Die Häufigkeit des Auftretens dieser Erkrankung ist regional unterschiedlich. In Europa ist eines von 2000 Neugeborenen betroffen.Cystic fibrosis is the most common congenital metabolic disease and the most common lethal genetic disorder with hundreds of thousands of deaths worldwide each year. The frequency of occurrence of this disease varies from region to region. In Europe, one in every 2000 newborns is affected.
Bei der cystischen Fibröse handelt es sich um eine autosomal- rezessive StoffWechselstörung, welche mit einer generalisierten Fehlfunktion exokriner Drüsen einhergeht. Durch einen erhöhten Elektrolytgehalt des Sekrets von Schweißdrüsen kommt es zu Flüssigkeits- und Elektrolytverlusten. Weiterhin ist die Viskosität von Sekreten erhöht, so daß es zu schweren Komplikationen im Bereich der Atemwege und des Verdauungstraktes mit sekundärer Cystenbildung kommt. Die Erkrankung bedingt eine erhebliche Verkürzung der Lebenserwartung.Cystic fibrosis is an autosomal recessive metabolic disorder that is associated with a generalized malfunction of exocrine glands. An increased electrolyte content in the secretions of sweat glands leads to fluid and electrolyte losses. Furthermore, the viscosity of secretions is increased, so that there are serious complications in the area of the respiratory tract and digestive tract with secondary cyst formation. The disease causes a significant reduction in life expectancy.
Einer der pathophysiologischen Mechanismen bei cystischer Fibröse (CF) ist eine gestörte Regulation vorwiegend epithe- lialer Cl~-, Na+- und K+-Kanäle. Insbesondere sind Cloridka- näle betroffen, die Cl~-Ionen aus der Zelle hinaustranspor- tieren, die sog. auswärts rektifizierenden Chloridkanäle (outwardly rectifying Chloride Channels - ORCC) .One of the pathophysiological mechanisms in cystic fibrosis (CF) is a disturbed regulation of predominantly epithelial Cl ~ , Na + and K + channels. Chloride channels are particularly affected, which transport Cl ~ ions out of the cell. animals, the so-called outwardly rectifying chloride channels (ORCC).
Die krankheitsbedingte fehlende Aktivierbarkeit dieser Chloridkanäle und die gestörte Regulation von Na+- und Wasserresorbierenden Transportprozessen führt zu einer verminderten epithelialen Sekretion von Flüssigkeiten und Bicarbonat. Dies bedingt die zähen Sekrete in Lunge, Pankreas und Darm in betroffenen Patienten, welche zu einer Verlegung von Atemwegen, Pankreasgängen und Darm führen. Hierdurch werden pulmonale Infektionen begünstigt und eine Malabsorption hervorgerufen.The disease-related inability to activate these chloride channels and the impaired regulation of Na + and water-absorbing transport processes lead to a reduced epithelial secretion of liquids and bicarbonate. This results in the viscous secretions in the lungs, pancreas and intestine in affected patients, which lead to obstruction of the airways, pancreatic ducts and intestine. This promotes pulmonary infections and causes malabsorption.
Die cystische Fibröse wird durch eine Vielzahl unterschiedlicher Mutationen des CFTR-Gens hervorgerufen, welches für Chloridkanäle und/oder Regulatoren von Chloridkanälen, Na+- Kanälen und K+-Kanälen kodiert. Die Mutationen führen zu einem gestörten transepithelialen Transport mit herabgesetzter Cl~- und HC03 "-Sekretion und gesteigerter Na+-Resorp- tion.The cystic fibrosis is caused by a large number of different mutations in the CFTR gene, which codes for chloride channels and / or regulators of chloride channels, Na + channels and K + channels. The mutations lead to a disturbed transepithelial transport with reduced Cl ~ and HC0 3 " secretion and increased Na + absorption.
Die Erfindung stellt sich die Aufgabe, Substanzen und Verfahren bereitzustellen, die die geschilderten, bei CF-Patienten gestörten Prozesse positiv beeinflussen. Beispielsweise soll die Flüssigkeitssekretion in den Epithelien von CF-Patienten angeregt und so der Krankheitsverlauf zumindest gelindert werden.The object of the invention is to provide substances and methods which have a positive influence on the processes described which are disturbed in CF patients. For example, the fluid secretion in the epithelia of CF patients is to be stimulated, thus at least alleviating the course of the disease.
Diese Aufgabe wird gelöst durch die Verwendung von Ceramiden und/oder Substanzen mit Ceramid-Baustein gemäß dem Anspruch 1 und den abhängigen Ansprüchen 2 bis 4. Die Aufgabe wird ebenfalls gelöst durch die Verwendung von biologischen Vorläufermolekülen gemäß Anspruch 5 und Aktivatoren gemäß Anspruch 6. Pharmazeutische Zusammensetzungen, welche mindestens einen dieser wirksamen Stoffe beinhalten, sind in den Ansprüchen 7 bis 12 beansprucht. Die Ansprüche 13 bis 17 betreffen allgemein die Verwendung der genannten Stoffe bei der Behandlung von Krankheiten, die mit gestörten Transportprozessen in Zusammenhang stehen. Der Wortlaut sämtlicher Ansprüche wird hiermit durch Bezugnahme zum Inhalt dieser Beschreibung gemacht.This object is achieved by the use of ceramides and / or substances with a ceramide building block according to claim 1 and dependent claims 2 to 4. The object is also achieved by the use of biological precursor molecules according to claim 5 and activators according to claim 6. Pharmaceutical Compositions containing at least one of these active substances are in claims 7 claimed to 12. Claims 13 to 17 generally relate to the use of the substances mentioned in the treatment of diseases which are associated with disrupted transport processes. The wording of all claims is hereby incorporated by reference into the content of this description.
Chloridkanäle in Zellen von gesunden Menschen, nicht aber in Zellen von CF-Patienten, können durch cAMP (zyklisches Adeno- sinmonophosphat) aktiviert werden [Rieh et al, Nature 347: 358-363, 1990]. Nach dem Stand der Technik war dementsprechend davon auszugehen, daß die Chloridkanäle in Zellen von CF-Patienten nicht zu aktivieren sind. Dieser Defekt bedingt aber gerade die fehlende Flüssigkeitssekretion der Zellen, welche eine wichtige Ursache für die Symptome der cystischen Fibröse ist. Vor kurzem konnte gezeigt werden, daß die auswärts gleichrichtende Chloridkanäle (ORCC-Kanäle) in Zellen aus gesunden Patienten durch Ceramide aktiviert werden können [Szabo et al, Acad. Sei. USA, 95(11): 6169-6174, 1998].Chloride channels in cells from healthy people, but not in cells from CF patients, can be activated by cAMP (cyclic adenosine monophosphate) [Rieh et al, Nature 347: 358-363, 1990]. According to the prior art, it was accordingly to be assumed that the chloride channels in cells of CF patients cannot be activated. This defect is due to the lack of fluid secretion of the cells, which is an important cause of the symptoms of cystic fibrosis. It has recently been shown that the outwardly rectifying chloride channels (ORCC channels) in cells from healthy patients can be activated by ceramides [Szabo et al, Acad. Be. USA, 95 (11): 6169-6174, 1998].
Überraschende Ergebnisse, die die Basis für die vorliegende Erfindung darstellen, zeigen nun, daß auch die Chloridkanäle in Lymphozyten von CF-Patienten durch Ceramide aktiviert werden können, obwohl die reguläre Aktivierbarkeit durch cAMP (zyklisches Adenosinmonophosphat) gestört ist. Die durch Ceramide aktivierten Kanäle bewirken eine Flüssigkeitssekretion der Zellen aus CF-Patienten, weshalb durch die Verwendung von Ceramiden die Symptome der cystischen Fibröse gebessert werden.Surprising results, which form the basis for the present invention, now show that the chloride channels in lymphocytes of CF patients can also be activated by ceramides, although the regular activability is impaired by cAMP (cyclic adenosine monophosphate). The channels activated by ceramides cause fluid secretion of the cells from CF patients, which is why the use of ceramides improves the symptoms of cystic fibrosis.
Als Ceramide werden bekanntlich körpereigene Stoffe bezeichnet, die insbesondere in der Gehirnsubstanz und im Myelin des Zentralnervensystems vorkommen. Es handelt sich um lipophile Amide. Im Organismus liegen die Ceramide als Cholinphosphat- ester oder als Glykoside vor. Die Ceramide bilden dabei die Bausteine der auch als Sphingomyeline bezeichneten Cholin- phosphatester oder der als sog. Cerebroside, Ganglioside und Sulfatide vorkommenden Glykoside. Die beiden genannten Gruppen von Stoffen mit Ceramiden als Bausteinen werden Sphingo- lipide genannt. Chemisch handelt es sich hier um Lipide, die an Stelle von Glycerin (bei Fetten und Ölen) als Alkohol-Komponente das in der Natur nicht frei vorkommende Sphingosin (4-Sphingenin) enthalten.The body's own substances are known to be ceramides, which occur particularly in the brain and in the myelin of the central nervous system. They are lipophilic amides. The ceramides are found in the organism as choline phosphate ester or as glycosides. The ceramides form the building blocks of the choline phosphate esters, also known as sphingomyelines, or of the glycosides which occur as so-called cerebrosides, gangliosides and sulfatides. The two groups of substances mentioned with ceramides as building blocks are called sphingolipids. Chemically, these are lipids that contain, instead of glycerin (in fats and oils) as an alcohol component, sphingosine (4-sphingenine), which is not naturally available.
Die Erfindung beinhaltet die Verwendung von Ceramiden und/ oder den anderen genannten Stoffen zur Behandlung der cystischen Fibröse. Durch Verabreichung einer wirksamen Menge derartiger Stoffe kann vorzugsweise die Flüssigkeitssekretion in Epithelien von Patienten stimuliert werden. Insbesondere die Lungen-, Pankreas- und Darmepithelien von CF-Patienten sind hier zu nennen. Weiterhin umfaßt die Erfindung die Behandlung von cystischer Fibröse durch Verabreichung von Ceramiden und/oder den anderen genannten Stoffen sowie die Verwendung aller dieser Stoffe zur Herstellung von Arzneimitteln, insbesondere von Arzneimitteln zur Behandlung der cystischen Fibröse.The invention includes the use of ceramides and / or the other substances mentioned for the treatment of cystic fibrosis. By administering an effective amount of such substances, fluid secretion in epithelia of patients can preferably be stimulated. The lung, pancreas and intestinal epithelia of CF patients are particularly worth mentioning here. The invention further includes the treatment of cystic fibrosis by administration of ceramides and / or the other substances mentioned and the use of all of these substances for the production of medicaments, in particular medicaments for the treatment of cystic fibrosis.
Die erfindungsgemäß verwendeten Ceramide oder Substanzen mit Ceramid-Baustein sind in einer bevorzugten Ausführungsform längerkettige Ceramide, wie beispielsweise das C12-Ceramid, bzw. deren Derivate, die als häufig natürlich vorkommende Stoffe direkt aus Zellmaterial isoliert werden können. Die eingesetzten Ceramide/Substanzen können auch durch enzymati- sche Behandlung von entsprechenden Vorstufen, die aus Zellmaterial isolierbar sind, gewonnen werden. In einer weiteren Ausführungsform der Erfindung werden Ceramide/Substanzen, die mit Hilfe von dem Fachmann bekannten molekularbiologischen Methoden gewonnen sind, verwendet. In einer besonders bevor- zugten Ausführungsform werden entsprechende, auf synthetischem Weg hergestellte Ceramide/Substanzen erfindungsgemäß eingesetzt. Eingesetzt werden hier insbesondere die C2- oder C6-Ceramide bzw. Substanzen mit diesen Ceramiden als Baustein.In a preferred embodiment, the ceramides or substances with a ceramide building block used according to the invention are longer-chain ceramides, such as C12 ceramide, or their derivatives, which can be isolated directly from cell material as frequently occurring substances. The ceramides / substances used can also be obtained by enzymatic treatment of corresponding precursors which can be isolated from cell material. In a further embodiment of the invention, ceramides / substances which are obtained with the aid of molecular biological methods known to the person skilled in the art are used. In a particularly preferred embodiment, corresponding, synthetically produced ceramides / substances are used according to the invention. In particular, the C2 or C6 ceramides or substances with these ceramides as building blocks are used here.
Erfindungsgemäß können auch Derivate von Ceramiden eingesetzt werden, die in ihren Ceramid-Bausteinen chemisch oder biologisch modifiziert sind. Diese Derivate entfalten hinsichtlich der cystischen Fibröse eine ähnliche, bevorzugt bessere, Wirkung als Ceramide und/oder weisen ähnliche oder bessere pharmazeutische Eigenschaften auf. Hierbei kann es sich beispielsweise um langsamere Abbauraten des Wirkstoffes im Körper oder um bessere Aufnahmeraten in die Zellen handeln.According to the invention, it is also possible to use derivatives of ceramides which are chemically or biologically modified in their ceramide building blocks. With regard to the cystic fibrosis, these derivatives have a similar, preferably better, effect than ceramides and / or have similar or better pharmaceutical properties. This can be, for example, slower breakdown rates of the active ingredient in the body or better uptake rates in the cells.
In einer bevorzugten Ausführungsform sind die erfindungsgemäß eingesetzten Stoffe biologische Vorläufermoleküle von Ceramiden oder von Substanzen mit Ceramid-Baustein, die, beispielsweise durch eine enzymatische Spaltung durch Sphingomyelina- sen, in der Zelle in die aktiven Ceramide umgewandelt werden.In a preferred embodiment, the substances used according to the invention are biological precursor molecules of ceramides or of substances with a ceramide building block, which are converted into the active ceramides in the cell, for example by an enzymatic cleavage by sphingomyelinas.
In einer weiteren bevorzugten Ausführungsform der Erfindung wirken die erfindungsgemäß verwendeten Stoffe auf Stoffwechselwege, die zur Bildung von Ceramiden führen. Insbesondere werden Aktivatoren von Sphingomyelinasen, welche die Spaltung von Vorlaufermolekülen zu den aktiven Ceramiden bewirken, eingesetzt.In a further preferred embodiment of the invention, the substances used according to the invention act on metabolic pathways which lead to the formation of ceramides. In particular, activators of sphingomyelinases, which cause the cleavage of precursor molecules to the active ceramides, are used.
Weiterer möglicher Angriffspunkt für Aktivatoren sind Zellkomponenten, vorzugsweise Enzyme, die den Ceramiden in der Signalkette nachgeschaltet sind. Insbesondere kommen hierfür Kinasen in Betracht, die durch Ceramide aktiviert werden. Neben der direkten Verabreichung der oben aufgeführten wirksamen Stoffe wird das Einbringen von Nukleinsäuren, welche für diese Substanzen kodieren, ebenfalls von der Erfindung umfaßt.Another possible point of attack for activators are cell components, preferably enzymes, which are connected downstream of the ceramides in the signal chain. Kinases which are activated by ceramides are particularly suitable for this purpose. In addition to the direct administration of the active substances listed above, the introduction of nucleic acids which code for these substances is also encompassed by the invention.
Die Erfindung umfaßt weiterhin pharmazeutische Zusammensetzungen, welche mindestens ein Ceramid und/oder die anderen genannten Stoffe sowie vorzugsweise zusätzlich einen pharmazeutisch akzeptablen Träger enthalten. Ob ein und ggf. welcher pharmazeutische Träger verwendet wird, ist von der Applikationsform des Arzneimittels abhängig.The invention further comprises pharmaceutical compositions which contain at least one ceramide and / or the other substances mentioned and preferably additionally a pharmaceutically acceptable carrier. Whether or not which pharmaceutical carrier is used depends on the form of administration of the drug.
Die Verabreichung aller genannten Stoffe bzw. der erfindungsgemäßen pharmazeutischen Zusammensetzungen kann systemisch erfolgen. In einer bevorzugten Ausführungsform wird die pharmazeutische Zusammensetzung über den Verdauungstrakt verabreicht. Besonders bevorzugt ist auch eine Applikation durch Inhalation, wobei der Wirkstoff schnell und direkt in die Lunge eingebracht wird. Diese Verabreichungsform ist insbesondere bei der Behandlung von cystischer Fibröse sinnvoll, da es bei dieser Krankheit zu schweren Komplikationen im Bereich der Atmungsorgane kommt.All of the substances mentioned or the pharmaceutical compositions according to the invention can be administered systemically. In a preferred embodiment, the pharmaceutical composition is administered via the digestive tract. Administration by inhalation is also particularly preferred, the active ingredient being introduced quickly and directly into the lungs. This form of administration is particularly useful in the treatment of cystic fibrosis, since this disease leads to serious complications in the area of the respiratory system.
Auch bei anderen Krankheiten, die mit einer gestörten Regulation von molekularen Transportprozessen vorzugsweise an Membranen einhergehen, kann eine Behandlung mit Ceramiden und/ oder den anderen genannten Stoffen positive Ergebnisse erzielen. Deshalb wird die Verwendung aller dieser Stoffe bei der Behandlung solcher Krankheiten mit von der Erfindung umfaßt. Vorzugsweise sollen die Transportprozesse bei der Erfindung an Ionenkanälen stattfinden, insbesondere an nach außen (aus einer Zelle oder Zellorganelle heraus) gerichteten, Ionenkanälen, vorzugsweise Chloridkanälen. Die beschriebenen Merkmale und weitere Merkmale der Erfindung ergeben sich aus der nachfolgenden Beschreibung von bevorzugten Ausführungsformen in Verbindung mit den Unteransprüchen und dem Beispiel. Hierbei können die einzelnen Merkmale jeweils für sich oder zu mehreren in Kombination miteinander verwirklicht sein.Treatment with ceramides and / or the other substances mentioned can also achieve positive results in the case of other diseases which are associated with a disturbed regulation of molecular transport processes, preferably on membranes. Therefore, the use of all of these substances in the treatment of such diseases is included in the invention. In the case of the invention, the transport processes should preferably take place on ion channels, in particular on ion channels, preferably chloride channels, directed outwards (out of a cell or cell organelle). The described features and further features of the invention result from the following description of preferred embodiments in conjunction with the subclaims and the example. The individual features can be implemented individually or in combination with one another.
In den Abbildungen zeigen:The pictures show:
Fig. 1 Beeinflussung der Chloridkanäle in normalen Lymphocyten (A, B) und in CF-Ly phocyten (C, D) durch cAMP,1 influence of the chloride channels in normal lymphocytes (A, B) and in CF-Ly phocytes (C, D) by cAMP,
Fig. 2 Beeinflussung der Chloridkanäle in CF-Lymphocyten durch Ceramid,2 influencing the chloride channels in CF lymphocytes by ceramide,
Fig. 3 Beeinflussung der Chloridkanäle in normalen Lymphocyten (A, B) und CF-Lymphocyten (C, D) durch die Tyrosinkinase Lck56.FIG. 3 Influence of the chloride channels in normal lymphocytes (A, B) and CF lymphocytes (C, D) by the tyrosine kinase Lck 56 .
Beispielexample
Die überraschend stimulierende Wirkung von C2/C6-Ceramid auf Chloridkanäle konnte in den folgenden Experimenten nachgewiesen werden. In diesen Versuchen wurde die dem Fachmann bekannte Patch-Clamp-Technik eingesetzt. Mit dieser Methode kann die Leitfähigkeit an biologischen Membranen nachgewiesen werden. In diesem Zusammenhang wird auch auf die bereits genannte Schrift von Szabo et al, Acad. Sei. USA, 95(11): 6169-6174, 1998, verwiesen, deren Inhalt diesbezüglich zum Inhalt dieser Beschreibung gemacht wird.The surprisingly stimulating effect of C2 / C6 ceramide on chloride channels could be demonstrated in the following experiments. The patch clamp technique known to the person skilled in the art was used in these experiments. This method can be used to demonstrate the conductivity on biological membranes. In this context, reference is also made to the aforementioned document by Szabo et al, Acad. Be. USA, 95 (11): 6169-6174, 1998, the content of which in this regard is made the content of this description.
Hierbei wird bekanntermaßen mit einer Mikropipette eine Membran angesaugt und durch Unterdruck an dem Rand der Mikropi- pette versiegelt. Die auf diese Weise fixierte Membran als Teil einer intakten Zelle oder als isoliertes Membranstück wird in ein geeignetes Elektrolytbad getaucht und die Leitfähigkeit zwischen diesem und der Elektrolytlösung innerhalb der Pipette (Patch-Clamp-Elektrode) gemessen. Durch Zugabe verschiedener Agenzien in die Elektrolytlösungen kann der Öffnungszustand von Ionenkanälen der Membran beeinflußt werden. Eine solche Beeinflussung ist durch eine sprunghafte Änderung der Leitfähigkeit nachweisbar.As is known, a membrane is sucked in with a micropipette and vacuum at the edge of the micropipette pette sealed. The membrane fixed in this way as part of an intact cell or as an isolated membrane piece is immersed in a suitable electrolyte bath and the conductivity between it and the electrolyte solution inside the pipette (patch clamp electrode) is measured. The opening state of the ion channels of the membrane can be influenced by adding various agents to the electrolyte solutions. Such an influence can be demonstrated by a sudden change in the conductivity.
Im vorliegenden Fall wurden T-Lymphocyten von normalen und von an cystischer Fibröse (CF) erkrankten Patienten isoliert, in eine Perfusionskammer eingebracht und mit einer Elektrolytlösung überströmt (145 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 2 M CaCl , 10 mM Glucose, 10 mM Hepes/NaOH, pH 7,4). Unter dem inversen Mikroskop wurden Patch-Clamp-Elektroden (gefüllt mit 160 mM Cs-Glutamat, 2 mM MgCl2, 0 , 1 mM CaCl2 , 1,1 mM EGTA, 4 Na2ATP, 10 mM Hepes/NaOH, pH 7,2) an die Zellmembran gebracht und durch Einsaugen der Zellmembran eine Verbindung zum Intrazellulärraum hergestellt (whole cell patch clamp) oder die Leitfähigkeit des angesaugten Membranflecks gemessen (cell attached patch clamp) . Damit wurde eine kontinuierliche Registrierung der Zellmembranleitfähigkeit möglich. Nach einer Kontrollperiode wurden die Zellen entweder extrazellulär mit cAMP (200 μM) , extrazellulär mit C2-Ceramid (50 μM) oder intrazellulär mit der Tyrosinkinase Lck56 (10 U/ml) stimuliert und die Leitfähigkeit kontinuierlich gemessen.In the present case, T-lymphocytes were isolated from normal and from patients suffering from cystic fibrosis (CF), placed in a perfusion chamber and flooded with an electrolyte solution (145 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 2 M CaCl, 10 mM Glucose, 10mM Hepes / NaOH, pH 7.4). Patch clamp electrodes (filled with 160 mM Cs-glutamate, 2 mM MgCl 2 , 0.1 mM CaCl 2 , 1.1 mM EGTA, 4 Na 2 ATP, 10 mM Hepes / NaOH, pH 7) were examined under the inverted microscope , 2) brought to the cell membrane and a connection to the intracellular space is established by sucking in the cell membrane (whole cell patch clamp) or the conductivity of the aspirated membrane spot is measured (cell attached patch clamp). This made a continuous registration of the cell membrane conductivity possible. After a control period, the cells were stimulated either extracellularly with cAMP (200 μM), extracellularly with C2-ceramide (50 μM) or intracellularly with the tyrosine kinase Lck 56 (10 U / ml) and the conductivity was measured continuously.
Die Elektroden wurden über geeignete Vorverstärker mit einem Patch-Clamp-Verstärker verbunden (EPC-9) , mit dessen Hilfe das Potential zwischen Patch-Clamp-Elektrode und einer Referenzelektrode im Bad von -100 mV bis +100 mV variiert wurde. Die jeweiligen Ströme über die Zellmembran zwischen Patch- Clamp-Elektrode und Referenzelektrode wurden mit Hilfe des Patch-Clamp-Verstärkers aufgezeichnet. Die Wahl der Lösungen ermöglichte eine ausschließliche Analyse von Strömen durch Chloridkanäle.The electrodes were connected to a patch clamp amplifier (EPC-9) by means of suitable preamplifiers, with the aid of which the potential between the patch clamp electrode and a reference electrode in the bath was varied from -100 mV to +100 mV. The respective currents across the cell membrane between patch The clamp electrode and reference electrode were recorded using the patch clamp amplifier. The choice of solutions allowed an exclusive analysis of currents through chloride channels.
Die in den einzelnen Teilen der Fign. 1 und 3 dargestellten Kurvenverläufe spiegeln die Veränderung der Leitfähigkeit (whole cell) innerhalb eines mehrminütigen Zeitraums nach der Applikation der jeweiligen Substanzen wider. Die in Fig. 2 dargestellten Daten zeigen die Leitfähigkeit vor Applikation von Ceramid (cell attached) und 7 Minuten nach Applikation von Ceramid. Die Applikation von cAMP und Lck56 erfolgte in die Pipette, so daß sich aus den in Fig. 1 und 3 dargestellten Kurven, die zu verschiedenen Zeiten aufgenommen wurden, die Kinetik der Aktivierung der Chloridkanäle nicht direkt ableiten läßt. Vielmehr ist diese Zeitabhängigkeit auf die Diffusion der zugeführten Substanzen innerhalb der Pipette zurückzuführen und also von der gewählten Methodik abhängig. Für die Interpretation ist hauptsächlich der Endpunkt der Leitfähigkeitsveränderung entscheidend, d.h. die jeweils oberste Kurve.The in the individual parts of Figs. 1 and 3 curve curves reflect the change in conductivity (whole cell) within a period of several minutes after the application of the respective substances. The data shown in FIG. 2 show the conductivity before application of ceramide (cell attached) and 7 minutes after application of ceramide. The application of cAMP and Lck 56 was carried out in the pipette, so that the kinetics of the activation of the chloride channels cannot be derived directly from the curves shown in FIGS. 1 and 3, which were recorded at different times. Rather, this time dependency is due to the diffusion of the substances supplied within the pipette and is therefore dependent on the method chosen. The end point of the change in conductivity, ie the top curve in each case, is decisive for the interpretation.
Ceramid wurde außerhalb der Pipette appliziert. Daher spielte bei dem Versuch, dessen Ergebnisse in Fig. 2 dargestellt sind, eine Diffusion keine Rolle. In der gewählten Darstellung ist deshalb nur der Kontrollwert (ohne Ceramid) und die veränderte Leitfähigkeit nach 7 Minuten Inkubation mit Ceramid aufgetragen.Ceramide was applied outside the pipette. Therefore, diffusion was of no importance in the experiment, the results of which are shown in FIG. 2. In the selected illustration, therefore, only the control value (without ceramide) and the changed conductivity after 7 minutes of incubation with ceramide are plotted.
Wie Fig. 1 zeigt, führte die Applikation von cAMP erwartungsgemäß zu einer Aktivierung des Cl~-Stromes in normalen Lymphocyten (Fig. 1 A, B) , nicht aber in CF-Lymphocyten (Fig. 1 C, D) . Dementsprechend liegen bei den Fig. 1 A, C, D die im zeitlichen Abstand bestimmten Meßkurven jeweils übereinander, bei Fig. 1 B jedoch nicht. Im Gegensatz dazu und überraschenderweise führte Ceramid in CF-Lymphocyten (Fig. 2) zu einer Aktivierung der Chloridkanäle. (Vergleich Kurve (vor Applikation) - Kurve (nach Applikation) ) . Weiterhin führte die Zugabe der Tyrosinkinase Lck56, die bekanntermaßen durch Ceramide aktivierbar ist, zu einer Aktivierung von Chloridkanälen in normalen (Fig. 3 A, B) sowie in CF-Lymphocyten (Fig. 3 C, D) . Die Kurven liegen bei Fig. 3 A und 3 C übereinander, bei den Fig. 3 B und 3 D nicht. Dies steht im Gegensatz zu den Ergebnissen mit cAMP.As shown in FIG. 1, the application of cAMP, as expected, led to an activation of the Cl ~ current in normal lymphocytes (FIG. 1A, B), but not in CF lymphocytes (FIG. 1 C, D). Accordingly, in FIGS. 1 A, C, D the measurement curves determined at intervals are each one above the other, but not in FIG. 1B. In contrast, and surprisingly, ceramide in CF lymphocytes (Fig. 2) led to activation of the chloride channels. (Comparison curve (before application) - curve (after application)). Furthermore, the addition of tyrosine kinase Lck 56 , which is known to be activated by ceramides, led to an activation of chloride channels in normal (FIG. 3A, B) and in CF lymphocytes (FIG. 3 C, D). The curves lie one above the other in FIGS. 3A and 3C, but not in FIGS. 3A and 3D. This is in contrast to the results with cAMP.
Diese Ergebnisse zeigen deutlich, daß der Defekt in CF-Zel- len, also die gestörte Aktivierbarkeit von Chloridkanälen, durch die erfindungsgemäße Verwendung von Ceramiden positiv beeinflußt werden kann. These results clearly show that the defect in CF cells, ie the impaired ability to activate chloride channels, can be positively influenced by the use of ceramides according to the invention.

Claims

Patentansprüche claims
1. Verwendung mindestens eines Ceramids und/oder mindestens einer Substanz, die ein Ceramid als Baustein enthält, zur Behandlung von cystischer Fibröse.1. Use of at least one ceramide and / or at least one substance which contains a ceramide as a building block for the treatment of cystic fibrosis.
2. Verwendung nach Anspruch 1, dadurch gekennzeichnet, daß das Ceramid ein C2- oder ein C6-Ceramid ist bzw. die Substanz ein solches Ceramid als Baustein enthält.2. Use according to claim 1, characterized in that the ceramide is a C2 or a C6 ceramide or the substance contains such a ceramide as a building block.
3. Verwendung nach Anspruch 1 oder 2 , dadurch gekennzeichnet, daß das Ceramid und/oder die Substanz mit Ceramid- Baustein aus biologischem Zellmaterial, insbesondere aus molekularbiologisch verändertem Zellmaterial, isoliert sind.3. Use according to claim 1 or 2, characterized in that the ceramide and / or the substance with ceramide building block made of biological cell material, in particular of molecular biologically modified cell material, are isolated.
4. Verwendung nach Anspruch 1 oder 2 , dadurch gekennzeichnet, daß das Ceramid und/oder die Substanz mit Ceramid- Baustein synthetisch hergestellt sind.4. Use according to claim 1 or 2, characterized in that the ceramide and / or the substance with a ceramide building block are synthetically produced.
5. Verwendung mindestens eines biologischen Vorläufers eines Ceramids oder einer Substanz , die ein Ceramid als Baustein enthält, zur Behandlung von cystischer Fibröse.5. Use of at least one biological precursor of a ceramide or a substance which contains a ceramide as a building block for the treatment of cystic fibrosis.
6. Verwendung mindestens eines Aktivators, insbesondere mindestens eines biologischen Aktivators, der auf ein Ceramid, auf eine Substanz, die ein Ceramid als Baustein enthält, oder auf einen biologischen Vorläufer dieses Ceramids oder dieser Substanz aktivierend wirkt, zur Behandlung von cystischer Fibröse. 6. Use of at least one activator, in particular at least one biological activator, which has an activating action on a ceramide, on a substance which contains a ceramide as a building block, or on a biological precursor of this ceramide or this substance, for the treatment of cystic fibrosis.
7. Pharmazeutische Zusammensetzung, umfassend eine wirksame Menge mindestens eines Ceramids und/oder mindestens einer Substanz, die ein Ceramid als Baustein enthält, sowie vorzugsweise einen pharmazeutischen Träger.7. A pharmaceutical composition comprising an effective amount of at least one ceramide and / or at least one substance containing a ceramide as a building block, and preferably a pharmaceutical carrier.
8. Pharmazeutische Zusammensetzung nach Anspruch 7, dadurch gekennzeichnet, daß das Ceramid ein C2- oder ein C6- Ceramid ist bzw. die Substanz ein solches Ceramid als Baustein enthält.8. Pharmaceutical composition according to claim 7, characterized in that the ceramide is a C2 or a C6 ceramide or the substance contains such a ceramide as a building block.
9. Pharmazeutische Zusammensetzung nach Anspruch 7 oder 8, dadurch gekennzeichnet, daß das Ceramid und/oder die Substanz mit Ceramid-Baustein aus biologischem Zellmaterial, insbesondere aus molekularbiologisch verändertem Zellmaterial, isoliert sind.9. Pharmaceutical composition according to claim 7 or 8, characterized in that the ceramide and / or the substance with ceramide building block from biological cell material, in particular from molecular biologically modified cell material, are isolated.
10. Pharmazeutische Zusammensetzung nach Anspruch 7 oder 8, dadurch gekennzeichnet, daß das Ceramid und/oder die Substanz mit Ceramid-Baustein synthetisch hergestellt sind.10. Pharmaceutical composition according to claim 7 or 8, characterized in that the ceramide and / or the substance with a ceramide building block are synthetically produced.
11. Pharmazeutische Zusammensetzung, umfassend eine wirksame Menge mindestens eines biologischen Vorläufers eines Ceramids oder einer Substanz , die ein Ceramid als Baustein enthält, und vorzugsweise mindestens einen pharmazeutischen Träger.11. A pharmaceutical composition comprising an effective amount of at least one biological precursor of a ceramide or a substance containing a ceramide as a building block, and preferably at least one pharmaceutical carrier.
12. Pharmazeutische Zusammensetzung, umfassend eine wirksame Menge mindestens eines Aktivators, vorzugsweise mindestens eines biologischen Aktivators, der auf ein Ceramid, auf eine Substanz, die ein Ceramid als Baustein enthält, oder auf einen biologischen Vorläufer dieses Ceramids oder dieser Substanz aktivierend wirkt, und vorzugsweise mindestens einen pharmazeutischen Träger. 12. A pharmaceutical composition comprising an effective amount of at least one activator, preferably at least one biological activator, which activates a ceramide, a substance containing a ceramide as a building block, or a biological precursor of this ceramide or substance, and preferably at least one pharmaceutical carrier.
13. Verwendung mindestens eines Ceramids und/oder mindestens einer Substanz, die ein Ceramid als Baustein enthält, zur Behandlung von Krankheiten, die mit einer gestörten Regulation von Transportprozessen an Membranen im Zusammenhang stehen.13. Use of at least one ceramide and / or at least one substance which contains a ceramide as a building block for the treatment of diseases which are associated with a disturbed regulation of transport processes on membranes.
14. Verwendung mindestens eines biologischen Vorläufers eines Ceramids oder einer Substanz , die ein Ceramid als Baustein enthält, zur Behandlung von Krankheiten, die mit einer gestörten Regulation von Transportprozessen an Membranen im Zusammenhang stehen.14. Use of at least one biological precursor of a ceramide or a substance which contains a ceramide as a building block for the treatment of diseases which are associated with a disturbed regulation of transport processes on membranes.
15. Verwendung mindestens eines Aktivators, insbesondere mindestens eines biologischen Aktivators, der auf ein Ceramid, auf eine Substanz, die ein Ceramid als Baustein enthält, oder auf einen biologischen Vorläufer dieses Ceramids oder dieser Substanz aktivierend wirkt, zur Behandlung von Krankheiten, die mit einer gestörten Regulation von Transportprozessen an Membranen im Zusammenhang stehen.15. Use of at least one activator, in particular at least one biological activator, which has an activating action on a ceramide, on a substance which contains a ceramide as a building block, or on a biological precursor of this ceramide or this substance, for the treatment of diseases which are associated with a disturbed regulation of transport processes on membranes.
16. Verwendung nach einem der Ansprüche 13 bis 15, dadurch gekennzeichnet, daß die Transportprozesse Ionenkanäle betreffen, insbesondere auswärts gleichrichtende Chloridkanäle.16. Use according to one of claims 13 to 15, characterized in that the transport processes relate to ion channels, in particular chloride channels which rectify outward.
17. Verwendung nach einem der Ansprüche 13 bis 16, dadurch gekennzeichnet, daß es sich bei dem Ceramid um ein Ceramid mit einem Merkmal nach einem der Ansprüche 2 bis 4 handelt. 17. Use according to one of claims 13 to 16, characterized in that the ceramide is a ceramide with a feature according to one of claims 2 to 4.
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