EP1427393A2 - Liposomes containing active substances - Google Patents

Liposomes containing active substances

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Publication number
EP1427393A2
EP1427393A2 EP01933914A EP01933914A EP1427393A2 EP 1427393 A2 EP1427393 A2 EP 1427393A2 EP 01933914 A EP01933914 A EP 01933914A EP 01933914 A EP01933914 A EP 01933914A EP 1427393 A2 EP1427393 A2 EP 1427393A2
Authority
EP
European Patent Office
Prior art keywords
active ingredient
liposomally encapsulated
liposomes
substances
phosphatidylcholine
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Withdrawn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
EP01933914A
Other languages
German (de)
French (fr)
Inventor
Jürgen Ebert
Gerd Berger
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.)
Pharmacept GmbH
Original Assignee
Pharmacept GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmacept GmbH filed Critical Pharmacept GmbH
Publication of EP1427393A2 publication Critical patent/EP1427393A2/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin

Definitions

  • the present invention relates to liposomes which contain active substances, in particular pharmaceutical active substances, a process for their preparation and the use of these liposomes for the production of pharmaceutical products.
  • active pharmaceutical ingredients in particular of large active ingredient molecules
  • the active ingredient is distributed in different amounts over the individual areas of the body, often only small amounts of the active ingredient reaching the target organ.
  • the active substances can be metabolized due to reactions with substances present in the body and undesirable side effects for the whole organism can occur.
  • Liposomes are used as carrier substances, in particular for relatively large active substance molecules.
  • the active substances are enclosed in the liposomes and cannot be attacked by other substances.
  • the liposomes can be built up in such a way that the drugs enclosed in them direct a specific structure of the liposome envelope into certain target organs.
  • Liposomes are artificially produced spherical lipid vesicles that consist of one or more concentric lipid bilayers with an aqueous interior. They can be produced by mechanical fine distribution (dispersion) of phospholipids in aqueous media. A distinction is made between multilamellar vesicles with a diameter of approximately 0.1 to 5 ⁇ m and unilamellar vesicles with a size of 0.02 to 0.05 ⁇ m or by approximately 1 ⁇ m. The liposomes play an important role particularly in the treatment of tumors as carrier systems.
  • An active ingredient that is administered encapsulated liposomally is e.g. B. Paclitaxel.
  • Paclitaxel shows only a low solubility in water and is therefore often used in combination with solubilizers such as Cremophor (polyethoxylated castor oil).
  • solubilizers such as Cremophor (polyethoxylated castor oil).
  • Cremophor polyethoxylated castor oil
  • the dilution of such solutions with physiological saline for application has the disadvantage that paclitaxel in physiological saline does not have sufficient stability.
  • International patent application WO 93/18751 discloses paclitaxel-liposome combinations, of which in particular vesicles with a positive charge have been produced based on cardiolipin, phosphatidylcholine and cholesterol.
  • the liposomes produced had to be applied in animal experiments on four consecutive days, from which it can be concluded that the desired antitumor effect could not be achieved after a single administration.
  • the present invention was accordingly based on the object of providing a liposomal system in which the activity of active substances in the treatment of diseases or the accumulation of active substances and / or pharmaceutical substances which are to be accumulated in a specific target organ, such as e.g. Contrast agents from imaging processes etc. can be accumulated.
  • a specific target organ such as e.g. Contrast agents from imaging processes etc.
  • the effectiveness of such systems in the treatment of diseases etc. should be increased and, preferably, the number of side effects should also be reduced.
  • liposomally encapsulated active substances such as anti-tumor active substances
  • the effect of liposomally encapsulated active substances can be increased many times over if the active substance is not completely liposomally encapsulated, i.e. if there are still free active substance molecules in the solution / emulsion to be applied outside the liposomes.
  • the present invention accordingly relates to a liposomally encapsulated active substance, which is characterized in that it is encapsulated in a proportion of 1% by weight to 90% by weight, based on the amount of active substance used.
  • the proportion of the encapsulation means that encapsulated active substance molecules are located in the center of the liposomes or in the membrane in the
  • Active substances which can be used in the present invention are pharmaceuticals and pharmacologically active substances, nutrients, cosmetic substances, diagnostic substances and contrast agents for imaging processes.
  • the term active ingredient also includes the pharmacologically acceptable salts of the active ingredients.
  • Suitable compounds are lipophilic and amphiphilic molecules, i.e. Substances which are insoluble in water or only slightly soluble in water and which can be prepared in the form of emulsions or micellar preparations.
  • Suitable drugs are e.g. Anti-cancer agents, anti-tumor agents, antibiotics, antimicotic, antivirual, anthelmintic and anti-parasitic compounds.
  • Anthracyclines such as doxorubicin, daunorubicin, karinomycin, N-acetylatriamycin, rubidacon, 5-imidodaunomycin, N-acetyldaunomycin, pirarubicin and epirubicin, alkaloids such as vincristine, vinbline, vinblastine, vinblastine, vinblastine, vinblastine, vinblastine, vinblastine, vinblastine be used. Further suitable substances are 5-fluorouracil, taxanes such as paclitaxel (Taxol ®), and derivatives of paclitaxel z.
  • Taxotere Docetaxol ®
  • Mitrotan platinum compounds such as cisplatin, carboplatin, lobaplatin and phenestrin).
  • anti-inflammatory agents examples include steroidal and non-steroidal and other anti-inflammatory compounds such as prednisone, methyl-prednisolone, paramethazone, 11-fluorocortisol, triamciniolone, betamethasone and dexamethasone, ibuprophen, piroxicam, beclomethasone Methotrexate, acaridine, etritinate, anthralin, psoraline, salycilate such as aspirin, and immunosuppressants such as cyclosporin.
  • anti-inflammatory corticosteids and the anti-inflammatory and immunosuppressive cyclosporin are lipophilic compounds which can be used particularly advantageously in the present invention.
  • anesthetics such as methoxyflurane, isoflurane, N-flurane, halothane and bezocaine
  • antiulcerative agents such as cimetidine
  • anticonvulsant agents such as barbiturates, azothioprine, an immunosuppressant and anti-rheumatic agent
  • muscle relaxants such as dantrolene and diazepam.
  • contrast agents for ultrasound, X-ray and NMR processes.
  • Radioisotopes or compounds containing such radioisotopes such as e.g. Iodine, octane, halogenated hydrocarbon and renograph.
  • Suitable contrast agents (gadolinium) that can be used in NMR methods are lipid-soluble paramagnetic compounds.
  • Dietary supplements that can be incorporated into the liposomal system according to the present invention are amino acids, sugars, proteins, carbohydrates, fat-soluble vitamins, fats (lipids). Combinations of different nutritional supplements are also suitable.
  • the active ingredients used can be encapsulated in a manner known per se with the addition of liposomes. In order to set the desired degree of encapsulation, it is possible to encapsulate the active ingredient only partially.
  • the partial encapsulation can be carried out using appropriate starting materials or suitable process conditions.
  • a completely or almost completely liposomally encapsulated active substance can be mixed with the corresponding amount of unencapsulated active substance or their solutions / emulsions, so that the desired degree of encapsulation is obtained.
  • the degree of encapsulation can be adjusted by simple process changes.
  • the liposome has a closed structure, which consists of a lipid bilayer that surrounds an aqueous inner core.
  • the liposomes used in accordance with the invention are not limited to certain liposomes; both neutral, negatively or positively charged liposomes can be used which are unilamellar, i.e. from a lipid bilayer, or polylamellar, i.e. be composed of several lipid bilayers and can be produced by methods known to the person skilled in the art.
  • the liposomes are usually produced from phospholipids and, if appropriate, cholesterol and cholesterol derivatives and / or one or more hydrophilic, lipophilic or amphiphilic component (s).
  • suitable phospholipids are phosphatidylcholine (PC), distearoylphsphatidylcholine (DSPC), soy phosphatidylcholine (soybean PC), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine (HEPCcholine) (HEPCcholine), diphenylpholine (HEPC) DPPC), dimyristoylphosphatidylcholine (DMPC) as well as any mixtures thereof, whereby the substances can be synthetic, semi-synthetic or natural products.
  • PC phosphatidylcholine
  • DSPC distearoylphsphatidylcholine
  • phospholipids are phosphatidylglycerides (PG) and phosphatidic acid, such as dimynstoylphosphatidylglycerid (DMPG), dilaurylphosphatidylglycerid (DLPG), dipalmitoylphosphatidylglycerid (DPPG), distearoylphosphatidylglycerid (DSPGilidic acid, DSistGilid) , Distearoylphosphatidic acid (DSPA), and phosphatidylethanolamines, phosphatidylinositols and phosphatidic acid, which contain residues of lauric acid, myristic acid, stearic acid and / or palmitic acid.
  • DMPG dimynstoylphosphatidylglycerid
  • DLPG dilaurylphosphatidylglycerid
  • DPPG dipalmitoylphosphatidylglycerid
  • Preferred phospholipids are HSPC, DSPC and HEPC.
  • positively charged liposomes can be formed from a solution containing phosphatidylcholine, cholesterol and stearylamine.
  • Negatively charged liposomes can be obtained, for example, from solutions containing phosphatidylcholone, cholesterol and phosphatidylserine or, preferably, cardiolipin. It is known to the person skilled in the art that other additives can also be added in order to modify the properties of the liposomes obtained.
  • liposomes can be changed, for example, by adding -Tocopherol. Good results are also achieved with so-called PEG liposomes, i.e. Liposomes with polyethylene glycol chains (PEG) in the lipid layer, the PEG either being bound in the molecule of the phospholipids or being present as a free substance.
  • PEG polyethylene glycol chains
  • the molecular weight of the PEG chains is preferably between 400 and 20,000, particularly preferably between 600 and 10,000 and in particular between 600 and 5,000.
  • liposomes can also be used as liposomes.
  • Preferred liposomes are e.g. B. from HSPC, DSPC and / or HEPC as phospholipid, cholesterol and / or N- (O-methyl-poly (ethylene glycol) -1, 2-distearoyl-sn-glycero-3-phosphoethanolamine and optionally polyethylene glycol.
  • the ratio of lipid to active ingredient can be adjusted within a wide range and generally depends on the active ingredient used and the lipid used for liposome production and the other components.
  • the final encapsulated product contains lipid and active ingredient in a ratio of 5: 1 to 100: 1, preferably 10: 1 to 40: 1, in particular 15: 1 to 25: 1.
  • the molar ratio of lipid to cholesterol is preferably from 3: 1 to 1: 3, preferably from 2: 1 to 1: 2.
  • Polyethylene glycol is preferably used in an amount of 1 to 50 mol%. , in particular from 5 to 20 mol%, based on the lipid.
  • the size of the active ingredients encapsulated liposomally according to the invention is preferably below 200 nm, particularly preferably below 150 nm.
  • microspheres are preferably produced from polysaccharides or polysaccharide derivatives and are particularly preferably degradable.
  • the polysaccharides are preferably selected from starch and starch derivatives, gelatin, albomine, collagen, dextran, dextane derivatives or similar materials. Lyophilized or degradable starch or gelatin particles are particularly preferred.
  • microspheres preferably have a water-insoluble, but hydrophilic, water-swellable, three-dimensional network of polysaccharide molecules or corresponding derivatives. Suitable microspheres are described in DE-US-25 24 279, WO 88/09163 and WO 89/03207.
  • the diameter of the microspheres is preferably between 0.1 and 200 ⁇ m, in particular between 10 and 100 ⁇ m.
  • the liposomal systems produced according to the invention can be added directly to the bloodstream of the person to be treated, and intraperitoneal, subcutaneous or inhalation administration is also possible.
  • the liposomes used according to the invention can be produced by any method known from the prior art.
  • the liposomes in be dissolved in a suitable solvent, usually in a non-polar or only slightly polar solvent which can be removed without leaving harmful substances, such as ethanol, methanol, chloroform, butanol or acetone.
  • a suitable solvent usually in a non-polar or only slightly polar solvent which can be removed without leaving harmful substances, such as ethanol, methanol, chloroform, butanol or acetone.
  • active ingredient mixtures are used, the individual solutions can also be mixed with one another.
  • the lipophilic material is also dissolved and mixed with the solution containing the active ingredient. After the solvent has been removed, for example by a lyophilization process, the lipid film remains on the active ingredient.
  • the mixture can be stored in this form, optionally under an inert gas atmosphere, with low storage temperatures, such as at -20 ° C., being particularly preferred.
  • the liposomes are usually formed by adding a suitable solution to the lipid film.
  • suitable solutions are polar solutions, preferably aqueous salt solutions, such as isotonic saline. Liposomes are formed, for example, by mixing and vortexing. If small vesicles, such as unilamellar vesicles, are produced, the solution can also be treated with ultrasound.
  • mixtures of multilamellar vesicles and unilamellar vesicles can also be used.
  • the liposomes produced can be administered directly to the patient or stored under suitable conditions.
  • the lipophilic active substances are preferably stored at about -20 ° C. as dry substances to which lipid films have been applied. After hydration, liposome suspensions to which a suitable amount of unencapsulated active ingredient has been added can be stored in buffered or neutral saline solutions for a period of a few hours to months, depending on the temperature and the ingredients.
  • the liposomal drug system according to the present invention can be administered in amounts of about 5 to 150 mg of drug / kg body weight of the patient within a period of 1 minute to 5 hours.
  • This liposome dispersion is shaken at room temperature for 24 h, the resulting multilayer vesicles (MLV) are subjected to sonication (proboscis, 6x4 min with 50% intensity, Branson B225 sonifer; Branson, Carouge-Geheve, Switzerland) and then centrifuged (3000 rpm) 20 min) to remove the titanium abrasion from the liposome dispersion.
  • sonication proboscis, 6x4 min with 50% intensity, Branson B225 sonifer; Branson, Carouge-Geheve, Switzerland
  • 5-fluorouracil (Riboluor ® 1000; 50 mg / ml Infusion solution Ribosepharm GmbH, Kunststoff, Germany) and carboplatin (Ribocarabo ® 50 mg / ml Infusion solution Ribosepharm GmbH, Kunststoff, Germany) was used.
  • the vesicle size was determined with a photon correlation spectrometer (Coulter Counter N4 MD modeil and the AccuComp ® system, Coulter Electronics Inc., Hialeah, US) and moved for both active ingredients in the order of 100 ⁇ 50 nm.
  • NOE Nuclear Overhauser Effect
  • the active substances were administered intravenously and intraarterially as well as in encapsulated form with and without starch microspheres (Spherex ® , Pharmacia Upjohn), generally for the treatment of tumors in liver tumors.
  • Spherex ® starch microspheres
  • the tumor was targeted by liposomally encapsulating the cytostatics and adding starch microspheres.
  • the SUV PEG used Liposomes have a particle size of 113 nm ⁇ 25 nm, the starch microspheres on average 45 ⁇ m.
  • the drug concentrations in the tumor and in the liver were measured in AUC (Area under the Curve). The results are shown in Table 1.

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Abstract

The invention relates to liposomally encapsulated active substances, which are characterized in that the active substance(s) is/are encapsulated in a proportion of 1 wt. % to 90 wt. % with regard to the amount of active substances used. These active substances are particularly well-suited for treating tumors, as food supplements, for producing contrast media for imaging methods and for producing diagnostic agents for diseases.

Description

Patentanmeldung Patent application
Wirkstoffhaltige LiposomeActive ingredient-containing liposomes
Die vorliegende Erfindung betrifft Liposome, die Wirkstoffe, insbesondere pharmazeutische Wirkstoffe enthalten, ein Verfahren zu Ihrer Herstellung sowie die Verwendung dieser Liposomen zur Herstellung von pharmazeutischen Produkten.The present invention relates to liposomes which contain active substances, in particular pharmaceutical active substances, a process for their preparation and the use of these liposomes for the production of pharmaceutical products.
Die Verabreichung von pharmazeutischen Wirkstoffen, insbesondere von großen Wirkstoffmolekülen, ist häufig problematisch. Der Wirkstoff verteilt sich in Abhängigkeit von seiner Löslichkeit in Wasser oder in lipophilen Lösungsmittel in unterschiedlichen Mengen über die einzelnen Körperbereiche, wobei häufig nur geringe Mengen an Wirkstoff das Zielorgan erreichen. Ferner können die Wirkstoffe aufgrund von Reaktionen mit im Körper vorhandenen Stoffen metabolisiert werden und es kann zu unerwünschten Nebeneffekten für den Gesamtorganismus kommen.The administration of active pharmaceutical ingredients, in particular of large active ingredient molecules, is often problematic. Depending on its solubility in water or in lipophilic solvents, the active ingredient is distributed in different amounts over the individual areas of the body, often only small amounts of the active ingredient reaching the target organ. Furthermore, the active substances can be metabolized due to reactions with substances present in the body and undesirable side effects for the whole organism can occur.
Insbesondere für relativ große Wirkstoffmoleküle werden Liposomen als Trägersubstanzen eingesetzt. Die Wirkstoffe sind in den Liposomen eingeschlossen und können von anderen Stoffen nicht angegriffen werden. Ferner lassen sich die Liposomen derart aufbauen, dass die darin eingeschlossenen Medikamente über einen spezifischen Aufbau der Liposomenhülle in bestimmte Zielorgane dirigieren.Liposomes are used as carrier substances, in particular for relatively large active substance molecules. The active substances are enclosed in the liposomes and cannot be attacked by other substances. Furthermore, the liposomes can be built up in such a way that the drugs enclosed in them direct a specific structure of the liposome envelope into certain target organs.
Liposome sind künstlich hergestellte kugelförmige Lipid-Vesikel, die aus einer oder mehreren konzentrischen Lipid-Doppelschichten mit wässerigem Innenraum bestehen. Sie lassen sich durch mechanische Feinverteilung (Dispergierung) von Phospholipiden in wässerigen Medien herstellen. Man unterscheidet multilamellare Vesikel mit einem Durchmesser von etwa 0,1 bis 5 μm von unilamellaren Vesikeln mit einer Größe von 0,02 bis 0,05 μm beziehungsweise um ca. 1 μm. Die Liposome spielen insbesondere bei der Behandlung von Tumoren als Trägersysteme eine wichtige Rolle. Ein Wirkstoff, der liposomal verkapselt verabreicht wird, ist z. B. Paclitaxel. Paclitaxel zeigt nur eine geringe Löslichkeit in Wasser und wird daher häufig in Kombination mit Lösungsvermittlern, wie Cremophor (polyethoxyliertes Rizinusöl) verwendet. Der Einsatz dieser Kombination führt jedoch zu erheblichen Nebenwirkungen wie z.B. zu anaphylaktoiden Reaktionen. Die Verdünnung solcher Lösungen mit physiologischer Kochsalzlösung zur Applikation hat den Nachteil, dass Paclitaxel in physiologischer Kochsalzlösung keine ausreichende Stabilität besitzt. In der internationalen Patentanmeldung WO 93/18751 werden Paclitaxel-Liposomen- Kombinationen offenbart, von denen insbesondere Vesikel mit positiver Ladung auf Basis von Cardiolipin, Phosphatidylcholin und Cholesterol hergestellt wurden. Die hergestellten Liposomen mussten, wie die Beispiele zeigen, jedoch in Tierversuchen an vier aufeinander folgenden Tagen appliziert werden, woraus sich schließen lässt, dass nach einer einmaligen Gabe nicht die gewünschte Antitumorwirkung erreicht werden konnte.Liposomes are artificially produced spherical lipid vesicles that consist of one or more concentric lipid bilayers with an aqueous interior. They can be produced by mechanical fine distribution (dispersion) of phospholipids in aqueous media. A distinction is made between multilamellar vesicles with a diameter of approximately 0.1 to 5 μm and unilamellar vesicles with a size of 0.02 to 0.05 μm or by approximately 1 μm. The liposomes play an important role particularly in the treatment of tumors as carrier systems. An active ingredient that is administered encapsulated liposomally is e.g. B. Paclitaxel. Paclitaxel shows only a low solubility in water and is therefore often used in combination with solubilizers such as Cremophor (polyethoxylated castor oil). However, the use of this combination leads to considerable side effects such as anaphylactoid reactions. The dilution of such solutions with physiological saline for application has the disadvantage that paclitaxel in physiological saline does not have sufficient stability. International patent application WO 93/18751 discloses paclitaxel-liposome combinations, of which in particular vesicles with a positive charge have been produced based on cardiolipin, phosphatidylcholine and cholesterol. However, as the examples show, the liposomes produced had to be applied in animal experiments on four consecutive days, from which it can be concluded that the desired antitumor effect could not be achieved after a single administration.
Auch bei der liposomalen Verkapselung von anderen Wirkstoffen tritt häufig das Problem auf, dass die Liposomen zum Teil zu Nebenwirkungen führen oder diese direkt in die Leber gelangen, wo sie umgehend eliminiert werden.Also with the liposomal encapsulation of other active substances, the problem often arises that the liposomes sometimes lead to side effects or these get directly into the liver, where they are immediately eliminated.
Auch umfangreiche Forschungen, die die Kapazität von Liposomen erhöhen sollen, führen nicht zu den gewünschten Wirkungen, da die Liposomen selbst nicht das Zielorgan erreichen.Even extensive research to increase the capacity of liposomes does not lead to the desired effects, since the liposomes themselves do not reach the target organ.
Der vorliegenden Erfindung lag demgemäß die Aufgabe zugrunde, ein liposomales System zur Verfügung zu stellen, worin die Aktivität von Wirkstoffen bei der Behandlung von Krankheiten beziehungsweise die Akkumulation von Wirkstoffen und/oder pharmazeutischen Substanzen, die in einem bestimmten Zielorgan akkumuliert werden sollen, wie z.B. Kontrastmittel von Bild gebenden Verfahren etc. akkumuliert werden können. Es sollten die Wirksamkeit derartiger Systeme bei der Behandlung von Krankheiten etc. erhöht und vorzugsweise auch die Anzahl der Nebenwirkungen verringert werden.The present invention was accordingly based on the object of providing a liposomal system in which the activity of active substances in the treatment of diseases or the accumulation of active substances and / or pharmaceutical substances which are to be accumulated in a specific target organ, such as e.g. Contrast agents from imaging processes etc. can be accumulated. The effectiveness of such systems in the treatment of diseases etc. should be increased and, preferably, the number of side effects should also be reduced.
Überraschenderweise wurde festgestellt, dass sich die Wirkung von liposomal verkapselten Wirkstoffen, wie Antitumorwirkstoffen, um ein Vielfaches erhöhen lässt, wenn der Wirkstoff nicht vollständig liposomal verkapselt ist, d.h. wenn außerhalb der Liposomen noch freie Wirkstoffmoleküle in der zu applizierenden Lösung/Emulsion vorliegen.Surprisingly, it was found that the effect of liposomally encapsulated active substances, such as anti-tumor active substances, can be increased many times over if the active substance is not completely liposomally encapsulated, i.e. if there are still free active substance molecules in the solution / emulsion to be applied outside the liposomes.
Gegenstand der vorliegenden Erfindung ist demgemäß ein liposomal verkapselter Wirkstoff, der dadurch gekennzeichnet ist, dass er in einem Anteil von 1 Gew.-% bis 90 Gew.-%, bezogen auf die eingesetzte Wirkstoffmenge, verkapselt ist.The present invention accordingly relates to a liposomally encapsulated active substance, which is characterized in that it is encapsulated in a proportion of 1% by weight to 90% by weight, based on the amount of active substance used.
Im Sinne der vorliegenden Erfindung bedeutet der Anteil der Verkapselung, dass verkapselte Wirkstoffmoleküle sich im Zentrum der Liposomen oder in der Membran befinden, imFor the purposes of the present invention, the proportion of the encapsulation means that encapsulated active substance molecules are located in the center of the liposomes or in the membrane in the
Gegensatz zu freien Wirkstoffmolekülen, die sich in dem die Liposomen umgebenden Lösungsmittel aufhalten oder durch van-der-Waal'sche Wechselwirkungen auf der Oberfläche der Lipidmembranen locker gebunden sind.Contrast to free active substance molecules that are in the surrounding liposomes Stop solvents or are loosely bound by Van der Waal's interactions on the surface of the lipid membranes.
Als aktive Wirkstoffe, die in der vorliegenden Erfindung eingesetzt werden können, sind Arzneimittel und pharmakologisch aktive Substanzen, Nährstoffe, kosmetische Substanzen, diagnostische Substanzen und Kontrastmittel für bildgebende Verfahren. Der Ausdruck Wirkstoff umfasst auch die pharmakologisch annehmbaren Salze der Wirkstoffe. Geeignete Verbindungen sind lipophile und amphiphile Moleküle, d.h. Substanzen, die wasserunlöslich oder nur eine geringe Wasserlöslichkeit aufweisen, und die in Form von Emulsionen oder micellaren Zubereitungen hergestellt werden können. Geeignete Arzneimittel sind z.B. Antikrebsmittel, Antitumormittel, Antibiotika, antimikotische, antiviruale, anthelminthische und antiparasitische Verbindungen.Active substances which can be used in the present invention are pharmaceuticals and pharmacologically active substances, nutrients, cosmetic substances, diagnostic substances and contrast agents for imaging processes. The term active ingredient also includes the pharmacologically acceptable salts of the active ingredients. Suitable compounds are lipophilic and amphiphilic molecules, i.e. Substances which are insoluble in water or only slightly soluble in water and which can be prepared in the form of emulsions or micellar preparations. Suitable drugs are e.g. Anti-cancer agents, anti-tumor agents, antibiotics, antimicotic, antivirual, anthelmintic and anti-parasitic compounds.
Bei der Behandlung von Tumoren oder Krebswachstum können als geeignete Verbindungen Anthracycline, wie Doxorubicin, Daunorubicin, Karinomycin, N-Acetylatriamycin, Rubidacon, 5-lmidodaunomycin, N-Acetyldaunomycin, Pirarubicin und Epirubicin, Alkaloide wie Vincristin, Vinblastin, Etoposid, Ellepticin und Damptothecin, eingesetzt werden. Weitere geeignete Substanzen sind 5-Fluorouracil, Taxane, wie Paclitaxel (Taxol®) und Derivate von Paclitaxel, z. B. Taxotere (Docetaxol®) oder Mitrotan, Platinverbindungen, wie Cisplatin, Carboplatin, Lobaplatin und Phenestrin).Anthracyclines, such as doxorubicin, daunorubicin, karinomycin, N-acetylatriamycin, rubidacon, 5-imidodaunomycin, N-acetyldaunomycin, pirarubicin and epirubicin, alkaloids such as vincristine, vinbline, vinblastine, vinblastine, vinblastine, vinblastine, vinblastine, vinblastine, vinblastine be used. Further suitable substances are 5-fluorouracil, taxanes such as paclitaxel (Taxol ®), and derivatives of paclitaxel z. B. Taxotere (Docetaxol ® ) or Mitrotan, platinum compounds such as cisplatin, carboplatin, lobaplatin and phenestrin).
Beispiele für entzündungshemmende Mittel, die in der vorliegenden Erfindung eingesetzt werden können, sind steroide und nicht-steroide und andere entzündungshemmende Verbindungen, wie Prednison, Methyl-Prednisolon, Paramethazon, 11-Fluorocortisol, Triamciniolon, Betamethason und Dexamethason, Ibuprophen, Piroxicam, Beclomethason, Methotrexat, Acaridin, Etritinat, Anthralin, Psoraline, Salycilate, wie Aspirin, und Immunsupresiva, wie Cyclosporin. Insbesondere entzündungshemmende Corticosteoride und das entzündungshemmende und immunsupresiv wirkende Cyclosporin sind lipophile Verbindungen, die in der vorliegenden Erfindung besonders vorteilhaft eingesetzt werden können.Examples of anti-inflammatory agents that can be used in the present invention are steroidal and non-steroidal and other anti-inflammatory compounds such as prednisone, methyl-prednisolone, paramethazone, 11-fluorocortisol, triamciniolone, betamethasone and dexamethasone, ibuprophen, piroxicam, beclomethasone Methotrexate, acaridine, etritinate, anthralin, psoraline, salycilate such as aspirin, and immunosuppressants such as cyclosporin. In particular, anti-inflammatory corticosteids and the anti-inflammatory and immunosuppressive cyclosporin are lipophilic compounds which can be used particularly advantageously in the present invention.
Weitere pharmakologisch aktive Substanzen, die in der vorliegenden Erfindung eingesetzt werden können, sind Anästhetika, wie Methoxyfluran, Isofluran, N-Fluran, Halothan und Bezocain, antiulcerative Mittel wie Cimetidin, krampflösende Mittel wie Barbiturate, Azothioprin, ein Immunsupresivum und antirheumatisches Mittel, sowie Muskelrelaxanzien, wie Dantrolen und Diazepam. Verfahren zur Herstellung von lipophilen Derivaten, die in Form von Liposomen oder Mycellen hergestellt werden können, sind dem Fachmann gut bekannt.Other pharmacologically active substances that can be used in the present invention are anesthetics such as methoxyflurane, isoflurane, N-flurane, halothane and bezocaine, antiulcerative agents such as cimetidine, anticonvulsant agents such as barbiturates, azothioprine, an immunosuppressant and anti-rheumatic agent, and muscle relaxants such as dantrolene and diazepam. Methods of making lipophilic derivatives that can be made in the form of liposomes or mycelles are well known to those skilled in the art.
Beispiele für Komponenten, die in bildgebenden Verfahren als Kontrastmittel eingesetzt werden können, sind Kontrastmittel für Ultraschall-, Röntgen- und NMR-Verfahren. Insbesondere für Röntgen-Verfahren eignen sich Radioisotope oder Verbindungen, die derartige Radioisotope enthalten wie z.B. Jod, Octane, Halogenkohlenwasserstoff und Renografin. Geeignete Kontrastmittel (Gadolinium), die in NMR-Verfahren eingesetzt werden können sind lipidlösliche paramagnetische Verbindungen.Examples of components that can be used as contrast agents in imaging processes are contrast agents for ultrasound, X-ray and NMR processes. Radioisotopes or compounds containing such radioisotopes, such as e.g. Iodine, octane, halogenated hydrocarbon and renograph. Suitable contrast agents (gadolinium) that can be used in NMR methods are lipid-soluble paramagnetic compounds.
Nahrungsergänzungsmittel, die in das liposomale System gemäß der vorliegenden Erfindung eingearbeitet werden können, sind Aminosäuren, Zucker, Proteine, Kohlenhydrate, fettlösliche Vitamine, Fette (Lipide). Kombinationen aus unterschiedlichen Nahrungsergänzungsmitteln sind ebenfalls geeignet.Dietary supplements that can be incorporated into the liposomal system according to the present invention are amino acids, sugars, proteins, carbohydrates, fat-soluble vitamins, fats (lipids). Combinations of different nutritional supplements are also suitable.
Weitere geeignete Substanzen sind Immunmodulatoren und Vakzine.Other suitable substances are immunomodulators and vaccines.
Die eingesetzten Wirkstoffe können in an sich bekannter Weise unter Zusatz von Liposomen verkapselt werden. Um den gewünschten Verkapselungsgrad einzustellen, ist es möglich, den Wirkstoff nur teilweise zu verkapseln. Die teilweise Verkapselung kann durch Verwendung entsprechender Ausgangsmaterialien oder geeigneter Verfahrensbedingungen erfolgen. In einer weiteren Ausführungsform kann ein vollständig oder nahezu vollständig liposomal verkapselter Wirkstoff mit der entsprechenden Menge an unverkapseltem Wirkstoff beziehungsweise deren Lösungen/Emulsionen vermischt werden, so daß der gewünschte Verkapselungsgrad erhalten wird. Insgesamt läßt sich der Verkapselungsgrad durch einfache Verfahrensänderungen einstellen.The active ingredients used can be encapsulated in a manner known per se with the addition of liposomes. In order to set the desired degree of encapsulation, it is possible to encapsulate the active ingredient only partially. The partial encapsulation can be carried out using appropriate starting materials or suitable process conditions. In a further embodiment, a completely or almost completely liposomally encapsulated active substance can be mixed with the corresponding amount of unencapsulated active substance or their solutions / emulsions, so that the desired degree of encapsulation is obtained. Overall, the degree of encapsulation can be adjusted by simple process changes.
Das Liposom hat eine geschlossene Struktur, das aus einer Lipid-Doppelschicht, die einen wässerigen inneren Kern umschließt, besteht. Die erfindungsgemäß eingesetzten Liposome sind nicht auf bestimmte Liposome beschränkt, es können sowohl neutrale, negativ oder positiv geladene Liposome eingesetzt werden, die unilamellar, d.h. aus einer Lipid- Doppelschicht, oder polylamellar, d.h. aus mehreren Lipid-Doppelschichten, aufgebaut sein und nach dem Fachmann bekannten Methoden hergestellt werden können .The liposome has a closed structure, which consists of a lipid bilayer that surrounds an aqueous inner core. The liposomes used in accordance with the invention are not limited to certain liposomes; both neutral, negatively or positively charged liposomes can be used which are unilamellar, i.e. from a lipid bilayer, or polylamellar, i.e. be composed of several lipid bilayers and can be produced by methods known to the person skilled in the art.
Die Liposome werden üblicherweise aus Phospholipiden sowie ggf. Cholesterol und Cholesterolderivaten und/oder einer oder mehreren hydrophilen, lipophilen oder amphiphilen Komponente(n) hergestellt. Als Phospholipide kommen beispielsweise Phosphatidylcholin (PC), Distearoylphsphatidyl- cholin (DSPC), Sojaphosphatidylcholin (Soja-PC), hydriertes Sojaphosphatidylcholin (HSPC), Ei-Phosphatidylcholin (Ei-PC), hydriertes Ei-Phosphatidylcholin (HEPC), Dipal- mitoylphosphatidylcholin (DPPC), Dimyristoylphosphatidylcholin (DMPC) sowie deren beliebigen Gemische in Betracht, wobei die Subsanzen synthetische, halbsynthetische oder natürliche Produkte sein können.The liposomes are usually produced from phospholipids and, if appropriate, cholesterol and cholesterol derivatives and / or one or more hydrophilic, lipophilic or amphiphilic component (s). Examples of suitable phospholipids are phosphatidylcholine (PC), distearoylphsphatidylcholine (DSPC), soy phosphatidylcholine (soybean PC), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine (HEPCcholine) (HEPCcholine), diphenylpholine (HEPC) DPPC), dimyristoylphosphatidylcholine (DMPC) as well as any mixtures thereof, whereby the substances can be synthetic, semi-synthetic or natural products.
Weitere geeignete Phospholipide sind Phosphatidylglyceride (PG) und Phosphatidsäure, wie Dimynstoylphosphatidylglycerid (DMPG), Dilaurylphosphatidylglycerid (DLPG), Dipalmitoyl- phosphatidylglycerid (DPPG), Distearoylphosphatidylglycerid (DSPG), Dimyristoylphos- phatidsäure (DMPA), Dilaurylphosphatidsäure (DLPA), Dipalmitoylphosphatidsäure (DPPA), Distearoylphosphatidsäure (DSPA), sowie Phosphatidylethanolamine, Phosphatidylinositole und Phosphatidsäure, die Reste der Laurinsäure, Myristinsäure, Stearinsäure und/oder Palmitinsäure enthalten.Further suitable phospholipids are phosphatidylglycerides (PG) and phosphatidic acid, such as dimynstoylphosphatidylglycerid (DMPG), dilaurylphosphatidylglycerid (DLPG), dipalmitoylphosphatidylglycerid (DPPG), distearoylphosphatidylglycerid (DSPGilidic acid, DSistGilid) , Distearoylphosphatidic acid (DSPA), and phosphatidylethanolamines, phosphatidylinositols and phosphatidic acid, which contain residues of lauric acid, myristic acid, stearic acid and / or palmitic acid.
Bevorzugte Phospholipide sind HSPC, DSPC und HEPC.Preferred phospholipids are HSPC, DSPC and HEPC.
Positiv geladene Liposome können beispielsweise aus einer Lösung, die Phosphatidylcholin, Cholesterol und Stearylamin enthält, gebildet werden. Negativ geladene Liposome können beispielsweise aus Lösungen, die Phophatidylcholon, Cholesterol und Phosphatidylserin oder bevorzugt Cardiolipin, erhalten werden. Dem Fachmann ist es bekannt, dass auch andere Additive zugegeben werden können, um die Eigenschaften der erhaltenen Liposome zu modifizieren.For example, positively charged liposomes can be formed from a solution containing phosphatidylcholine, cholesterol and stearylamine. Negatively charged liposomes can be obtained, for example, from solutions containing phosphatidylcholone, cholesterol and phosphatidylserine or, preferably, cardiolipin. It is known to the person skilled in the art that other additives can also be added in order to modify the properties of the liposomes obtained.
Die Eigenschaften Liposome können beispielsweise durch Zugabe von -Tocopherol verändert werden. Gute Ergebnisse werden auch mit sog. PEG-Liposomen, d.h. Liposomen mit Polyethylenglykolketten (PEG) in der Lipidschicht, wobei das PEG entweder im Molekül der Phospholipide gebunden oder als freie Substanz vorliegen kann, erhalten. Das Molekulargewicht der PEG-Ketten liegt vorzugsweise zwischen 400 und 20.000, besonders bevorzugt zwischen 600 und 10.000 und insbesondere zwischen 600 und 5.000.The properties of liposomes can be changed, for example, by adding -Tocopherol. Good results are also achieved with so-called PEG liposomes, i.e. Liposomes with polyethylene glycol chains (PEG) in the lipid layer, the PEG either being bound in the molecule of the phospholipids or being present as a free substance. The molecular weight of the PEG chains is preferably between 400 and 20,000, particularly preferably between 600 and 10,000 and in particular between 600 and 5,000.
Ferner können als Liposome auch die in der WO96/05821 genannten Verkapselungsmittel eingesetzt werden. Bevorzugt eingesetzte Liposomen werden z. B. aus HSPC, DSPC und/oder HEPC als Phospholipid, Cholesterol und/oder N-(O-methyl-poly(ethylenglykol)-1 ,2-distearoyl-sn- glycero-3-phosphoethanolamin sowie ggf. Polyethylenglykol gebildet.The encapsulants mentioned in WO96 / 05821 can also be used as liposomes. Preferred liposomes are e.g. B. from HSPC, DSPC and / or HEPC as phospholipid, cholesterol and / or N- (O-methyl-poly (ethylene glycol) -1, 2-distearoyl-sn-glycero-3-phosphoethanolamine and optionally polyethylene glycol.
Das Verhältnis von Lipid zu Wirkstoff kann in weiten Bereichen eingestellt werden und hängt in der Regel vom verwendeten Wirkstoff sowie dem zur Liposomherstellung eingesetztem Lipid und den weiteren Komponenten ab. In einer möglichen Ausführungsform liegen im fertigen verkapselten Produkt Lipid und Wirkstoff in einem Mengenverhältnis von 5 : 1 bis 100 : 1, vorzugsweise von 10 : 1 bis 40 : 1, insbesondere von 15 : 1 bis 25 : 1 vor. Ist auch Cholesterol bzw. ein Cholesterolderivat vorhanden so beträgt das molare Verhältnis von Lipid zu Cholesterol vorzugsweise von 3 : 1 bis 1 : 3, vorzugsweise von 2 : 1 bis 1 : 2. Polyethylenglykol wird vorzugsweise in einer Menge von 1 bis 50 Mol-%, insbesondere von 5 bis 20 Mol-%, bezogen auf das Lipid, eingesetzt.The ratio of lipid to active ingredient can be adjusted within a wide range and generally depends on the active ingredient used and the lipid used for liposome production and the other components. In one possible embodiment, the final encapsulated product contains lipid and active ingredient in a ratio of 5: 1 to 100: 1, preferably 10: 1 to 40: 1, in particular 15: 1 to 25: 1. If cholesterol or a cholesterol derivative is also present, the molar ratio of lipid to cholesterol is preferably from 3: 1 to 1: 3, preferably from 2: 1 to 1: 2. Polyethylene glycol is preferably used in an amount of 1 to 50 mol%. , in particular from 5 to 20 mol%, based on the lipid.
Die Größe der erfindungsgemäß liposomal verkapselten Wirkstoffe liegt vorzugsweise unter 200 nm, besonders bevorzugt unter 150 nm.The size of the active ingredients encapsulated liposomally according to the invention is preferably below 200 nm, particularly preferably below 150 nm.
Die physiologische Verträglichkeit und Wirksamkeit der erfindungsgemäß liposomal verkapselten Wirkstoffe kann durch Kombination mit geeigneten Arzneimittelträgern weiter verbessert werden Beispiele für besonders gut geeignete Arzneimittelträger sind zum Beispiel sogenannte Mikrosphären, welche vorzugsweise aus Polysachariden oder Polysacharidderivaten hergestellt werden und besonders bevorzugt abbaubar sind. Die Polysacharide sind vorzugsweise ausgewählt aus Stärke und Stärkederivaten, Gelatine, Albomin, Kolagen, Dextran, Dextanderivaten oder ähnlichen Materialien. Besonders bevorzugt sind lyopohilisierte oder abbaubare Stärke- oder Gelatinepartikel. Derartige Mikrosphären weisen vorzugsweise ein wasserunlösliches, jedoch hydrophiles, in Wasser quellbares, dreidimensionales Netzwerk von Polysacharidmolekülen oder entsprechenden Derivaten auf. Geeignete Mikrosphären werden in der DE-US-25 24 279, WO 88/09163 und WO 89/03207 beschrieben. Der Durchmesser der Mikrosphären liegt vorzugsweise zwischen 0,1 und 200 μm, insbesondere zwischen 10 und 100 μm.The physiological tolerance and effectiveness of the active ingredients encapsulated in accordance with the invention can be further improved by combination with suitable medicament carriers. Examples of particularly well-suited medicament carriers are, for example, so-called microspheres, which are preferably produced from polysaccharides or polysaccharide derivatives and are particularly preferably degradable. The polysaccharides are preferably selected from starch and starch derivatives, gelatin, albomine, collagen, dextran, dextane derivatives or similar materials. Lyophilized or degradable starch or gelatin particles are particularly preferred. Such microspheres preferably have a water-insoluble, but hydrophilic, water-swellable, three-dimensional network of polysaccharide molecules or corresponding derivatives. Suitable microspheres are described in DE-US-25 24 279, WO 88/09163 and WO 89/03207. The diameter of the microspheres is preferably between 0.1 and 200 μm, in particular between 10 and 100 μm.
Die erfindungsgemäß hergestellten liposomalen Systeme können direkt in den Blutkreislauf der zu behandelnden Personen gegeben werden, wobei auch die intraperitoneale, subkutane oder inhalative Verabreichung möglich ist.The liposomal systems produced according to the invention can be added directly to the bloodstream of the person to be treated, and intraperitoneal, subcutaneous or inhalation administration is also possible.
Die erfindungsgemäß eingesetzten Liposome können nach einem beliebigen aus dem Stand der Technik bekannten Verfahren hergestellt werden. Zum Beispiel können die Liposome in einem geeigneten Lösungsmittel gelöst werden, üblicherweise in einem unpolaren oder nur schwach polaren Lösungsmittel, welches ohne schädliche Substanzen zu hinterlassen, wieder entfernt werden kann, wie Ethanol, Methanol, Chloroform, Butanol oder Aceton. Wenn Wirkstoffgemische eingesetzt werden, können die einzelnen Lösungen auch miteinander vermischt werden. Auch das lipophile Material wird aufgelöst und mit der Wirkstoff-haltigen Lösung vermischt. Nach dem Entfernen des Lösungsmittels, beispielsweise durch ein Lyophilisierungsverfahren, verbleibt der Lipidfilm auf dem Wirkstoff. Das Gemisch kann in dieser Form, gegebenenfalls unter Inertgasatmospäre, gelagert werden, wobei niedrige Lagertemperaturen, wie bei -20°C, besonders bevorzugt sind. Die Bildung der Liposome erfolgt in der Regel durch Zugabe einer geeigneten Lösung zum Lipidfilm. Typische Lösungen sind polare Lösungen, vorzugsweise wässerige Salzlösungen, wie isotonische Kochsalzlösung. Die Bildung der Liposome erfolgt zum Beispiel durch Mischen wie durch Mischen im Wirbelstrom (Vortexing). Werden kleiner Vesikel, wie unilamellare Vesikel hergestellt, kann die Lösung auch mit Ultraschall behandelt werden.The liposomes used according to the invention can be produced by any method known from the prior art. For example, the liposomes in be dissolved in a suitable solvent, usually in a non-polar or only slightly polar solvent which can be removed without leaving harmful substances, such as ethanol, methanol, chloroform, butanol or acetone. If active ingredient mixtures are used, the individual solutions can also be mixed with one another. The lipophilic material is also dissolved and mixed with the solution containing the active ingredient. After the solvent has been removed, for example by a lyophilization process, the lipid film remains on the active ingredient. The mixture can be stored in this form, optionally under an inert gas atmosphere, with low storage temperatures, such as at -20 ° C., being particularly preferred. The liposomes are usually formed by adding a suitable solution to the lipid film. Typical solutions are polar solutions, preferably aqueous salt solutions, such as isotonic saline. Liposomes are formed, for example, by mixing and vortexing. If small vesicles, such as unilamellar vesicles, are produced, the solution can also be treated with ultrasound.
In einer weiteren Ausführungsform der vorliegenden Erfindung können auch Gemische aus multilamellaren Vesikeln und unilamellaren Vesikeln eingesetzt werden.In a further embodiment of the present invention, mixtures of multilamellar vesicles and unilamellar vesicles can also be used.
Die hergestellten Liposome können unmittelbar dem Patienten verabreicht werden oder bei geeigneten Bedingungen gelagert werden. Vorzugsweise werden die lipophilen Wirkstoffe als mit Lipidfilmen beaufschlagte trockene Substanzen bei etwa -20°C gelagert. Nach der Hydratisierung können Liposomsuspensionen, denen eine geeignete Menge an unverkapseltem Wirkstoff zugesetzt worden ist, in gepufferten oder neutralen Kochsalzlösungen in Abhängigkeit von der Temperatur und den Inhaltsstoffen über einen Zeitraum von einigen Stunden bis zu Monaten aufbewahrt werden. Das liposomale Wirkstoffsystem gemäß der vorliegenden Erfindung kann in Mengen von etwa 5 bis 150 mg Wirkstoff/kg Körpergewicht des Patienten innerhalb eines Zeitraums von 1 Minute bis 5 Stunden verabreicht werden.The liposomes produced can be administered directly to the patient or stored under suitable conditions. The lipophilic active substances are preferably stored at about -20 ° C. as dry substances to which lipid films have been applied. After hydration, liposome suspensions to which a suitable amount of unencapsulated active ingredient has been added can be stored in buffered or neutral saline solutions for a period of a few hours to months, depending on the temperature and the ingredients. The liposomal drug system according to the present invention can be administered in amounts of about 5 to 150 mg of drug / kg body weight of the patient within a period of 1 minute to 5 hours.
Weitere Gegenstände der vorliegenden Erfindung sind die Verwendung eines wie oben beschriebenen liposomal verkapselten Wirkstoffs zur Herstellung eines Medikaments zur Behandlung von Tumoren, als Nahrungsergänzungsmittel, zur Herstellung eines Kontrastmittels für bildgebende Verfahren und zur Herstellung eines Diagnostikmittels für Krankheiten. BeispieleFurther objects of the present invention are the use of a liposomally encapsulated active substance as described above for the manufacture of a medicament for the treatment of tumors, as a food supplement, for the manufacture of a contrast agent for imaging methods and for the manufacture of a diagnostic agent for diseases. Examples
Herstellung der Liposomen: Hydriertes Ei-Phosphatidylcholin (HEPC, 50 mg/ml; Nattermann Phospholipid GmbH, Köln, Deutschland), Cholesterol (CH, 24,8 g/ml; Merck, Darmstadt, Deutschland) und Polyethylenglykol (MPEG-DSPE, 3000, 5,4 mg/ml; Sygena LTD, Liestal, Schweiz) werden im molaren Verhältnis 1:1:0,1 in Chloroform gelöst. Die organische Phase wird am Rotationsverdampfer entfernt und der entstandene, gut getrocknete Lipidfilm mit einer Wirkstofflösung sowie einer Gd-DTPA-FS-Lösung resuspendiert. Diese Liposomendispersion wird 24 h bei Raumtemperatur geschüttelt, die dabei entstandenen Multischichtvesikel (MLV) einer Beschallung unterworfen (Rüsselbeschaller, 6x4 min mit 50 % Intensität, Branson B225 sonifer; Branson, Carouge-Geheve, Schweiz) und anschließend zentrifugiert (3000 U/min über 20 min), um von der Liposomendispersion den Titanabrieb zu entfernen.Preparation of the liposomes: hydrogenated egg phosphatidylcholine (HEPC, 50 mg / ml; Nattermann Phospholipid GmbH, Cologne, Germany), cholesterol (CH, 24.8 g / ml; Merck, Darmstadt, Germany) and polyethylene glycol (MPEG-DSPE, 3000 , 5.4 mg / ml; Sygena LTD, Liestal, Switzerland) are dissolved in a molar ratio of 1: 1: 0.1 in chloroform. The organic phase is removed on a rotary evaporator and the resulting, well-dried lipid film is resuspended with an active ingredient solution and a Gd-DTPA-FS solution. This liposome dispersion is shaken at room temperature for 24 h, the resulting multilayer vesicles (MLV) are subjected to sonication (proboscis, 6x4 min with 50% intensity, Branson B225 sonifer; Branson, Carouge-Geheve, Switzerland) and then centrifuged (3000 rpm) 20 min) to remove the titanium abrasion from the liposome dispersion.
Als Wirkstoffe wurden 5-Fluorouracil (Riboluor® 1000; 50 mg/ml Infusionslösung, Ribosepharm GmbH, München, Deutschland) und Carboplatin (Ribocarabo® 50 mg/ml Infusionslösung, Ribosepharm GmbH, München, Deutschland) verwendet.As active ingredients, 5-fluorouracil (Riboluor ® 1000; 50 mg / ml Infusion solution Ribosepharm GmbH, Munich, Germany) and carboplatin (Ribocarabo ® 50 mg / ml Infusion solution Ribosepharm GmbH, Munich, Germany) was used.
Die Vesikelgröße wurde mit einem Photonenkorrelationsspektrometer (Coulter Counter N4 MD modeil and the AccuComp® System, Coulter Electronics Inc., Hialeah, US) bestimmt und bewegte sich für beide Wirkstoffe in einer Größenordnung von 100 ± 50 nm.The vesicle size was determined with a photon correlation spectrometer (Coulter Counter N4 MD modeil and the AccuComp ® system, Coulter Electronics Inc., Hialeah, US) and moved for both active ingredients in the order of 100 ± 50 nm.
Die 5-Fluorouracil-Liposome wurden ferner mittel Kemresonanzsprektroskopie, nämlich H1- NMR- und NOESY-Spektroskopie, untersucht (NOE = Nuklear Overhauser Effekt). Das H1- NMR-Spektrum des liposomal verkapselten 5-Fluorouracil (c = 50 mg/ml) zeigte neben dem H6-Proton des 5-FU bei 7.5 ppm zwischen 2.5 und 4.0 ppm Signale der liposomalen PEG- Matrix. Im 1 D-NOE-Spektrum (Selektive Anregung des H6-Protons bei 7.5 ppm) ergab eine positive NOE-Resonanz bei 3.5 ppm, was eine räumliche Nähe des Wirkstoffs zum Liposom, d.h. eine partielle Verkapselung des Wirkstoffs, bedeutet.The 5-fluorouracil liposomes were also examined by nuclear magnetic resonance spectroscopy, namely H1-NMR and NOESY spectroscopy (NOE = Nuclear Overhauser Effect). The H1 NMR spectrum of the liposomally encapsulated 5-fluorouracil (c = 50 mg / ml) showed, in addition to the H6 proton of the 5-FU at 7.5 ppm, between 2.5 and 4.0 ppm signals of the liposomal PEG matrix. In the 1 D NOE spectrum (selective excitation of the H6 proton at 7.5 ppm) there was a positive NOE resonance at 3.5 ppm, which indicates that the active ingredient is spatially close to the liposome, i.e. a partial encapsulation of the active ingredient.
Die Wirkstoffe wurden jeweils in freier Form intravenös und intraarteriell sowie in verkapselter Form mit und ohne Stärkemikrosphären (Spherex®, Pharmacia Upjohn) i.a. zur Tumorbehandlung bei Lebertumoren verabreicht.The active substances were administered intravenously and intraarterially as well as in encapsulated form with and without starch microspheres (Spherex ® , Pharmacia Upjohn), generally for the treatment of tumors in liver tumors.
In dem antitumoralen System erfolgte das Tumortargeting durch liposomale Verkapselung der Zytostatika und Zusatz von Stärkemikrossphären. Die verwendeten SUV-PEG- Liposomen haben eine Partikelgröße von 113 nm ± 25 nm, die Stärkemikrosphären im Mittel 45 μm.In the antitumor system, the tumor was targeted by liposomally encapsulating the cytostatics and adding starch microspheres. The SUV PEG used Liposomes have a particle size of 113 nm ± 25 nm, the starch microspheres on average 45 μm.
Die Wirkstoffkonzentrationen wurden im Tumor und in der Leber wurden gemessen in AUC (Area under the Curve). Die Ergebnisse sind in Tabelle 1 dargestellt.The drug concentrations in the tumor and in the liver were measured in AUC (Area under the Curve). The results are shown in Table 1.
Tabelle 1Table 1

Claims

Patentansprüche claims
1. Liposomal verkapselter Wirkstoff, der dadurch gekennzeichnet ist, dass er in einem Anteil von 1 Gew.-% bis 90 Gew.-%, bezogen auf die eingesetzte Wirkstoffmenge, verkapselt ist.1. Liposomally encapsulated active ingredient, which is characterized in that it is encapsulated in a proportion of 1% by weight to 90% by weight, based on the amount of active ingredient used.
2. Liposomal verkapselter Wirkstoff nach Anspruch 1, dadurch gekennzeichnet, dass der Wirkstoff in einem Anteil von 5 bis 85 Gew.-%, bezogen auf die eingesetzte Wirkstoffmenge, verkapselt ist.2. Liposomally encapsulated active ingredient according to claim 1, characterized in that the active ingredient is encapsulated in a proportion of 5 to 85 wt .-%, based on the amount of active ingredient used.
3. Liposomal verkapselter Wirkstoff nach einem der Ansprüche 1 oder 2, dadurch gekennzeichnet, dass der Wirkstoff ausgewählt ist aus Arzneimitteln und pharmakologisch aktiven Substanzen, Nährstoffen, kosmetischen Substanzen, diagnostischen Substanzen und Kontrastmitteln für bildgebende Verfahren.3. Liposomally encapsulated active ingredient according to one of claims 1 or 2, characterized in that the active ingredient is selected from drugs and pharmacologically active substances, nutrients, cosmetic substances, diagnostic substances and contrast agents for imaging processes.
4. Liposomal verkapselter Wirkstoff nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, dass die Liposome gebildet sind aus Phospholipiden sowie ggf. Cholesterol und Cholesterolderivaten und/oder einer oder mehreren hydrophilen, lipophilen oder amphiphilen Komponente(n).4. Liposomally encapsulated active ingredient according to one of claims 1 to 3, characterized in that the liposomes are formed from phospholipids and optionally cholesterol and cholesterol derivatives and / or one or more hydrophilic, lipophilic or amphiphilic component (s).
5. Liposomal verkapselter Wirkstoff nach Anspruch 4, dadurch gekennzeichnet, dass das Phospholipid ausgewählt ist aus Phosphatidylcholin (PC), Distearoylphsphatidylcholin (DSPC), Sojaphosphatidylcholin (Soja-PC), hydriertes Sojaphosphatidylcholin (HSPC), Ei-Phosphatidylcholin (Ei-PC), hydriertes Ei-Phosphatidylcholin (HEPC), Dipalmitoyl- phosphatidylcholin (DPPC), Dimyristoylphosphatidylcholin (DMPC) sowie deren beliebigen Gemischen.5. Liposomally encapsulated active ingredient according to claim 4, characterized in that the phospholipid is selected from phosphatidylcholine (PC), distearoylphsphatidylcholine (DSPC), soy phosphatidylcholine (soy PC), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidyl choline (egg PC) hydrogenated egg phosphatidylcholine (HEPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC) and their mixtures.
6. Verwendung des liposomal verkapselten Wirkstoffs nach einem der Ansprüche 1 bis 5 zur Herstellung eines Medikaments zur Behandlung von Tumoren.6. Use of the liposomally encapsulated active ingredient according to one of claims 1 to 5 for the manufacture of a medicament for the treatment of tumors.
7. Verwendung nach Anspruch 6, dadurch gekennzeichnet, dass der Wirkstoff in Kombination mit einem Arzneimittelträger eingesetzt wird, insbesondere mit Mikrosphären, welche vorzugsweise aus Polysachariden oder Polysacharidderivaten hergestellt werden und besonders bevorzugt abbaubar sind. 7. Use according to claim 6, characterized in that the active ingredient is used in combination with a medicament carrier, in particular with microspheres, which are preferably produced from polysaccharides or polysaccharide derivatives and are particularly preferably degradable.
8. Verwendung des liposomal verkapselten Wirkstoffs nach einem der Ansprüche 1 bis 5 als Nahrungsergänzungsmittel.8. Use of the liposomally encapsulated active ingredient according to one of claims 1 to 5 as a dietary supplement.
9. Verwendung des liposomal verkapselten Wirkstoffs nach einem der Ansprüche 1 bis 5 zur Herstellung eines Kontrastmittels für bildgebende Verfahren.9. Use of the liposomally encapsulated active substance according to one of claims 1 to 5 for the production of a contrast agent for imaging processes.
10. Verwendung des liposomal verkapselten Wirkstoffs nach einem der Ansprüche 1 bis 5 zur Herstellung eines Diagnostikmittels für Krankheiten. 10. Use of the liposomally encapsulated active ingredient according to one of claims 1 to 5 for the production of a diagnostic agent for diseases.
EP01933914A 2000-05-02 2001-05-02 Liposomes containing active substances Withdrawn EP1427393A2 (en)

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