DE19839515B4 - New pharmaceutical preparation containing colloidal polymer-drug associations, especially for mucosal drug administration - Google Patents

Abstract

A colloidal associate containing a polyol ester-drug complex wherein the polyol ester is derived from a linear polyol having more than six hydroxyl groups as the central molecule to which biodegradable hydroxy carboxylic acid ester moieties are attached.

Description

The invention relates to novel pharmaceutical formulations, in colloidal form, consisting of at least one biocompatible and biodegradable polymer in combination with at least one active ingredient, their Wirkort-, Wirkart- and Wirkstoffadaptierter production, and their use, in particular for mucosal drug administration.

[State of the art]

In modern pharmacotherapy, drug formulations and drug combinations are increasingly important, which bring the (the) drug (s) in an administrable form and thereby especially the drug stability, its biodistribution, bioavailability and / or absorption have a positive influence. Due to major advances in molecular biology, gene and biotechnology is increasingly an increasing number of pharmacologically active agents such. As proteins, glycoproteins, peptides, oligonucleotides and DNA constructs and growth factors, accessible. However, the human or veterinary use of these substances without suitable Tragersysteme by many undesirable side effects difficult or even partially not possible.

In particular, for vaccines, such. B. tetanus and diphtheria toxoid, and viral antigens, currently lacking suitable carrier systems, a simple, cheap, more comfortable for the patient and especially with regard to the accessibility of patients, eg. In the third world, enable reliable immunization.

Literature-written experiments show that by parenteral biopolymer depot or Retardierungsformulierungen in implant or microparticle form, although a fundamental improvement can be achieved, yet so far remain clear success for these systems, which is not least due to the lack of suitable biopolymer carriers.

Therefore, in many places, another, more targeted way for the administration of pharmacologically relevant drugs was taken. It is already known from the literature that particulate systems, when applied to mucous membranes, can be resorbed in the best case by specific structures of the mucosal-associated lymphoid tissues (MALT: mucosa-associated lymphoid tissues). However, to date only systems have been used in which the observed transport phenomena indicate a largely unspecific absorption. However, it appears that a particle size dependency could be present.

The first literature-based attempts to transport pharmacologically active, low-molecular substances associated with such colloidal systems into the body in this way have already shown promising results.

On the other hand, macromolecular, mostly hydrophilic active substances have not hitherto been accessible to this invasive process, which is due, in particular, to the lack of suitable carrier polymers and to the hitherto known, too "aggressive" particle production technologies.

Problems in the association of mostly hydrophilic, macromolecular, pharmacologically active agents with polymeric or particulate systems are thus on the one hand in their often too low stability compared to the usual methods of preparation, for. As to aggressive polymerization or dispersion, such as high-pressure homogenization or ultrasonic dispersion, or the conditions prevailing in these processes (temperature increase, contact with lipophilic solvents, etc).

However, other problems are also particularly in the case of hitherto insufficient compatibility and interaction between carrier (polymer) and active ingredient. Although biocompatible polymers have already been proposed for use in retardation formulations (depot systems), claims or exemplary embodiments in the sense of the present invention are still lacking. In particular, the deliberate adaptation of all components, ie in particular carrier polymer (s), active substance (s), method of application (s) and site (s), etc., to the particular requirements of such applications has hitherto not been targeted.

OBJECT OF THE INVENTION

• 1. die Fahigkeit, die meist makromolekularen, hydrophilen Wirkstoffe in biologisch aktiver Form unbeschadet zu assoziieren.
• 2. die Fahigkeit, die unter 1. genannten Assoziate, wenn erforderlich, steuerbar zum therapeutisch sinnvollen Zielort zu führen.
• 3. die Fahigkeit gemaß 1. und 2., dass dieser Zielort der mucosale Zielort ist.
• 4. eine geringere Zahl notwendiger Verfahrensschritte während der Herstellung.
• 5. Biokompatibilitat und Bioabbaubarkeit des Trägerpolymers.
• 6. eine geringere toxische Wirkung auf das umgebende Gewebe.
• 7. eine ausreichend lange Verweilzeit, gemäß 3. auf mukosalen Oberflachen.
• 8. eine Wirkstoffanreicherung, gemäß 3. auf mukosalen Oberflachen.
• 9. eine eventuell erforderliche ausreichende zelluläre Aufnahme.
• 10. gemäß 3., die Fahigkeit zur Induktion einer Immunantwort, wenn der relevante Wirkstoff ein Antigen zur Immunisierung ist.
• 11. gemäß 10., die Fahigkeit zur Induktion einer Immunantwort, wenn der relevante Wirkstoff ein DNA Konstrukt ist.

It is therefore an object of the present invention to provide pharmaceutical formulations consisting of at least one biocompatible and biodegradable polymer (component 1) and at least consist of an active ingredient (component 2) and as colloidal systems (ie, particulate in the micron and submicron size range) are present, which is distinguished from known Tragerystemen by

• 1. the ability to safely associate the most macromolecular, hydrophilic agents in biologically active form.
• 2. the ability to lead, if necessary, controllably to the therapeutically meaningful destination, the associates mentioned under 1. above.
• 3. the ability according to 1. and 2. that this destination is the mucosal target.
• 4. a smaller number of necessary process steps during manufacture.
• 5. Biocompatibility and biodegradability of the carrier polymer.
• 6. a lower toxic effect on the surrounding tissue.
• 7. a sufficiently long residence time, according to 3. on mucosal surfaces.
• 8. an active agent enrichment, according to 3. on mucosal surfaces.
• 9. a possibly required sufficient cellular uptake.
• 10. according to 3., the ability to induce an immune response if the relevant drug is an antigen for immunization.
• 11. according to 10, the ability to induce an immune response if the relevant drug is a DNA construct.

Therefore, it is a further object of the present invention to provide gentle processes for the preparation of such pharmaceutical compositions which meet the requirements of one or more of the sub-aspects 1 to 11, and for the preparation of which at least one carrier polymer (component 1) and at least one relevant biologically active ingredient (component 2), which may be present alone or in a physical or chemical form, to be combined to the colloidal carrier system.

For this purpose, it is a further object of the present invention to provide suitable carrier polymers available, which correspond to the above tasks / aspects.

Solution to the Invention

The discovery of suitable carrier polymers according to the present invention is achieved by branched-chain polyol esters consisting of a central molecule (charge-modified and uncharged polyols) to which short-chain biodegradable hydroxycarboxylic ester groups are attached.

Surprisingly, it has been found that the adjustability of the amphiphilic character, ie the balance of hydrophobic, hydrophilic and possibly charged components, of the carrier polymers likewise unexpectedly is suitable for associating them with sufficient amounts of relevant active ingredients, as well as these associates then in colloidal form, for. To, on and through cell barriers.

The properties of the polymers, which surprisingly turned out to be adjustable, include, among other things, their solubility in addition to the amphiphilic character. It was suitable for the wide range of water solubility to solubility in organic solvents, with the preferred range according to the invention containing toxicologically harmless solvents: of water via alcohols (such as, for example, ethanol) and esters (such as, for example, ethyl acetate) to ketones (such as acetone), as well as their combinations.

Surprisingly, the solubility in water was not only soluble in the range from good to poorly soluble, but a lower critical demixing temperature (LCST), which was adjustable as a function of the overall polymer composition, was found.

• (I) Im Falle von definiert eingestellter Löslichkeit der Polymere in Wasser, besteht die Herstellungstechnologie fur die pharmazeutischen Formulierungen nach Teilaufgabe 2 darin, dass die Herstellung des erfindungsgemaßen Tragersystems durch uberraschend gefundene spontane in-situ Ausbildung von somit ebenfalls erfindungsgemäßen kolloidalen Polymer-Wirkstoff-Komplexen erfolgt, wenn eine Lösung A mit Komponente B vereinigt wird. Dabei muss Losung A ein erfindungsgemäßes Polymer enthalten und Komponente B ist wenigstens ein relevanter Wirkstoff oder enthalt diese(n) in chemisch gebundener oder physikalisch assoziierter Form. Komponente B kann dabei a) selbst Bestandteil von Losung A oder b) eine zweite Lösung sein.
• (II) Ist, z. B. aus Kompatibilitätsgründen zum Wirkstoff, eine hohere Lipophilie der Polymere erwunscht, wird die Loslichkeit der Polymere definiert auf Loslichkeit in untoxischen organischen Losungsmitteln bzw. auch Losungsmittelgemischen, wie z. B. Estern, Ethern, Alkoholen, Ketonen eingestellt und dann erfindungsgemäß als Herstellungstechnologie eine modifizierte Solvent Deposition Methodik bevorzugt, bei dem erfindungsgemaße Trägersysteme zeitgleich (in-situ) oder in einem nachgeschalteten Schritt adsorptiv oder chemisch mit wenigstens einem relevanten Wirkstoff assoziiert werden.

Inventive carrier systems consist of at least one biodegradable and biocompatible polyol ester and at least one relevant active ingredient (that is, components 1 and 2). The properties of the carrier polymers (component 1) can be synthetically optimized both with regard to their transport behavior in vivo and their compatibility with or interaction with active ingredients. Furthermore, these properties also allow the selection of the gentle manufacturing technology to be used of the carrier system according to the invention (according to the second-mentioned task).

• (I) In the case of defined adjusted solubility of the polymers in water, the production technology for the pharmaceutical formulations according to subtask 2 is that the preparation of the inventive carrier system by surprisingly found spontaneous in-situ formation of thus also inventive colloidal polymer-drug complexes takes place when a solution A with Component B is combined. In this case, solution A must contain a polymer according to the invention and component B is at least one relevant active substance or contains this substance (s) in chemically bound or physically associated form. Component B may be a) itself a component of solution A or b) a second solution.
• (II) Is, for. B. for compatibility reasons for the active ingredient, a higher lipophilicity of the polymers desired, the solubility of the polymers is defined to solubility in non-toxic organic solvents or solvent mixtures such. For example, esters, ethers, alcohols, ketones and then according to the invention as a production technology preferred a modified solvent deposition method, are associated with the invention carrier systems at the same time (in situ) or in a subsequent step adsorptive or chemical with at least one relevant drug.

Component 1: Polymers and their preparation

The branched polyol ester thus used as a component of the carrier system according to the invention, consisting of a central molecule (modified and unmodified polyols) which is property-modified and property-optimized by the targeted synthetic attachment of short-chain biodegradable hydroxycarboxylic ester groups, are preferably prepared by so-called melt grafting.

Melt grafting preferred according to the invention refers to the reaction of one or more hydroxycarboxylic acids in dimer or lactone form (also referred to below as monomers) above one of their melting points in the presence of the central molecule (s) using a catalyst suitable for ring-open polymerization.

Catalysts may be all suitable for ring-opening polymerization substances, but are preferably non-toxic and z. As the FDA approved catalysts such as tin and its salts, zinc and its salts, and hydrides such. B. calcium hydride.

A special feature of this reaction for the specific adjustment of the properties of the carrier polymers according to the invention is, on the one hand, that the central polyols must dissolve in a defined, sufficient extent in accordance with the invention preferred melt grafting in the melt of the monomers. Then, by suitable selection of the synthesis conditions (quantitative ratios, reaction temperature and reaction time, choice of the central polyol, etc.), the required properties of the central polyol can be manipulated specifically and tailored to the requirements of the carrier system.

• a) lineare synthetische Polymere mit > 6 bis 500 Hydroxylgruppen, insbesondere Polyvinylalkohole (PVA) mit Polymerisationsgraden von > 6 bis 500 (und Verseifungsgraden von 70 bis 98%, sofern durch Hydrolyse von Poly(vinylacetat) erhalten);
• b) lineare synthetische Vinylcopolymere, insbesondere des Polyvinylalkohols, mit > 6 bis 500 Hydroxylgruppen, insbesondere mit Vinylacetat, Vinylpyrrolidonen, Vinylaminen, Vinylimidazolen, Vinylpyridinen, Vinylsulfonsauren, Vinylphosphorsauren.
• c) lineare, verzweigte oder cyclische, geladene oder ungeladene Polysaccharide, insbesondere Starken oder Bestandteile, Glycogene, Cellulosen oder Bestandteile, Dextrane, Tunicine, Inuline, Chitine, Alginate, Pektine, Mannane, Galactane, Xylane und andere Polyosen, Chondroitinsulfate, Heparin, Hyaluronsaure und andere Glykosaminoglykane, Mureine, Dextrine, Cyclodextrine, Chitosane, sowie insbesondere deren teilhydrophobisierte Derivate, wobei Hydrophobisierung durch Methyl- und/oder Ethyl-Etter, -Ester und/oder -Urethan-Verknüpfung erreicht ist.
• d) insbesondere oben aufgefuhrte Polyole, mit der Besonderheit, dass sie zusätzlich eine oder mehrere Carboxyl-, Sulfobutyl- oder Sulfopropyl-Gruppe(n) und/oder Butylamin-, Propylamin- oder Ethylamin-(primäres, sekundares, tertiares oder quärtares Amin, wobei entsprechende N-Substituenten insbesondere -H, -CH3 und/oder -C2H5-Gruppe(n) darstellen)-Gruppe(n) enthalten, die in Form von Ether-, -Ester- oder -Urethan-Verknupfung an das Polyol gebunden sind.

Central polyols are therefore preferred:

• a) linear synthetic polymers having> 6 to 500 hydroxyl groups, in particular polyvinyl alcohols (PVA) with degrees of polymerization of> 6 to 500 (and saponification degrees of 70 to 98%, if obtained by hydrolysis of poly (vinyl acetate));
• b) linear synthetic vinyl copolymers, in particular of the polyvinyl alcohol, having> 6 to 500 hydroxyl groups, in particular with vinyl acetate, vinylpyrrolidones, vinylamines, vinylimidazoles, vinylpyridines, vinylsulfonic acids, vinylphosphoric acids.
• c) linear, branched or cyclic, charged or uncharged polysaccharides, in particular starches or constituents, glycogens, celluloses or constituents, dextranes, tunicines, inulins, chitins, alginates, pectins, mannans, galactans, xylans and other polyoses, chondroitin sulfates, heparin, hyaluronic acid and other glycosaminoglycans, mureins, dextrins, cyclodextrins, chitosans, and especially their partially hydrophobized derivatives, wherein hydrophobization is achieved by methyl and / or ethyl etter, ester and / or urethane linkage.
• d) in particular polyols listed above, with the particularity that they additionally contain one or more carboxyl, sulfobutyl or sulfopropyl group (s) and / or butylamine, propylamine or ethylamine (primary, secondary, tertiary or quaternary amine, wherein corresponding N-substituents in particular -H, -CH3 and / or -C2H5 group (s) represent) group (s) which are bonded in the form of ether, ester or urethane Verknupfung to the polyol ,

In the case of polyols bearing charged and / or protonatable and / or deprotonatable groups, also their alkali metal or halogen salts, in particular their sodium or chloride salts.

Since the preferred central polyols according to the invention also include polysaccharides, sufficient solubility and stability in the melt of the monomers must be ensured when they are used. For non-charge modified polysaccharides this z. B. achieved by literature known targeted partial acylation.

In the case of charge-modified polysaccharides, the additionally introduced hydrophilicity, owing to the charge-carrying group (s) and / or group (s), is overcompensated by suitable aliphatic lipophilic spacer chain lengths. This is z. B. as already stated above by sulfobutyl or sulfopropyl ether, ester or urethane groups and / or butylamine or propylamine (primary, secondary, tertiary and / or quaternary amine, with corresponding N-substituents preferably - H, -CH ₃ and / or -C ₂ H ₅ group) esters, esters or urethane groups.

Only if this solubility of the central polyols is guaranteed, z. B. About Syntheseeinwaagagemengen, choice and amount of catalyst (system) s, reaction temperature (s) and time (s) the inventively required properties of the carrier polymers (component 1) are adjusted adequately, z. Over the length of the hydroxycarboxylic acid moieties. A criterion that then z. B. with known spectroscopic analysis methods - such as NMR spectroscopy - can be easily checked.

A special feature of this reaction for the specific adjustment of the properties of the carrier polymers according to the invention is furthermore that only the combination of suitable central polyol or its properties and the choice of the monomer (s) and the reaction conditions with respect to later active ingredient (s) whose association with the polymeric carrier and its gentle production and its in-vivo transport behavior leads to a promising carrier system.

The Hydroxycarbonsaureestergruppierungen preferred for property adaptation of the central polyols are preferably prepared by reaction products of dimers / anhydrides / lactones of hydroxycarboxylic acids (monomers). In this case, all come into question whose polymerization products are considered biologically harmless. Preferred in accordance with the invention are L-lactide, D, L-lactide and glycolide, since these can be easily metabolized in vivo to give lactic acid and glycolic acid.

In this case, it has surprisingly been found, by way of example, that in particular ratios of polyol-OH group to acid building unit in the range from 0.6: 1 to 6: 1, in particular in the range from 1: 1 to 3: 1 lead to the formation of water-soluble carrier polymers, which in In addition, the traps of the examples have a lower critical demixing temperature in the water.

Furthermore, it has been found that - if the Hydroxycarbonsäuregruppierungen the central polyol are each composed of 1 to 100, and in particular 5 to 50 building blocks - a solubility in z. As acetone results.

Although the literature proposes polyol esters based on a polyol with poly (hydroxycarboxylic acid) side chains for biomedical applications. Targeted studies on the synthetic adjustment of the polymer properties and in particular the adjustment of the polymer properties to the special requirements of the claims according to the invention are, however, woolly.

By way of example, this literature findings are explained here:
EP 0 407 617 A1 Although describes reaction products of poly (hydroxy carboxylic acid esters) and saccharides, also describe DE 34 30 852 A1 and DE 40 21 517 A1 However, polymers of poly (hydroxycarboxylic acid esters) and polyols, claims or exemplary embodiments in the sense of the colloidal carrier systems according to the invention are wanting.

WO 95/23175 A1 describes poly (hydroxycarboxylic acid) polyols and their use as a depot matrix system for the retardation of pharmacologically active substances. Claims or application examples in the sense of the colloidal carrier system according to the invention, in particular for the mucosal administration of active substance, are missing. Furthermore, the specific control of the polymer properties is not described, as well as the basic possibility to doubt the examples given there.

US 4,745,160 A describes biodegradable amphipathic copolymers, US 5 210 108 A degradable foam materials, exemplary embodiments or claims are wanting woolly.

US 5 612 412 A such as US 5 712 334 A describe the synthesis of lactone-modified polyvinyl alcohol (PVA), the present invention corresponding claims or exemplary embodiments but completely missing.

US 5 331 045 A describes the synthesis of poly (hydroxycarboxylic acid ester) modified polyvinyl alcohols (PVA) by polycondensation of PVA with monomeric hydroxycarboxylic acids. A method that According to the current state of the art impurities in the form of poly (hydroxycarboxylic acid) homopolymers does not exclude, which makes such a preparation for the inventive colloidal carrier systems as undesirable.

Component 2: Active substances

Surprisingly, it has been found that all relevant and in particular hydrophilic active ingredients can be used according to the invention. Therefore, according to the invention, all active substances which are selected from the group are preferred: active peptides, proteins, enzymes, enzyme inhibitors, antigens, cytostatics, anti-inflammatory agents, antibiotics, DNA constructs and growth factors.

However, inventive carrier systems are also used in particular also - but without being restricted - according to sub-aspects 3 or 8 to 11 of the first task as a colloidal carrier for mucosal or transmucosal administration of relevant hydrophilic active ingredients, such as. As proteins, peptides, oligonucleotides and DNA constructs as well as lipophilic drugs such. Corticosteroids and steroidal sex hormones.

Pharmaceutical formulations according to the invention for mucosal administration can be administered in different ways, eg. Sublingual, buccal, oral, nasal, pulmonary, vaginal, ocular or gastrointestinal.

Such formulations are of interest to all pharmaceutically-relevant biologically active substances when drug delivery to, into or through the mucosa is desired. They are particularly advantageous for active substances which are destroyed or inactivated without the use of a colloidal carrier system according to the invention in sublingual, buccal, oral, nasal, pulmonary, vaginal, ocular or gastrointestinal administration, or for which a high local mucosal concentration is desired.

• 1) Gruppe der hydrophilen makromolekularen pharmakologisch relevanten WS: 1.1 Impfstoffe, die ausgewahlt werden der Gruppe: Lebend-Impfstoffe, Tot-Impfstoffe, Toxoid-Impfstoffe, DNA Konstrukte sowie deren adjuvierte Zubereitung und gerichtet sind gegen pathogene Keime bzw. deren pathogenen Metaboliten, von denen literaturmaßig der mukosale Penetrationsweg bekannt ist bzw. die vorwiegend auf mukosalen Oberflachen kolonisieren, wie z. B. Helicobacter pylori, typhiforme Salmonellen, Vibrio cholerae, Neisserien (N. gonorrhoee, N. meningitidis), Entamoeba histolytica, Haemophilus influenzae, Bordetella Pertussis, Hepatitis A-Viren, colon- bzw. vagina-kolonisierende Staphylokokken, Streptokokken Typ A & B, Klebstellen (K. pneumoniae, K. oxytoca, K. rhinoscleromatis), Shigellen, Yersinien, Vibrionen, Pseudomonas aeroginosa, Legionellen, Korynebakterium diphtheriae, Bacillus anthracis, Mycobakterien (M. tuberkulosis, M. leprae), Treponema palli-dum, Mycoplasma pneumoniae, Chlamydien (C. trachomatis, C. pneumoniae), Polio-Viren, Rhino-Viren, Myxoviren (Masern-V., Mumps-V., Parainfluenza-V.), Röteln-Virus, Rotaviren, Adeno-Viren, Influenzaviren, Herpes-Viren (Varizellen-Zoster-Virus, Zytomegalievirus, Herpes Simplex Viris, EBV), HAV, HEV. 1.2 Impfstoffe, die ausgewählt werden aus der Gruppe: Lebend-Impfstoffe, Tot-Impfstoffe, Toxoid-Impfstoffe, DNA Konstrukte sowie deren adjuvierte Zubereitung und gerichtet sind gegen pathogene Keime bzw. deren pathogenen Metaboliten, bei denen bekanntermaßen ein parenteraler Infektionsweg vorliegt, aber eine mukosale Applikation systemische, spezifische Immunglobulintiter hervorruft, wie z. B. Clostridium tetani, Clostridium botulinum, Yersinia pestis, Borrelia burgdorferi, Tollwut-Virus, HIV-1 & HIV-2, HBV, HCV, HDV
• 2) Gruppe der lipophilen pharmakologisch relevanten Wirkstoffe, wie z. B. Corticosteroide, steroidale Sexualhormone, Cyclosporin A, Nystatin, Amphotericin B, Clotrimazol, Econazol, Miconazol, Fluconazol, Itraconazol, Bifonazol, sowie Antibiotika

According to the invention for mucosal administration preferred active ingredients (WS) are therefore:

• 1) Group of hydrophilic macromolecular pharmacologically relevant WS: 1.1 vaccines selected from the group: live vaccines, dead vaccines, toxoid vaccines, DNA constructs and their adjuvated preparation and are directed against pathogenic germs or their pathogenic metabolites, from which literaturmaßig the mucosal penetration path is known or colonize mainly on mucosal surfaces, such. B. Helicobacter pylori, typhiform Salmonella, Vibrio cholerae, Neisseria (N. gonorrhoee, N. meningitidis), Entamoeba histolytica, Haemophilus influenzae, Bordetella pertussis, hepatitis A viruses, colon or vagina-colonizing staphylococci, streptococci type A & B , Adhesive sites (K. pneumoniae, K. oxytoca, K. rhinoscleromatis), Shigella, Yersinia, Vibrios, Pseudomonas aeroginosa, Legionella, Korynebacterium diphtheriae, Bacillus anthracis, Mycobacteria (M. tuberculosis, M. leprae), Treponema pallidum, Mycoplasma pneumoniae, chlamydia (C. trachomatis, C. pneumoniae), polio viruses, rhino viruses, myxoviruses (measles v., mumps v., parainfluenza v.), rubella virus, rotaviruses, adenoviruses, influenza viruses , Herpes viruses (Varicella Zoster Virus, Cytomegalovirus, Herpes Simplex Viris, EBV), HAV, HEV. 1.2 Vaccines selected from the group consisting of live vaccines, dead vaccines, toxoid vaccines, DNA constructs and their adjuvated preparation, and directed against pathogenic microorganisms or their pathogenic metabolites known to have a parenteral route of infection, but one mucosal application systemic, specific immunoglobulin causes such. Clostridium tetani, Clostridium botulinum, Yersinia pestis, Borrelia burgdorferi, rabies virus, HIV-1 & HIV-2, HBV, HCV, HDV
• 2) group of lipophilic pharmacologically relevant agents such. Corticosteroids, steroidal sex hormones, cyclosporin A, nystatin, amphotericin B, clotrimazole, econazole, miconazole, fluconazole, itraconazole, bifonazole, as well as antibiotics

Preparation of colloidal associates

[I] Polymer-active substance complexes (Solution Problem 2 - Method 1)

The invention relates to pharmaceutical formulations consisting in the form of polymer-active substance complexes in colloidal form of at least one polymer and at least one active ingredient. The active ingredient may itself be a partner in the complex or bound to a partner of the complex. It was surprisingly found that inventive polymers with relevant active ingredients spontaneously self-assembling associates, also called complexes here, and that their properties could be manipulated and optimized via simple parameter variations.

It is already known from the literature that charged macromolecules with ions of opposite charge form ionic compounds, which often precipitate from aqueous solutions, depending on the charge distribution, the molecular weight and the hydrophobicity of the end product. In this case, low molecular weight ions of the same charge are displaced by the higher molecular weight compound, phenomena that are summarized under the generic term "polyelectrolyte effect". State of the art include the formation of gels (combining alginate solutions and Ca ²⁺ ) or protein precipitations. Such polyelectrolyte complexes may in principle consist either of a macromolecular, multiply charged component of one polarity and many low molecular weight ions of the other polarity, or of two macromolecular, respectively multiply charged, partners of different polarity. Hollow capsules prepared from such polyelectrolyte complexes are described, for example, in US Pat BE 9 01 704 A1 . EP 0 127 989 A2 . EP 0 188 309 A2 . EP 0 127 713 A2 . DE 32 09 127 A1 . EP 0 152 898 A2 . US 4 487 758 A . DE 32 09 098 A1 . US 4 409 331 A , It is also known from the literature, for. In DE 40 13 110 A1 described that polyelectrolyte complexes can be prepared in microparticulate form, thereby enabling an improvement in the therapeutic usability and breadth of an active ingredient.

Surprisingly, it has now been found that even the inventive biodegradable, thus toxicologically woolly harmless polymers, even without clear Polyelektrolyteigenschaften complexes with pharmacologically relevant drugs spontaneously, due to hydrophilic-hydrophobic interaction form. Furthermore, the easy controllability of this interaction by targeted carrier polymer manipulation of hydrophilic-hydrophobic interaction up to interionic interaction is also surprising. Thus, these polymers also show in particular to a large extent as vehicles substances properties that not only meet the requirements profile of biocompatible and biodegradable polymer systems, but also by slight variation in the production of the special requirements of a therapeutic, especially for mucosal drug administration can be easily adjusted.

Surprisingly, even for the unmodified, thus no clear Polyelektrolyteigenschaften possessing, colloids according to the invention high drug association (loadings) found that were not expected, furthermore, the degree of loading could be manipulated and optimized by targeted selection of the carrier polymer (component 1).

It was also surprising that in the preparation of such complexes fine particles with controllable sizes from nm to the micron range formed and not - as was to be expected - only a clumped mass.

In this case, particle sizes, deliberately kept in the range of below 100 nm to a few microns, and particle size / distribution easily adjustable, for example on the choice of / the carrier polymers, the concentrations used, salt concentrations, ionic strength, pH, temperatures, etc. The required parameters can easily be determined in appropriate preliminary experiments and adapted to the desired requirements of a therapeutic agent, in particular also for mucosal administration of active ingredient.

Furthermore, it was surprising that with the help of such complex-like associates high local drug concentrations can be generated on relevant cell tissues.

According to the invention, such self-assembling colloidal systems can be prepared particularly well by polymer-active substance complexes according to the invention. External influences, such as solution equilibria, charge interactions, pH changes, etc. can furthermore, provided that, from a therapeutic point of view, a slow decomplexation, ie drug release, be achieved in vivo. These release conditions can also be controlled as the consistency properties over the complex composition.

The advantages of such spontaneously forming aqueous solutions systems are obvious, u. a. No special manufacturing technologies are necessary, it is not mandatory, the properties of the therapeutic agent-reducing auxiliaries, such. As organic solvents, emulsifiers, etc. necessary, which inventively a faster, reproducible and cost-effective way was found to obtain efficient therapeutics, especially with regard to mucosal drug administration.

[II] Preparation and Loading of Colloidal Carrier Systems According to the Invention by Modified Solvent Deposition (Solution Problem 2 - Method 2, for More Lipophilic Polymers)

Since, as already stated, most production techniques are particularly unsuitable for sensitive active substances, an adapted and optimized so-called solvent deposition method is further preferred for producing the inventive carrier system, can be prepared easily and reproducibly with the inventive colloids, provided the use of a more lipophilic, thus no longer water-soluble carrier polymer is desired or required.

This manufacturing method, which differs from the nanoparticle production method introduced by Fessi in 1986 [ US 5 118 528 A . US 5 049 322 A ], based on the controlled precipitation of a dissolved polymer in a non-solvent, thus allowing the preparation of particulate systems in the micron and submicron size without preceding emulsification or dispersion steps. It has been optimized for carrier systems according to the invention, in particular also for mucosal drug administration, by choosing the appropriate polymer / solvent / non-solvent / (emulsifier) system.

Particle sizes according to the invention were deliberately adjusted to a range from below 100 nm to a few μm. The width of the particle size distribution is easily adjustable, for example, on the concentration of the polymer solution used, the rate of addition, emulsifier and by appropriate selection of a cosolvent added in defined proportions. These parameters can also be easily determined in appropriate screenings and adapted to the desired requirements, in particular they could thereby be optimized for the mucosal administration of the active compound according to the invention.

It was found that the large range of less than 500 nm in diameter according to the invention is particularly well suited for mucosal application, an increased drug concentration on relevant cells, as well as a corresponding immune response could be achieved in vivo.

With polyol esters according to the invention, such colloidal carrier systems can be prepared particularly well. Due to the specifically manipulated properties of the carrier polymers, it was also surprisingly found that the use of additional emulsifiers for stabilization could be omitted woolly or as far as possible, which enabled not only the simultaneous maintenance of the specifically set surface properties of the carrier system according to the invention, but also the number of components to be used / Reduced procedural steps and thus simplifies the production and cheaper.

• 1. durch Adsorption des Wirkstoffs aus einer Lösung, mit der die bereits hergestellten Polymer-Kolloide nach Losungsmittelentzug in Kontakt kommen;
• 2. durch in-situ-Adsorption des Wirkstoffs aus einer Losung, mit der die gerade hergestellten Polymer-Kolloide vor Losungsmittelentzug in Kontakt kommen;
• 3. durch in-situ-Einschluss des Wirkstoffs in die Polymermatrix wahrend der Partikelbildung und anschließendem Losungsmittelentzug;
• 4. durch kovalente Bindung, wobei der Wirkstoff chemisch an mindestens eine funktionelle Gruppierung des eingesetzten Polymers gebunden ist;
• 5. durch kovalente Bindung an die Partikeloberfläche, wobei der Wirkstoff chemisch an mindestens eine funktionelle Gruppierung der Partikel gebunden ist.

The association of this system with active substance (s) can be carried out in at least five ways:

• 1. by adsorption of the active ingredient from a solution with which the already prepared polymer colloids come into contact after solvent removal;
• 2. by in situ adsorption of the active ingredient from a solution with which the polymer colloids just prepared come into contact with solvent deprivation;
• 3. by in situ inclusion of the active ingredient in the polymer matrix during particle formation and subsequent solvent deprivation;
• 4. By covalent bonding, wherein the active ingredient is chemically bonded to at least one functional grouping of the polymer used;
• 5. by covalent bonding to the particle surface, wherein the active ingredient is chemically bound to at least one functional grouping of the particles.

Because of the one hand far variable, on the other hand, very specific adjustable properties show inventive polymer colloid-active ingredient formulations property profiles that they pradestinate especially for a mucosal application.

It is summarized for both Kolloidherstellungsweisen [(I) and (II)] a special feature of the invention that the respective biocompatible and biodegradable carrier polymer (1) is deliberately made of metabolizable and thus non-toxic building blocks and targeted (2) to the particular requirements of the / the drug molecules, (3) could be adapted to the desired site of action or absorption as well as mechanism of action and (4) to the possible use of a gentle particle production process.

ad (1)

For the synthesis of the carrier polymer (component 1), at least one biocompatible component (polyols) as well as polyester fragments linked thereto, which decompose by simple hydrolysis in the body to form fragments that can be metabolized in the citric acid cycle, are used.

ad (2) and (3)

By suitably and selectively selecting or modifying the polyols (eg type, molecular weight, number of esterifiable hydroxyl groups, number and type of charge-carrying groups, distance of the charge-carrying groups from the polyol base skeleton, etc.), the properties of the polyol predetermined thereby are systematically enforced targeted attachment of polyester building blocks adapted to the requirements of the colloidal system of the invention. Examples include solubility, molecular weight, structure, charge density, particle surface charge, etc.

If a desired hydrophilic-hydrophobic interaction is desired for the desired active substance-carrier polymer interaction (such as, for example, active substance stabilization, degree of loading, etc.), this is achieved by the targeted synthetic adjustment of the polymer amphiphilia. If an interionic interaction is desired, this is achieved the selection and / or modification of a corresponding electrolyte polyol achieved.

ad (4)

• [II] If a stable particulate carrier is to be associated or combined with active ingredients in the case of more lipophilic active ingredients during production or more hydrophilic active ingredients after the preparation of the carrier system according to the invention, the solubility of the carrier polymer is deliberately adjusted so that a modified and optimized gentle so-called Solvent deposition method can be used for particle generation.
• [I] If this technological production step can or should be dispensed with, the carrier polymer is deliberately made water-soluble, so that upon combination of active ingredient and carrier polymer spontaneously defined colloidal polymer-active substance complexes form in aqueous solution and their properties, such , B. Large, loading level, etc. can also be set specifically.

With the help of these inventive novel colloidal, biodegradable polymeric carrier systems, which achieve their special effectiveness only by combining all the above-mentioned points, on the one hand wollig new therapeutic applications are possible, on the other hand, conventional therapy concepts can be optimized, such. B. by increasing the therapeutic range and efficiency of an active ingredient.

Further advantages which result from the invention are the more convenient way of administering the patient, since it is not absolutely necessary to administer parenterally, ie via an infection, and the associated increased "patient" compliance. This can, compared to previous vaccination, a vaccination z. B. by taking a tablet or solution, similar to the poliomyelitis vaccine (polio) done. The resulting lower degree of contamination in the population has a not insignificant economic benefit, which also results from the application of inventive systems in veterinary medicine.

Examples

The invention is explained in more detail below, but without being limited by a few selected examples.

polymer synthesis

Zentralpolyol modification

The exemplary modification of an uncharged central polymer, here poly (vinyl alcohol), was comparable to [F. Dolle, J. Le Moigne and P. Gramain, Eur. Polym. J. 6 (1970) 1227-1232; Gramain and J. Le Moigne, Eur. Polym. J. 8 (1972) 703-709] via etherification of its activated hydroxyl groups with butanesulfone or with diethyaminoethyl chloride. Table 1 description Molecular weight [kg / mol] Degree of substitution [%] Substituent to -OH PVA ~ 6 (*) (20) (-O-COCH ₃ ) PVA ~ 15 (*) (12) (-O-COCH ₃ ) SB03PVA ~ 16 3 -O- (CH ₂ ) ₄ SO ₃ ^- SB10PVA ~ 19 10 -O- (CH ₂ ) ₄ SO ₃ ^- SB20PVA ~ 23 20 -O- (CH ₂ ) ₄ SO ₃ ^- SB30PVA ~ 27 30 -O- (CH ₂ ) ₄ SO ₃ ^- SB40PVA ~ 31 40 -O- (CH ₂ ) ₄ SO ₃ ^- SB50PVA ~ 35 50 -O- (CH ₂ ) 4SO ₃ ^- DEAE10PVA ~ 18 10 -O- (CH ₂ ) ₂ N (C ₂ H ₅ ) ₂ * Manufacturer's instructions

shows by way of example FT-IR spectroscopy (Nicolet 510 P FT-IR spectrometer) the introduction of the sulfobutyl groups in the originally uncharged central polymer and the increase in the intensities of the corresponding SO-related vibrations at 1190, 1040 and 610 cm ^-1 .

shows the corresponding modifications by NMR spectroscopy (Jeol GX400 Delta N FT-NMR spectrometer), e.g. By reducing the signal intensity of -CH ₂ -CH-OH (~ 1.7 ppm) and -CH ₂ -CH-OH (~ 4.1 ppm), increasing the signal intensities -CH ₂ -CH-OC and -CH -O-CH ₂ - (~ 1.9 ppm) and the sulfobutyl group itself at ~ 3.08 and ~ 3.7 ppm.

Melt grafting to produce the carrier polymers (component 1) by attaching hydroxycarboxylic acid groups to the central polyols

Exemplary of the lactones of lactic and glycolic acid: D, L-lactide (dimeric anhydride of lactic acids) and glycolide (dimeric anhydride of glycolic acid) are in defined molar amounts, eg. B. 1: 1, in the presence of defined amounts of the central polyol, for. B. 1 wt .-% PVA, at z. B. 130 ° C for z. B. for three hours under inert, anhydrous conditions with defined molar amounts of catalyst (eg., 0.2 mmol of stannous octoate). Subsequently, the reaction product is taken up in a suitable solvent and precipitated in a 10: 1 excess of a miscible polymer denier solvent for purification and dried to constant weight in vacuo. For this example, the crude product was dissolved in dichloromethane (DCM) and precipitated in ethanol. Depending on the solubility, however, combinations of acetone in water or inverse water in acetone also resulted. Further details s. Table 2. polymer D, L-LA: GA [mol: mol] Central polyol * / amount [% by weight] Reaction Temp. [° C] solvent PVAPLG1 1: 1 PVA / 1 130 DCM PVAPLG10 1: 1 PVA / 10 130 acetone PVAPLG33 1: 1 PVA / 33 140 Acetone / water PVAPLG50 1: 1 PVA / 50 140 water PVAPLG57.1 1: 1 PVA / 57.1 140 water PVAPLG62.5 1: 1 PVA / 62.5 140 water PVAPLG70 1: 1 PVA / 70 140 water PVAPLA50 * 1: 0 (* L-LA) PVA / 50 110 water SB03PVAPLG10 1: 1 SB0PVA / 10 140 acetone SB10PVAPLG10 1: 1 SB10PVA / 10 140 acetone SB20PVAPLG10 1: 1 SB20PVA / 10 140 acetone SB30PVAPLG10 1: 1 SB30PVA / 10 140 acetone SB40PVAPLG10 1: 1 SB40PVA / 10 140 acetone SB50PVAPLG10 1: 1 SB50PVA / 10 140 acetone DEAE10PVAPLG10 1: 1 DEAE10PVA / 10 150 acetone SB40PVAPLG33 1: 1 SB40PVA / 33 140 Acetone / water SB40PVAPLG50 1: 1 SB40PVA / 50 140 water SB10PVAPLG45 1: 1 SB10PVA / 45 140 water SB10PVAPLG50 1: 1 SB10PVA / 50 140 water Reaction time in each case 3 hours, catalyst in each case tin octoate
* according to table 1

Structure and solubility of the polyol esters (component 1)

shows by NMR spectroscopy the structural change of the polymers depending on the conditions of synthesis. Here, the hydroxycarboxylic acid moiety lengths at the central polymer were systematically shortened to particularly demonstrate solubility adjustability. While in PVAPLG10 the hydroxycarboxylic acid moieties attached to the OH groups of the central polymer still each consist of about 10 acid moieties, evidently e.g. B. on the ratio of lactide signal intensities -CH ₃ chain: -CH ₃ end group, ie at ~ 1.5 ppm to ~ 1.3 ppm, the groupings in PVAPLG50 are already shortened so that this Tragerpolymer well in cold water lost, but - surprisingly found - precipitated by temperature increase again and therefore has a lower critical demixing temperature.

shows by means of a combination of size exclusion chromatography and static light scattering (Merck-Hitachi system with RI 71 detector in combination with MiniDawn Light Scattering detector), how this structural change accordingly also on the molecular weight of the carrier polymer compared to an unmodified central polyol effect.

shows by means of photometric turbidity measurement (Shimadzu UV-VIS spectrophotometer UV 160), for example for uncharged carrier polymers, that the surprisingly found lower critical demixing temperatures can also be set in a defined manner as a function of the (hydroxycarboxylic acid grouping) chain lengths.

shows a further manipulation possibility of the position of the lower critical demixing temperature over the concentration of the polymer in the water by means of photometric turbidity measurement by way of example here for a charge-modified carrier polymer.

Using photon correlation spectroscopy [PCS, Malvern Zetasizer 4], exemplified here for a charge-modified carrier polymer, shows the size of the pure carrier polymer associates as a function of temperature, demonstrating a way to control the properties of the polymer-drug complexes. Particularly for very sensitive active ingredients (which may be mentioned by way of example growth factors), a particular carrier polymer associate in aqueous (comparable to a micellar system) can be offered to the active ingredient even at low temperatures, on the surface of which the active ingredient itself is then associated.

Spontaneously formed polymer-drug complexes in the aqueous

General procedure for the following examples: A defined amount of a Wirkstoffstammlosung is presented, diluted with a defined amount of buffer, then admixed with a defined amount of polymer stock solution, so that unless otherwise stated, a total volume of 1 ml results.

shows the possibility of controlling the size of such polymer-drug complexes over the polymer concentration used (active ingredient: fluorescently labeled model protein FITC-BSA (bovine serum albumin) 640 μg, polymer PVAPLG50, isotonic phosphate buffer pH 6.0 (PBS 6.0), large measurement per PCS ).

exemplifies the easy adjustability of polymer-drug complex properties via "process" parameters such as pH and / or ionic strength of aqueous solutions (active ingredient: tetanus toxoid, amount for this factorial design varies within the limits 332 μg to 949 μg / 200 ml, polymer: PVAPLG57.1 1.670 μg / 200 ml, size measurement by PCS, zeta potential measurement with Malvern Zetasizer 4).

shows by way of example that the addition of an emulsifier, if desired, also represents a possibility of influencing the polymer-drug complex properties (active ingredient: tetanus toxoid 3.367 mg, polymer: PVAPLG57.1 3.32 mg, in total 1.220 ml PBS) 6.0, emulsifier Pluronik F68 BASF).

shows by way of example how the overall system, ie polymer-drug complex, can be optimized for an active ingredient by selecting the carrier polymer and its amount used. If an uncharged central polyol with hydroxycarboxylic acid groups is provided synthetically, 90% of the model active substance used (FITC-BSA, 450 μg, total liquid volume: 1 ml PBS 6.0) are bound in the form of the polymer-active substance complexes according to the invention at a sufficiently high concentration of this carrier polymer in the water but if a central polyol with only 10% charge-carrying groups is used for the synthesis of component 1, the entire amount of the protein used is associated. Once the desired polymer has been chosen in such a way, beyond its amount used, any desired amount of the protein used can be associated in the form of the polymer-active substance complexes.

shows that - if therapeutically useful - also a decomplexing / release of drugs, here exemplified tetanus toxoid, can be achieved from the polymer-drug complexes. Releases, which were controlled here by way of example by the selection of the carrier polymer. Furthermore, this example shows that in particular mucosomal application of these systems according to the invention, no significant amounts of protein are released within the first few hours, so that the mucosal target site can be reached, and only there a release of the drug takes place (photometric content determination at 280 nm , Amount of component 1 (carrier polymers), each 10 mg, amount of component 2 (tetanus toxoid) in each case 1,992 μg, each in total 1,000 .mu.l PBS 6.0, after spontaneous formation of the polymer-drug complexes, they were centrifuged at indicated times at 44 ° C. the supernatant was examined photometrically, then the complexes were further incubated in fresh PBS 6.0 at 37 ° C).

shows here the example of a complex between a charge-carrying carrier polymer and the model protein FITC-BSA, the massive accumulation of the complexes on mucosalahnlichem cell tissue (Caco 2 cell culture), a phenomenon that can not be generated when using free drugs alone and thus clearly demonstrated the surprisingly found efficacy of the new carrier system according to the invention (640 μg protein, 10 mg polymer, together 1000 μl PBS 6.0)

colloids

When it is desired to use a carrier polymer whose hydrophilic-hydrophobic balance is more shifted to the lipophilic side, e.g. B. compatibility reasons for the active ingredient or if the relevant active ingredient should be exclusively defined on the surface of the colloidal carrier, then an acetone-soluble carrier polymer can be prepared with appropriate total polymer composition and converted according to modified solvent deposition methodology in the colloidal carrier according to the invention. Exemplary preparation description (production parameters may vary for property optimization thereof): 50 mg of polymer are dissolved in 5 ml of acetone (may contain cosolvent). The solution is added at a constant rate of addition in 50 ml of PBS (may contain proportional emulsifier). The resulting suspension is dispersed continuously. After the addition, the organic solvent is evaporated under stirring for 8 hours.

demonstrates using the example of an uncharged biodegradable polymer a way to vary the size of such particles produced by controlled precipitation, here on the proportionate addition of a toxicologically acceptable Kosolvenz (ethyl acetate) in the Tragerpolymeracetonlosung.

illustrates the morphology and size of such colloids (transmission electron microscopic (TEM) photography) using the example of a charged carrier polymer SB50PVAPLG10.

demonstrates, by way of example, the possibility, due to the amphiphilic character of the carrier polymers, of being able to work not only without addition of emulsifier (emulsifier here PVA (18.88), Hoechst) in the solvent deposition method, but also the possible preservation of the surface properties of the colloids.

shows by way of example that the colloid surface properties as a function of the carrier polymers, more precisely the choice of the central polyol used for melt grafting and its properties in combination with suitable Hydroxycarbonsauregruppierungsmodifikation, are adjustable - here exemplified listed: surface charge of the colloids (zeta potential measurement and Particle Charge Detection (PCD) Titration) and their hydrophobicity (Bengal adsorption method).

demonstrates that also the loading level of such relevant active ingredient colloids is a direct function of the carrier polymers, more specifically the choice of the central polyol used for melt grafting and its properties in combination with appropriate hydroxycarboxylic acid grouping modification.

shows the high effectiveness of the colloids according to the invention, here exemplarily for red-labeled SB30PVAPLG10 colloids in the cell culture model (confocal laser microscopy), not only on (apical), but also in and even basal one finds a high fluorescent association (optical cell section planes in from left to right about 0 μm, 10 μm, 20 μm).

further supports this finding, as the cell adsorbed and cell-adsorbed colloid amount again represents a function of the carrier polymers, more specifically the choice of the central polyol used for melt grafting and its properties in combination with appropriate hydroxy carboxylic acid moiety modification.

Toxicity and drug stability

demonstrates the toxicological safety of colloids according to the invention, according to MTT assay, a cell viability of 100% is maintained, ie no cell death by the colloids is recognizable, even for LDH release, the colloids behave neutral. In the propidium iodide experiment this finding is also substantiated, less than 0.2% of the cells died on incubation with the colloids, a value equal to that of pure aqueous buffer medium.

shows the result of gel electrophoresis (GE) experiments (Native PAGE and SDS PAGE), which demonstrate the Ververeitheit of the drug, both in the polymer-drug complex, as well as desorbed drug after 24 hours show no changes compared to free untreated drug (1 = free untreated model protein FITC-BSA; 2 = polymer-drug complex: SB10PVAPLG50-FITC-BSA complex; 3 = FITC-BSA desorbed from 2 to 24 h).

General method description Cell experiments

Determination of cytoadhesion and cytotoxicity

A prerequisite for the application of colloidal drug carriers, in particular also mucosal administration, besides a low toxic effect on the surrounding tissue (epithelial cells), also a sufficiently long residence time on mucosal surfaces (cytoadhasion) as well as a possibly required sufficient cellular uptake. To study the interaction with mucosal cells, the Caco 2 cell culture model was used. This in vitro model of the intestinal mucosa has already been shown in many studies to be useful in the study of intestinal absorption, cytoadhasion, and toxicity of drugs and drug delivery systems.

cell culture

Caco 2 cells of passages 42 to 45 were cultured in Dulbecco Modified Eagles medium with a glucose content of 4.5 g / l with the addition of 10% Fotalem calf serum, 2 mM L-glutamine and 1% nonessential amino acids in 10% CO ₂ atmosphere at 95% RH and 37 ° C.

toxicity studies

Caco 2 cells were seeded at a cell density of 6.5 x 10 ⁴ cells / cm ² on polystyrene multiwell plates. Medium change took place every 2 days. On day 21 after sowing, the cells have formed a differentiated monolayer and can be used for in vitro studies.

For this purpose, cells were washed twice with isotonic phosphate-buffered saline (PBS, pH 7.2) and corresponding colloid suspensions according to the invention (0.25, 2.5 mg / ml) applied to the cell surface. After several hours of incubation (time intervals as shown in the figures), the following toxicity tests were carried out.

1. Release of lactate dehydrogenase (LDH)

Principle: As a cytoplasmic enzyme LDH occurs in a damage of the plasma membrane from the cell interior into the external medium (buffer) and can be quantified by kinetic determination methods against a standard UV photometry. The toxic reference standard used is Triton X 100 (0.1% in PBS), whose LDH release corresponds to 70%.

2. Transformation of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide)

Principle: The tetrazole compound MTT is enzymatically reduced in mitochondria of still viable cells to a blue-colored formazan dye. The blue coloration is proportional to the viability of the cells and is detected photometrically, with PBS being the non-toxic reference.

3. Propidium iodide staining

Principle: Propidium iodide penetrates as a hydrophilic substance only damaged cell membranes and binds to nucleic acids of the nucleus, resulting in UV excitation to a red nuclear fluorescence, which is microscopically detectable. The evaluation is carried out by fluorescence microscopy with wavelength filter (380/550) against a non-toxic PBS standard.

Cytoadhesion and cellular uptake

Caco 2 cells were seeded at a cell density of 6.5 × 10 ⁴ cells / cm ² on polycarbonate membranes (Costar-Transwell). Medium change took place every 2 days. On day 21 after seeding, the cells have formed a differentiated monolayer and can be used for adhesion and uptake studies.

Colloids fluorescently labeled with Nile red (1%, w / w) and polymer-active substance complexes (active ingredient: fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA)) were applied to the apical surface of the cells and incubated at 37 ° C. for 120 min. 95% RH, 10% CO2 incubated. The colloid concentration was 250 μg / ml in both cases.

After incubation, the cell monolayers were washed 3 times with PBS and then fixed with formalin solution (4% in PBS) for 60 minutes. The filter membranes were excised by scalpel and embedded in glycerol gelatin. The preparations thus prepared were examined for adhasive and intracytoplasmic fluorescence by confocal laser scanning microscopy (CLSM).

In controls only with active ingredient without colloids or complexes, no fluorescence was detectable on or in the cells.

Claims (23)

A colloidal associate containing a polyol ester-drug complex wherein the polyol ester is derived from a linear polyol having more than six hydroxyl groups as the central molecule to which biodegradable hydroxyl carboxylic acid ester moieties are attached. A colloidal associate according to claim 1, characterized in that the linear polyol has charged and / or protonatable and / or deprotonatable groups attached to the linear polyol via a spacer and via an ether, ester or urethane linkage. A colloidal associate according to claim 1, characterized in that the active ingredient (s) is / are selected from the group consisting of active peptides, proteins, enzymes, enzyme inhibitors, antigens, cytostatics, anti-inflammatory agents, antibiotics, DNA constructs and growth factors. Colloidal associate according to claim 1, characterized in that the colloidal particles have an average particle size range of <10 μm. Colloidal associate according to claim 4, characterized in that the average particle size range is <1 μm. Colloidal associate according to claim 1, characterized in that the average particle size range is 10 to 10,000 nm. A process for the preparation of the colloidal associate according to claim 1, comprising the measures a) presentation of an aqueous solution of the polyol ester according to claim 1, optionally in combination with the active ingredient; b) increasing the temperature until a spontaneous in-situ formation of colloidal polyol ester or polyol ester-drug complex takes place, and c) optionally, contacting the colloidal polyol ester with the active agent to form the colloidal polyol ester-drug complex. A method according to claim 7, characterized in that the temperature after step b) above the lower critical demixing temperature is increased, which is in the temperature range of 0 ° C to 100 ° C. Process for the preparation of the colloidal association according to claim 2, comprising the measures: a) presentation of an aqueous solution of the polyol ester according to claim 2, and b) addition of an active substance which optionally has charged and / or protonatable and / or deprotonatable groups which are capable of forming complexes with the charged and / or protonatable and / or deprotonatable groups of the polyol ester. Polyol esters containing a linear polyol having more than six hydroxyl groups as the central molecule to which biodegradable hydroxycarboxylic ester groups are attached, the linear polyol having charged and / or protonatable and / or deprotonatable groups via a spacer and via an ether, ester or urethane Linkage are bound to the linear polyol. Polyol ester according to claim 10, characterized in that the spacer is an aliphatic group. Polyolester according to claim 10, characterized in that the polyol is selected as the central molecule from the group consisting of linear synthetic polymers having> 6 to 500 hydroxyl groups, in particular polyvinyl alcohols (PVA) with degrees of polymerization of> 6 to 500 (and saponification degrees of 70 to 98% when obtained by hydrolysis of poly (vinyl acetate). Polyol esters according to claim 10, characterized in that the polyol is selected from the group consisting of linear synthetic vinyl copolymers, in particular of polyvinyl alcohol, having> 6 to 500 hydroxyl groups, in particular vinyl acetate, vinylpyrrolidones, vinylamines, vinylimidazoles, vinylpyridines, vinylsulfonic acids, vinylphosphoric acids , Polyol ester according to claim 10, characterized in that the bonded via a spacer to the central molecule charged and / or protonatable and / or deprotonatable group is selected from the group consisting of carboxyl groups, sulfonic groups or primary, secondary, tertiary or quaternary amine groups. A polyol ester according to claim 14, characterized in that the groups bound via a spacer to the central molecule one or more carboxyl, sulfobutyl or sulfopropyl group (s) and / or butylamine, propylamine or ethylamine (primary, secondary, tertiary or quaternary amine, wherein corresponding N-substituents, in particular -H, -CH ₃ and / or C ₂ H ₅ group (s) represent) group (s) may contain. Polyol esters according to claim 10, characterized in that the biodegradable hydroxycarboxylic acid ester groups are derived from L-lactic acid, D-lactic acid and / or glycolic acid. A polyol ester according to claim 16, characterized in that the hydroxycarboxylic acid ester groups are derived from dimers of L-lactide, D-lactide, D, L-lactide and / or glycolide in any molar compositions. Polyol ester according to claim 10, characterized in that by introducing Hydroxycarbonsaureestergruppierungen in the polyols, the water-immunity is preserved. Polyol ester according to claim 10, characterized in that it has a lower critical demixing temperature in the temperature range of 0 ° C to 100 ° C in aqueous solution. Polyol ester according to claim 10, characterized in that the molar ratios of polyol-OH group to Saurebaueinheit in the range of 0.6: 1 to 6: 1, in particular in the range of 1: 1 to 3: 1 A polyol ester according to claim 10, characterized in that the Hydroxycarbonsauregruppierungen the central polyol are each composed of 1 to 100, and in particular 5 to 50, Hydroxycarbonsaurebausteinen. Use of the colloidal associate according to claim 1 for the preparation of pharmaceutical preparations, preferably for mucosal administration of active ingredients, in particular for sublingual, buccal, oral, nasal, pulmonary, vaginal, ocular and / or gastrointestinal drug administration. Use of the colloidal association of claim 1 for the preparation of a vaccine.

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