EP1112346A1 - Utilisation de nanocellules dans des produits finis sous forme de milieux de culture - Google Patents

Utilisation de nanocellules dans des produits finis sous forme de milieux de culture

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Publication number
EP1112346A1
EP1112346A1 EP99939894A EP99939894A EP1112346A1 EP 1112346 A1 EP1112346 A1 EP 1112346A1 EP 99939894 A EP99939894 A EP 99939894A EP 99939894 A EP99939894 A EP 99939894A EP 1112346 A1 EP1112346 A1 EP 1112346A1
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EP
European Patent Office
Prior art keywords
nanocell
vitamin
end products
culture media
component
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Withdrawn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
EP99939894A
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German (de)
English (en)
Inventor
Andreas Werner Supersaxo
Marc Antoine Weder
Hans Georg Weder
Hans Konrad Biesalski
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.)
Vesifact AG
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Vesifact AG
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Publication date
Application filed by Vesifact AG filed Critical Vesifact AG
Publication of EP1112346A1 publication Critical patent/EP1112346A1/fr
Withdrawn legal-status Critical Current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
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins

Definitions

  • the present invention relates to the use of NanoCells in culture media end products, culture media end products containing these NanoCells and the various uses of these culture media end products.
  • Culture media end products in the sense of the present invention are e.g. Media for the cultivation and investigation of cells, fungi, bacteria, viruses, bacteriophages, insects and plants, media for the in vitro or ex vivo cultivation and investigation of organs and tissues, media for freezing cells, fungi, bacteria, viruses, bacteriophages , Insects, plants, organs and tissues, embryo transfer freezing media, media for therapy (adoptive immunotherapy, cancer treatment), media for perfusion of organs such as Kidney or liver, media for storing organs until they are transplanted, and supplements for the above media.
  • the invention is based on the object of lipophilic, i.e. to incorporate poorly water-soluble or water-insoluble substances into end products of culture media in such a way that
  • vehicles such as micelles, mixed micelles, liposomes or finely emulsified emulsions are also used to introduce lipophilic substances.
  • these vehicles particularly in high concentrations, are often toxic to cells, fungi, bacteria, viruses, bacteriophages, insects, plants, tissues and organs or influence the function of the biological system kept in culture.
  • the functional influencing of the biological systems kept in culture makes the interpretation of pharmacological, toxicological, metabolic and regulatory examinations impossible or difficult.
  • the low loading capacity with hydrophobic substances as well as the insufficient stability in certain culture media are further reasons which can limit the use of these vehicles.
  • methylbetacyclodextrin has the decisive disadvantage that it "punctures" the cell membrane and, as has been repeatedly found, disregulates calcium flux; i.e. there is an influx of calcium into the cell with all subsequent processes activated by calcium.
  • the invention is also based on the object of incorporating lipophilic compounds into end products of culture media which are particularly suitable for organ storage until their transplantation.
  • Various lipid-soluble compounds are suitable for protecting cells and tissues against transient ischemia, but very particularly also against reperfusion.
  • reperfusion phase With changed oxygen supply.
  • This so-called ischemia / reperfusion due to the breakdown of ATP during ischemia, leads to an activation of enzyme systems which, depending on the availability of oxygen, then metabolize the breakdown products of ATP, with considerable amounts of reactive oxygen compounds being formed.
  • lipid-soluble antioxidants such as 3-carotene, vitamin E, but also water-soluble vitamin C are suitable for suppressing the formation of such reactive oxygen compounds, including their secondary products, which occur in ischemia / reperfusion syndrome.
  • the perfusion media used were either fat solutions or the donor organs were pre-orally given vitamins
  • a lipophilic constituent, lipophilic, ie water-insoluble or sparingly water-soluble substances can be introduced into end products of culture media, so that the cells, fungi, bacteria, viruses, bacteria, viruses, bacteriophages, insects, plants or tissues neither grow nor grow are adversely affected in their functions.
  • the particular advantage of this NanoCell is that it is an extremely physiological form of application that, unlike all other solubilizers of fat-soluble compounds, can also be used easily under in vivo conditions.
  • endothelial cells, but also other cellular compartments such.
  • B. fibroblasts or intestinal mucosa cells absorb vitamins well in this form, also allows use for the application of vitamins in the buccal mucosa.
  • lipophilic compounds such as Bring antioxidants in perfusion solutions that are suitable as
  • Organ preservation solutions to be used So far - available in vitro and in vivo studies, which have been carried out with such antioxidant formulations, show that in this way the antioxidative vitamins in the critical cell compartments, especially the endothelial cells, are actually accumulated. This accumulation was not expected since the cells are normally only supplied via lipo-protein-bound antioxidants. With the available experimental approaches it could also be shown that the infusion of antioxidant vitamins via the NanoCell technology leads to a first pass effect in the organ, i. H. leads in the critical cells of the perfused organ. With this, however, the organ is protected in a physiological manner with the help of the antioxidants so applied, so that the use of antioxidants in this form is suitable for improving organ preservation and preservation.
  • NanoCells are their high loading capacity with lipophilic substances as well as the good stability in the end products of culture media.
  • the present invention therefore relates to the use of a NanoCell containing, as preparative composition (a), a membrane-forming molecule,
  • the NanoCell preferably contains (a) as membrane-forming molecules, substances which are suitable for forming two-layer systems (so-called “bilayers”),
  • the NanoCell preferably contains a phospholipid, a hydrogenated or partially hydrogenated phospholipid, a lysophospholipid or mixtures of these compounds.
  • a phospholipid of the formula is very particularly preferred
  • R 1 C 10 -C 20 acyl
  • R 2 is hydrogen or
  • R3 is hydrogen, 2-trimethylamino-l-ethyl, 2-amino-l-ethyl, unsubstituted or by one or more
  • Carboxy, hydroxy or amino groups substituted C j -C ⁇ alkyl mean the inositol or glyceryl group, or salts of these compounds.
  • Straight chain with an even number of carbon atoms are, for example, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl or n-octadecanoyl.
  • Straight chain e i ner double bond and an even number of carbon atoms are 6-cis- or 6-trans-, for example, 9-cis- or 9-trans-dodecenoyl, -Tetradece- noyl, -Hexadecenoyl, -Octadecenoyl or -Icosenoyl, in particular 9-cis-octa-decenoyl (oleoyl), further 9,12-cis-octadecadienoyl or 9, 12, 15-cis-octadecatrienoyl.
  • a phospholipid of the formula (1) in which R is 2-trimethylamino-1-ethyl is designated with the trivial name lecithin and a phospholipid of the formula (1) in which R3 is 2-amino-1-ethyl is designated with the trivial name cephalin.
  • cephalin naturally occurring cephalin or lecithin are suitable, e.g. Kephalin or lecithin from soybeans or hen's egg with different or identical acyl groups or mixtures thereof.
  • the phospholipid of formula (1) can also be of synthetic origin.
  • synthetic phospholipid defines phospholipids which have a uniform composition with respect to R 1 and R 2.
  • Such synthetic phospholipids are preferably the lecithins and cephalins defined above, whose acyl groups R-L and R2 have a defined structure and are derived from a defined fatty acid with a degree of purity higher than approximately 95%.
  • R ⁇ and R2 can be the same or different and unsaturated or saturated.
  • R ⁇ is saturated, e.g. n-hexadecanoyl, and R unsaturated, e.g. 9-cis-octadecenoyl (oleoyl).
  • naturally occurring phospholipid defines phospholipids which have no uniform composition with respect to R ] _ and R2. Such natural phospholipids are also lecithins and kephalins, the acyl groups RL and R2 of which are derived from naturally occurring fatty acid mixtures.
  • the requirement of "essentially pure" phospholipid of the formula (1) defines a degree of purity of more than 90 % By weight, preferably more than 95% by weight. of the phospholipid of the formula (1), which can be detected using suitable determination methods, for example paper or thin-layer chromatography, using HPLC or an enzymatic staining test.
  • R 3 with the meaning C ⁇ -C ⁇ j alkyl, for example methyl or ethyl.
  • the ⁇ meaning methyl is preferred.
  • R with the meanings substituted by one or more carboxy, hydroxyl or amino groups C - ⁇ - C ⁇ alkyl are, for example, 2-hydroxyethyl, 2, 3-dihydroxy-n-propyl,
  • Carboxymethyl 1- or 2-carboxyethyl, dicarboxymethyl, 2-carboxy-2-hydroxyethyl or 3-carboxy-2, 3-dihydroxy-n-propyl, 3-amino-3-carboxy-n-propyl or 2-amino-2 -carboxy- n-propyl, preferably 2-amino-2-carboxyethyl.
  • Phospholipids of formula (1) with these groups can be used in salt form e.g. as sodium or potassium salt.
  • Phospholipids of the formula (1), in which R3 denotes the inositol or the glyceryl group, are known under the names phosphatidylinositol and phosphatidylglycerol.
  • acyl radicals in the phospholipids of the formula (1) the names given in parentheses are also common: 9-cis-dodecenoyl (lauroleoyl), 9-cis-tetradecenoyl (myristoleoyl), 9-cis-hexadecenoyl (palmitoleoyl), 6-cis - octadecenoyl (petroseloyl), 6-trans-octadecenoyl (petrose-laidoyl), 9-cis-octadecenoyl (oleoyl), 9-trans-octadecenoyl (elaidoyl), 9, 12-cis-octadecadienoyl (linoleoyl), 9.12, 15-cis-octadecatrienoyl (linolenoyl), 11-cis-octadecenoyl (
  • a salt of the phospholipid of formula (1) is acceptable. Salts are defined by the existence of salt-forming groups in the substituent R3 and by the free hydroxyl group on the phosphorus. The formation of internal salts is also possible. Alkali metal salts, in particular sodium salts, are preferred.
  • purified lecithin from soybeans of the quality LIPOID S 100 or S75 or a lecithin defined in the monograph of USP23 / NF 18 is used.
  • Component (a) is preferably used in a concentration of approximately 0.1 to 30% by weight, based on the total weight of components (a), (b) and (c).
  • An emulsifier or emulsifier mixtures which preferably form O / W structures are preferably used as component (b).
  • Particularly preferred emulsifiers are:
  • Alkali, ammonium and aminium salts of fatty acids are lithium, sodium, potassium, ammonium, triethylamine, ethanolamine, diethanolamine or triethanolamine salts.
  • the sodium, potassium or ammonium (NR2R2R3) salts are used, where R ⁇ , R and R3 independently of one another are hydrogen, C ⁇ -C ⁇ alkyl or C ⁇ -C ⁇ hydroxyalkyl.
  • Saturated and unsaturated alkyl sulfates such as e.g. Sodium docecyl sulfate and alkane sulfonates such as e.g. Sodium docecansulfona; Salts of bile acid such as Sodium cholate, sodium glycocholate and sodium taurocholate;
  • Invert soaps such as zetylpyridinium chloride; Partial fatty acid esters of sorbitan such as sorbitan monolaurate; Sugar esters such as sucrose monolaurate; Alkyl glucosides, such as n-octyl glucoside or n-dodecyl glucoside;
  • Alkyl maltosides such as n-dodecyl maltoside
  • Fatty acid partial glycerides such as e.g. Lauric acid monoglyceride;
  • Polyglycerol esters of fatty acids - propylene glycol esters of fatty acids;
  • Lactic acid esters of fatty acids such as Sodium stearoyl lactyl-2-lactate
  • Emulsifiers of the polyoxyethylene type are very particularly preferred. Examples of such emulsifiers are:
  • Polyethoxylated sorbitan fatty acid esters such as e.g. Polysorbate 80;
  • Vegetable oils such as polyoxyethylene glycolated natural or hydrogenated castor oils
  • Polyethoxylated fatty acid partial glycerides such as e.g.
  • Diethylene glycol monostearate - Polyethoxylated fatty alcohols such as Oleth-20,
  • Polyethoxylated fatty acids such as Polyoxyl 20 stearate,
  • Polyethoxylated lanolin and its derivatives such as e.g.
  • Block polymers of ethylene oxide and propylene oxide e.g. Poloxamer 188
  • Component (b) is present in the NanoCell used according to the invention in a concentration of approximately 1 to approximately 50% by weight, based on the total weight of components (a), (b) and (c).
  • Component (c) is preferably a natural, a synthetic or a partially synthetic di- or triglyceride, a lipophilic substance customary for culture media or mixtures of these substances.
  • suitable substances are e.g. B. essential, the growth of cells, fungi, bacteria, ⁇ viruses, bacteriophages, insects, plants, tissues, etc. supporting substances such as vitamins, amino acids, peptides, natural or recombinant proteins, carbohydrates, lipids, nucleic acids, ribonucleosides, deoxyribonucleosi- de, inorganic salts and trace elements; also natural, semi-synthetic or synthetic active ingredients such as antioxidants, antibiotics, antifungals, hormones; Mixtures of substances for screenings and markers for the investigation of cell biological functions.
  • vitamins are vitamin A, vitamin B and vitamin B 2 , vitamin B 6 , nicotinamide, pantothenic acid, vitamin B 12 , vitamin B 15 , folic acid, biotin, vitamin C, vitamin D, vitamin E, vitamin F, vitamin K and vitamin P.
  • amino acids and peptides are alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, Histidine, glycyl-L-glutamine, L-alanyl-L-glutamine and hydroxy-L-proline.
  • EGF epidermal growth factor
  • KGF keratinocyte growth factor
  • aFGF and bFGF acidic and basic fibroblast growth factor
  • IGF-I insulin-like growth factor-I and II
  • IGF-II Nerve Growth Factor
  • PDGFs Platelet-Derived Growth Factors
  • Chemotactic factors such as macrophage / monocyte chemotactic and activating factor (MCAF); Colony-stimulating factors such as granulocyte macro phage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF); Interferons and interleukins; Cell adhesion and extracellular matrix proteins such as collagens, fibronectins, integrins, laminins, merosins, proteoglycans, RGD adhesion peptides, tenascins, thrombospondins and vitronectins and enzymes such as collagenase, dispase and trypsin. Other examples of proteins are ⁇ insulin, albumin and transferrin.
  • carbohydrates examples include glucose, galactose, fructose, sucrose, maltose, ribose, deoxyribose, trehalose, tryptose and yeastolate.
  • Examples of lipids are saturated and unsaturated fatty acids and derivatives thereof.
  • Examples of saturated fatty acids are myristic acid, palmitic acid, stearic acid.
  • Examples of unsaturated fatty acids are omega-9 fatty acids such as e.g. Oleic acid, omega-6 fatty acids such as Linoleic acid, gamma-linolenic acid, dihommo-gamma-linolenic acid and arachidonic acid and omega-3 fatty acids such as e.g. alpha linolenic acid, stearidonic acid, eicosapentaenoic acid and docosahexaenoic acid.
  • Other examples of lipids are cholesterol and derivatives thereof.
  • nucleic acids examples include adenine, guanine, thymidine, uracil, adenosine, guanosine, cytidine, uridine, 2 '-deoxyadenosine, 2' -deoxyguanosine, 2 '-deoxycytidine, 2' -deoxythymidine, 5-methyl Deoxycytidine and 5-methylcytosine.
  • inorganic salts and trace elements are CaCl 2 , KC1, MgS0 4 , NaCl, NaHC0 3 , NaH 2 P0 4 , Fe (N0 3 ), FeS0 4 , KH 2 P0 4 , MgCl 2 , (NH 4 ) (Mo 7 0 24 ), CuS0 4 , KN0 3 , ZnS0 4 , NiCl 2 , Na 2 Si0 3 , H 2 Se0 3 , MnS0 4 and Ca (N0 3 ) 2 .
  • antioxidants examples include vitamin E and derivatives, tocotrienols, vitamin A and derivatives, vitamin C, beta-carotene, carotenoids, ubiquinones such as coenzyme Q10, flavonoi de, isoflavonoids, polyphenols, phytoestrogens, lycopenes, luteins, lipoic acids, glutathione.
  • antibiotics and antifungals examples include tylocin, gentamycin sulfate, kanamycin sulfate, neomycin sulfate, nystatin, polymixin B sulfate, streptomycin sulfate, actinomycin D, penicillin G, ampicillin, carbenicillin, amphote ⁇ ricin B and Mycophenolic acid.
  • hormones examples include hydrocortisone, progesterone, dexamethasone and triamzinolone.
  • mixtures of substances for screenings are vegetable or animal extracts with bioactive ingredients.
  • markers for examining cell biological functions are fluorescent dyes, immunohistochemical markers and vital markers.
  • substances for culture media are coenzymes such as diphosphopyridine nucleotide (DPN), flavin adenine dinucleotide (FAD), triphosphopyridine nucleotide (TPN) and uridine triphosphate (UTP), coenzyme A, cocarboxylase; Peptones such as meat peptone, casein peptone, soy peptone, bacto peptone and lactalbumin hydrolyzate as well as extracts such as yeast extract, beef pituitary extract, chicken embryo extract or beef extract.
  • DPN diphosphopyridine nucleotide
  • FAD flavin adenine dinucleotide
  • TPN triphosphopyridine nucleotide
  • UDP uridine triphosphate
  • coenzyme A cocarboxylase
  • Peptones such as meat peptone, casein peptone, soy peptone, bacto peptone and lactalbumin hydrolyzate
  • extracts such as yeast extract, beef pitu
  • substances for culture media are putrescine, phenol red, fumaric acid, malic acid, alpha-keto-glutaric acid, succinic acid, lactic acid, DL-68-thiocitinic acid, para-aminobenzoic acid, HEPES, calcium lactate, sodium succinate, Na -Pyruvate, sodium glucuronate, sodium acetate, sodium mucate, D-glucuron lactone, aminopterin, hypoxanthine, xanthine, choline chloride, choline bitartrate, taurine and salmon sulfate.
  • Component (c) is used according to the invention.
  • the NanoCell used according to the invention contains, as optional component (d), a solubilizer, preferably a C 2 -C 8 alcohol, such as ethanol or propylene glycol.
  • a solubilizer preferably a C 2 -C 8 alcohol, such as ethanol or propylene glycol.
  • the NanoCell used according to the invention contains only components (b) and (c).
  • a NanoCell composition with components (a), (b) and (c) is characterized by favorable phase properties when dispersed in an aqueous medium. With opalescence and transparency in the backlight, it can only be seen from an extremely low milky haze that the dispersion still shows physical differences compared to the ideal state of a real molecular solution. Electron microscopic images show that a population of more than 98% is in a Gaussian distribution as a suspension of particles (nanoparticles) with a particle size smaller than approx. 100 nm (nanodispersion), typically smaller than approx. 50 nm. This
  • Differences compared to a real solution are tolerable due to the particularly good homogeneity properties of the dispersion, for example, due to the surprisingly high storage stability, e.g. B. no segregation after storage for several months at temperatures up to room temperature (stability expected by extrapolation longer than two years) are detectable.
  • the two components (b) and (c) are mixed, optionally with heating, to give a homogeneous liquid phase.
  • component (a) is dissolved, if necessary with the aid of a solubilizer such as ethanol.
  • a solubilizer such as ethanol
  • Stirring is carried out using conventional stirrers, such as propellers, inclined blades or magnetic stirrers. Due to the special selection of components (a), (b) and (c), ultrafine, monodisperse NanoCells are created immediately.
  • rotor-stator or ultrasonic homogenizers which have high shear and cavitation can ⁇ to a homogenization by means of nozzles, generate, be dispensed with.
  • NanoCell pre-phase to the aqueous phase is usually carried out at a temperature ⁇ 50 ° C.
  • the NanoCells characterized by the described production process have an average particle diameter of less than 100 nm, typically less than 50 nm.
  • the particle distribution is monodisperse and obeys a Gaussian distribution.
  • Laser light scattering measurements and electron microscopic examinations (Cryo-TEM) confirm the very small size and excellent homogeneity of the NanoCells.
  • the NanoCells are used in end products of culture media.
  • These culture media end products are preferably liquid or semi-solid, but can also be solid.
  • culture media end products are animal sera such as fetal bovine serum, serum from newborn calf, calf serum, sera from other animal species, serous liquids from bovine; synthetic culture media such as serum-free / serum-reduced media, minus media for radioactive installation studies, media for molecular and cytogenetics, selection media, media for the plaque assay, media for the detection of mycoplasma, media for freezing, media for tissue, organ and Embryo transfer, media for therapy, media for perfusion of organs, media for storing organs until their transplantation, media for pharmacological logical, toxicological and metabolic studies, media for absorption and transport studies, media for the investigation of functional and regulatory mechanisms, supplements such as enzyme, amino acid and vitamin solutions; Lipid concentrates, growth stimulating additives, serum replacement products containing growth factors, vitamins, binding proteins, hormones, adhesion factors and trace elements, bouillons, peptones such as meat peptone, casein peptone, soy peptone, bacto peptone and lac
  • the NanoCells are incorporated into the aqueous portion of the end product.
  • the incorporation of the NanoCells is preferably carried out with stirring at room temperature, with conventional stirrers such as Propeller, inclined blade or magnetic stirrers are used.
  • the corresponding NanoCell pre-phase can also be incorporated into the aqueous portion of the culture media end products.
  • the NanoCell pre-phase is added to the aqueous portion of the end products with stirring and preferably at a temperature ⁇ 50 ° C.
  • Solid culture media end products such as Powder media
  • NanoCells are coated or loaded with NanoCells by spraying or soaking.
  • the culture media end products are preferably used for in vitro cultivation and examination of cells, fungi, bacteria, viruses, insects and plants, for in vitro or ex vivo cultivation and examination of tissues and organs, for freezing organs, tissues, cells, Fungi, bacteria, viruses, insects and plants, for the transfer of organs, tissues and embryos, for therapy ⁇ (adoptive immunotherapy, cancer treatment), for perfusion of organs such as kidneys or liver, for the storage of organs until their transplantation, for cytogenetic , molecular genetic, pharmacological, toxicological and metabolic studies, for admission and transport studies or for the investigation of functional and regulatory mechanisms.
  • NanoCell Polysorbate 80 and Miglyol 812 are mixed to a homogeneous liquid phase. The soy lecithin dissolved in ethanol is added to this phase. This results in a homogeneous solution, which is added to the phosphate buffer with stirring. The NanoCell obtained is then sterile filtered through a 0.2 ⁇ m filter.
  • Example 2 Egg lecithin 1.4% NanoCell
  • the NanoCell is produced in an analogous manner, for example 1.
  • NanoCell Polysorbate 80, Miglyol 812 and Vitamin E are mixed to a homogeneous liquid phase.
  • the soy lecithin dissolved in ethanol is added to this phase. This results in a homogeneous solution, which is added to the phosphate buffer with stirring.
  • the NanoCell obtained is then sterile filtered through a 0.2 ⁇ m filter.
  • Vitamin E acetate 2.00% soybean lecithin 0.49%
  • NanoCell is produced in an analogous way, for example 3.
  • Example 5 Vitamin E acetate 1.2% NanoCell
  • Vitamin E acetate 1.20%
  • Polysorbate 80 mixed to a homogeneous liquid phase. ⁇ The resulting clear solution is added to the water with stirring. The NanoCell obtained is then sterile filtered through a 0.2 ⁇ m filter.
  • Vitamin A palmitate (1.7 million IU / g) 1.80%
  • Vitamin E acetate 0.10%
  • Miglyol 812 1.80% polysorbate 80 3.15%
  • the NanoCell is produced in an analogous way, for example 3.
  • NanoCell is produced in an analogous way, for example 3.
  • Example 8 Omega-3 fatty acids 3.5% NanoCell
  • Vitamin E acetate 0.60%
  • the omega-3 fatty acids (Incromega E3322, CrodA), vitamin E acetate, polysorbate 80 and soy lecithin are mixed to form a homogeneous liquid phase.
  • the resulting clear solution is added to the phosphate buffer with stirring.
  • the NanoCell obtained is then sterile filtered through a 0.2 ⁇ m filter.
  • Ronoxan A (Röche) 0.02% 10 mM phosphate buffer, pH 6 ad 100.00%
  • NanoCell To produce the NanoCell, Miglyol 812, Polysorbate 80, Propylene Glycol, Ronoxan A and Soy Lecithin are mixed to a homogeneous liquid phase.
  • the coenzyme Q10 is added to this mixture with stirring and heating. This results in a clear liquid that is added to the phosphate buffer, which was previously heated to 50 ° C, with stirring (e.g. magnetic stirrer).
  • the NanoCell obtained is then sterile filtered through a 0.2 ⁇ m filter.
  • NanoCells The particle sizes of NanoCells are summarized in Table 1 below:
  • NanoCells also have excellent storage stability (Table 2) g:
  • the particle diameter and the particle size distributions were determined by means of laser light scattering (Nicomp 370 Submicron Particle Sizer, Volume weighting).
  • the content of vitamin E acetate was determined by means of HPLC Examples of culture media end products containing NanoCells
  • Example 10 Culture medium end product containing vitamin E acetate NanoCells
  • Vitamin E acetate Microvascular endothelial cells of the HMEC-1 cell line were grown to confluence, then washed once and incubated in a medium of the following composition:
  • Vitamin E acetate NanoCell according to example 4 0.0575%
  • the cellular amount of vitamin E was determined.
  • the cells were removed using
  • Vitamin E acetate Microvascular endothelial cells of the HMEC-1 cell line were grown to confluence, then washed once and incubated in a medium of the following composition for 96 hours: Vitamin E acetate NanoCell according to example 4 0.0575%
  • Vitamin E acetate was used to determine the free radicals generated in natural metabolism using a fluorometric method.
  • the cells preincubated in the above medium for 96 hours were loaded with a non-fluorescent dye (carboxy-H 2 DCFDA / AM, Molecular Probes) which reacts intracellularly with free radicals, in particular hydroperoxides, and thereby becomes fluorochrome.
  • the resulting relative fluorescence is a measure of the amount of radicals released.
  • the relative fluorescence was measured over a period of 150 minutes.
  • the results shown in Figure 2 show that the vitamin E acetate NanoCells is a very good antioxidant
  • Vitamin E acetate was added to the medium not in the form of the NanoCell, but as a solution in ethanol or DMSO, as a solubilizate in fetal calf serum or as a cyclodextrin complex.
  • the cell suspensions obtained were then supplemented with 5, 10, 25, 50 and 75 ⁇ M vitamin E acetate, in the form of the vitamin E acetate NanoCells according to Example 4, and incubated for 96 hours.
  • the cytotoxic effect was associated with
  • Microvascular endothelial cells of the HMEC-1 cell line were grown to confluence, then washed once and taken up in a medium of the following composition:
  • Example 11 Culture medium end product containing vitamin A palmitate NanoCells
  • Skin fibroblasts cellular uptake of vitamin A palmitate
  • Human dermal fibroblasts of the cell line HFP-1 were grown to confluence used, then washed once and incubated in egg ⁇ nem medium of the following composition:
  • Vitamin A palmitate NanoCell according to example 6 0.0095% 1% penicillin / streptomycin 0.0050%
  • the cellular amount of vitamin A palmitate was determined.
  • the cells were harvested at the times indicated, washed, extracted with hexane and the concentration of vitamin A palmitate in the hexane extract was determined by means of HPLC. The pellets remaining after the hexane extraction were used for DNA determination.
  • the results shown in FIG. 4 show that vitamin A palmitate is absorbed into the cells quickly and efficiently from the above medium.
  • the cellular vitamin A palmitate concentrations are given in an analogous experiment in which the same amount of vitamin A palmitate was added to the medium not as a NanoCell but as a cyclodextrin complex.
  • Bronchial epithelial cells Cellular uptake of vitamin A palmitate
  • the cellular amount of vitamin A palmitate was determined.
  • the cells were harvested at the specified times, washed, extracted with hexane and the concentration of vitamin A palmitate in the hexane extract was determined by means of HPLC. The pellets remaining after the hexane extraction were used for DNA determination.
  • the results shown in FIG. 5 show that vitamin A palmitate is rapidly and extremely efficiently absorbed into the cells from the above medium.
  • the cellular vitamin A palmitate concentrations are given in an analogous experiment in which the same amount of vitamin E was added to the medium not as a NanoCell but as a cyclodextrin complex.
  • Figure 1 Cellular intake of vitamin E resp. Vitamin E acetate from media, containing different vitamin E_ resp. Vitamin E acetate supplements.
  • the amounts of vitamin E taken up by the cells are expressed as pmol / ⁇ g DNA. The values given are mean values from four independent tests.
  • Figure 2 Antioxidative capacity of various vitamin E resp. Vitamin E acetate supplements against intracellularly generated radicals. The values given are mean values +/- SD from four independent tests.
  • Figure 3 Cytotoxicity of vitamin E acetate NanoCells against HMEC-1 cells. The values given are mean values from four independent tests.
  • Figure 4 Cellular uptake of vitamin A palmitate from media containing vitamin A palmitate NanoCells or vitamin A palmitate cyclodextrin complexes. The amount of vitamin A absorbed by the skin fibroblasts (HFP-1)
  • Palmitate are expressed as ⁇ mol / ⁇ g DNA. The values given are mean values +/- SD from three independent tests.
  • Figure 5 Cellular uptake of vitamin A palmitate from media containing vitamin A palmitate NanoCells or vitamin A palmitate cyclodextrin complexes.
  • the amounts of vitamin A palmitate taken up by the bronchial epithelial cells (NHBE) are expressed as ⁇ mol / ⁇ g DNA.
  • the values given are mean values +/- SD from three independent tests.

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Abstract

Utilisation d'une nanocellule qui contient, en tant que composition de préparation, (a) une molécule formant membrane, (b) un co-émulsifiant et (c) un constituant lipophile, dans des produits finis sous forme de milieux de culture. Les nanocellules selon la présente invention sont non toxiques et sont appropriées en tant qu'agents de solubilisation pour des substances lipophiles présentes dans des produits finis sous forme de milieux de culture.
EP99939894A 1998-09-14 1999-09-08 Utilisation de nanocellules dans des produits finis sous forme de milieux de culture Withdrawn EP1112346A1 (fr)

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CH186398 1998-09-14
CH186398 1998-09-14
PCT/CH1999/000420 WO2000015763A1 (fr) 1998-09-14 1999-09-08 Utilisation de nanocellules dans des produits finis sous forme de milieux de culture

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US6939376B2 (en) 2001-11-05 2005-09-06 Sun Biomedical, Ltd. Drug-delivery endovascular stent and method for treating restenosis

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CA2130357A1 (fr) * 1992-02-18 1993-08-19 Doron Friedman Compositions seches pour la preparation d'emulsions submicroniques
ES2094688B1 (es) * 1994-08-08 1997-08-01 Cusi Lab Manoemulsion del tipo de aceite en agua, util como vehiculo oftalmico y procedimiento para su preparacion.
EP0866689A1 (fr) * 1995-12-12 1998-09-30 Vesifact Ag Pulverisateur d'hydrocortisone pour administration topique
ATE240088T1 (de) * 1996-12-13 2003-05-15 Vesifact Ag Kosmetische präparate in form einer nanodispersion

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