EP0781294A1 - Polypeptides de ribonucleotides contenant des metaux - Google Patents

Polypeptides de ribonucleotides contenant des metaux

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Publication number
EP0781294A1
EP0781294A1 EP96926996A EP96926996A EP0781294A1 EP 0781294 A1 EP0781294 A1 EP 0781294A1 EP 96926996 A EP96926996 A EP 96926996A EP 96926996 A EP96926996 A EP 96926996A EP 0781294 A1 EP0781294 A1 EP 0781294A1
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EP
European Patent Office
Prior art keywords
rnp
culture
solution
leukocytes
cells
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.)
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Application number
EP96926996A
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German (de)
English (en)
Inventor
Josef Wissler
Enno Logemann
Stefan Kiesewetter
Ludwig Heilmeyer
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Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Original Assignee
Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
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Publication of EP0781294A1 publication Critical patent/EP0781294A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to metal-containing ribonucleotide polypeptides (RNP) and to a process for their preparation, their use and medicaments which contain ribonucleotide polypeptides or antibodies against ribonucleotide polypeptides and / or their molecular biological equivalence structures and / or parts and / or derivatives.
  • RNP metal-containing ribonucleotide polypeptides
  • Tissue homeostasis of the body depends on the regulatory mechanisms of angiogenesis (lateral and directional growth of the blood vessel capillaries). It influences tissue repair and wound healing, new tissue formation in embryogenesis and reproductive cycles as well as the growth, regression and destruction of tumors, transplants and vascularized and vascular-free tissues. So far, no non-mitogenic mediators have been found that can influence tissue homeostasis, ie induction and regulation of vascular growth.
  • the object of the invention is also to provide a method for producing the non-mitogenic mediators and a medicament which contains this non-mitogenic mediator.
  • bioactive ribonucleotide polypeptide according to claim 1, by a method according to claim 5 or 26, by a medicament according to claim 28 or 29 and by the use according to claim 30 or 31.
  • non-mitogenic cellular mediators based on nucleic acids with a defined sequence which can specifically cause the formation of blood vessels in vivo and in vitro and which are biologically specific, naturally acting non-mitogenic mediators of angiogenesis or the directional growth of blood vessel shoots.
  • the new class of cellular morphogens for endothelial cells which the inventors have demonstrated for the first time, is in the form of bioactive metal ribonucleotide peptides (RNP).
  • RNP bioactive metal ribonucleotide peptides
  • the metal can preferably be calcium, copper or zinc.
  • RNA part they contain the following sequence of the nucleotides or parts or derivatives thereof:
  • sequences are to be understood to include RNPs in which nucleotides and / or amino acids have been exchanged for the sequences shown above in the RNA part and / or protein part or that only parts of the above sequences are present.
  • the invention also relates to DNA coding for the above-mentioned amino acids, the DNA comprising:
  • RNA part The nucleotides contained in the RNA part were translated into DNA:
  • the RNP according to the invention are further characterized by the following properties: a) biological effects in vitro and in vivo:
  • Zn / Cu / Ca-RNP can develop regulated and selective nuclease effects and their mutual bioactivity can be modulated and regulated via Zn / Cu / Ca metal ion levels as "molecular switches”.
  • RNA ribonucleic acid
  • Acrylamide matrices anodic or as a wide distribution over the entire running distance (anomalous hydrodynamic behavior);
  • RNA part of the RNP contains modified bases in the RNA part of the RNP, at least one of which can be represented by isoguanosine;
  • the native protein consists of only one peptide unit (smallest common multiple of the RNP unit);
  • the bioactive RNPs of the invention are cellular inflammation and wound healing mediators with topobiochemically and biologically specific activity. Its biological task is to induce and regulate non-mitogenic vascular growth. This can lead to tissue neovascularization. They arise in vitro during the culture of leukocytes or in vivo when leukocytes accumulate at the site of inflammation in addition to a variety of other hormones and mediators.
  • bioactive RNP morphogens produced and obtained according to the invention are valuable endogenous active substances. They can be used, for example, to influence the vascular status of tissues (e.g.
  • Cardiac muscle tissue, skeletal muscle tissue, lungs, wound healing, reproductive cycles, embryogenesis and transplants can be used.
  • Another possible use is the production of inhibitors of undesired angiogenesis and neovascularization of tissues in the case of pathological symptoms in tuberculosis, diabetes, tumors, reproductive cycles and tissue transplantations.
  • paracrine mediators they can transfer genetic information from cell to cell ("shuttle”). They can also be used in genetic engineering for the transmission of genetic information with an optimized nucleic acid content (“horizontal transfer”) and with enzymatic effects (nuclease activity) to influence the nucleic acid content and the cellular functions (eg differentiation).
  • the bioactive RNP morphogens of the invention can be administered individually or as a mixture in the form of customary drugs locally in mammals, for example humans, in an amount of> 1 fmol in a concentration of> 10 pmol / l.
  • the threshold dose of activity in vivo is> 50 fmol, preferably 2.5 fmol.
  • These drugs are useful for specifically influencing angiomorphogenesis and vascular condition of tissue in a mammalian body.
  • these medicaments can also contain at least one anti-RNP immunoglobulin and / or molecular biological equivalence structures.
  • the invention also relates to a method for producing and obtaining the bioactive RNP morphogens of the cells of the reticulo-endothelial system, the leukocytes and the inflammatory tissue, which is characterized in that the cells, e.g. Leukocytes or inflammatory tissue are homogenized or leukocytes are cultivated and the resulting RNP morphogens are obtained from the homogenates or from the supernatant culture solution.
  • the cells e.g. Leukocytes or inflammatory tissue are homogenized or leukocytes are cultivated and the resulting RNP morphogens are obtained from the homogenates or from the supernatant culture solution.
  • the culture of the cells can in principle be carried out in any medium obtained from the cells (leukocytes).
  • serum for example calf serum or horse serum
  • serum-containing culture solution is to be worked up on proteins (mediators) which are generated by the culture, the production of the product proteins, which are usually only present in low concentrations, is very difficult because of the large number of foreign proteins originating from the serum.
  • the fully synthetic cell culture medium preferably used according to the invention contains the usual groups of substances, such as salts, sugars, amino acids, nucleosides and nucleoside bases, vitamins, vitaminoids, coenzymes and / or steroids in aqueous solution. It is characterized in that it additionally contains one or a mixture of several substances which prove to be particularly valuable for the viability and growth of the leukocytes and their ability to produce mediators. These substances include unsaturated fatty acids, flavonoids, ubiquinones, vitamin C and mevalolactone.
  • the cell culture medium is used for long-term cell or leukocyte culture, preferably without the addition of serum. Instead, it receives at least one defined protein, which in a particularly preferred embodiment is high-purity, molecularly uniform serum albumin.
  • the fully synthetic serum-free cell culture medium used according to the invention can contain further compounds from the substance classes of polyhydroxy compounds and sugars, amino acids, nucleosides, anionic compounds and / or vitamins, which are beneficial for the culture of leukocytes, the use of which is not common in the known culture media , contain.
  • the proportions of the constituents of the medium used according to the invention are adjusted to one another in such a way that the concentration of the components in the medium are largely adapted to the natural concentration ranges of the plasma; see. Ciby-Geigy AG (Editor) (1969) in Documenta Geigy, Scientific Tables, 7th edition Geigy SA, Basel.
  • the cell culture medium is preferably free of surfactants, heavy metal salts and dyes, which damage the cells and can disrupt the extraction of the desired cell products from the culture solution.
  • the culture of the cells / leukocytes in the method of the invention is the cell culture medium with the composition given in Table 1 below.
  • Water with ATM-1 quality is used to produce the medium; see. ASTM D-1193-70 Standard Specification for Reagent Water 1970; Annual Book of ASTM Standards, Easton Maryland, ASTM 1970. It is also exempt from possible endotoxin contamination by ultrafiltration on surfactant-free membranes with an exclusion limit of 10,000 daltons.
  • the finished medium is filter-sterilized on surfactant-free membranes with a pore size of ⁇ 0.2 ⁇ m.
  • either mixed cell / leukocyte populations or individual cell / leukocyte types are cultivated.
  • the production and culture of the cells or leukocytes must be carried out under sterile conditions. The culture is carried out for a sufficiently long time to obtain a satisfactory mediator yield.
  • a duration of about 10 to 50 hours has proven to be suitable for this. In the case of shorter times, the mediator yield is too low, so that the process is uneconomical. On the other hand, if the culture lasts about 50 hours, the medium is exhausted and the cells begin to die, so that an increase in the yield can no longer be expected.
  • the culture of the cells or leukocytes is carried out at a temperature of approximately 30 to 42 ° C., preferably approximately 37 ° C.
  • the culture process is unsatisfactory at lower temperatures, while at temperatures above 42 ° C the cells or leukocytes are damaged.
  • the culture is carried out at a concentration of about 10 6 to 5 ⁇ 10 8 cells / ml, preferably up to 10 7 to 10 8 cells / ml. At lower cell concentrations, the yield per unit volume of the culture solution is too low. The process becomes uneconomical due to excessive culture volumes. At cell concentrations above 5 ⁇ 10 8 cells / ml, the medium becomes very poor in nutrients.
  • the culture can be carried out in the atmosphere.
  • An increased carbon dioxide paral pressure which ranges up to about 10% by volume, in particular up to about 2% by volume, is preferably maintained via the culture can.
  • the oxygen supply to the culture is of great importance. It can be ensured, for example, by introducing air.
  • the air supplied is preferably sterilized and heat-decontaminated, ie freed of endotoxins and other organic components.
  • the solution can be stirred or shaken during culture.
  • a lectin preferably from Canavalia ensiformis (Con A), is preferably used as the cell stimulant.
  • the cells or leukocytes are centrifuged off from the culture solution, which is then worked up on the resulting mediators.
  • the culture is accelerated at a relatively low rate, i.e. about 300 to 400 x g, centrifuged.
  • the supernatant is expediently centrifuged again at higher acceleration in order to remove any remaining suspended particles.
  • the separated cells or leukocytes can either be re-cultivated, cryopreserved or used for another purpose.
  • the bioactive RNP morphogens of the invention can also be obtained from inflammatory tissue. There they arise from the accumulation of leukocytes as a result of the inflammatory process triggered by the tissue damage.
  • the inflammatory tissue can be obtained in a conventional manner and used for the preparation of the RNP. For this purpose, the inflammatory tissue is homogenized in buffer solution and the soluble components (exudate) are separated from the insoluble structural components of the fabric.
  • Inflamed infarcted myocardial tissue is preferably used, which is formed by ligation of the left anterior descending branch of the left coronary artery using a transfemoral catheter technique for 24 hours.
  • the inflamed heart muscle part containing leukocytes is separated from non-infarcted, healthy tissue at 0 to 4 ° C.
  • the culture solution contains the mixture of the components of the medium.
  • the proteins formed are separated from the components of the medium and at the same time from the large volume of the aqueous solution. This can be achieved by selectively salting out the proteins from the culture solution, which is achieved, for example, by adding a sulfate or phosphate. The precipitation of the proteins is described below using the example of salting out by adding ammonium sulfate to the culture solution.
  • the majority of the resulting proteins are extracted together with any serum albumin that may be present falls.
  • the substance deposit has been separated off, for example by centrifuging, it can be separated into its individual components in the manner described below and the bioactive RNP contained can be obtained.
  • the supernatant obtained also contains the part of the substances which is soluble in saturated ammonium sulfate solution, including part of the bioactive RNP. The supernatant is concentrated and the substances contained are obtained therefrom in the following manner. If the protein-containing culture solution is mixed with ammonium sulfate until saturation, the greater part of the accompanying proteins precipitates.
  • the protein mixture contained in the culture solution is therefore already separated into several fractions in the precipitation stage.
  • This separation into several protein fractions is possible because the individual proteins are precipitated at different ammonium sulfal concentrations.
  • the culture solution in the process of the invention is therefore gradually mixed with ammonium sulfate up to certain degrees of saturation, the fraction of the protein precipitating in each fraction whose solubility product is below the respective degree of saturation.
  • the culture solution is first mixed with ammonium sulfate up to a saturation of 35%.
  • the protein precipitate obtained is separated off.
  • the degree of saturation of the supernatant solution is then increased to 45%.
  • a protein precipitate forms again, which is separated off.
  • the supernatant solution is then adjusted to a degree of saturation of 90%.
  • the protein precipitate obtained is separated off at life.
  • the supernatant solution of this precipitation is concentrated, for example, by dewatering dialysis or ultrafiltration.
  • the salt precipitation of the proteins is preferably carried out at a temperature of approximately 0 to 10 ° C., in particular approximately 0 to 4 ° C.
  • the solutions used for cleaning have a pH between 5 and 9, in particular between 6 and 8.
  • a strong buffer e.g. 0.1 mol / l phosphate buffer added.
  • cysteine is preferably added to the solutions in an amount of 0.001 mol / l. Sterile conditions for protein purification are not required.
  • the proteins obtained in the salt precipitation can, after being dissolved in a medium which is not harmful to proteins, be fed directly to the purification and separation described below.
  • the supernatant from the last precipitation stage is concentrated, for example by dewatering dialysis or ultrafiltration. All compounds with a molecular weight of greater than about 300 to 500 Dal tons, ie also the proteins and peptides of this fraction, obtained quantitatively as retentate.
  • the protein fractions obtained in the step described above contain the bioactive RNP of the invention in a mixture with numerous foreign proteins (other secreted proteins, optionally serum albumin and optionally CON).
  • the foreign proteins are present in the mixtures in a predominant amount.
  • the bioactive RNP must be enriched and freed from the foreign proteins to such an extent that they no longer interfere with their molecular biological specificity.
  • the bioactive RNP itself is also a class of substances that is broken down into its individual, specific-acting individuals.
  • cleaning processes for protein bodies (proteins) and other natural products consist of a sequence of combined separation processes, which differ in molecular size, charge, shape, structural stability and molecular surface texture
  • purification steps which are known per se in biochemistry.
  • purification steps are: preparative and analytical molecular sieve filtration, anion and cation exchange chromatography, or one-pot adsorption process, chromatography on hydroxylapa tit, zone precipitation chromatography and circulatory or cascade molecular sieve filtration.
  • a considerable amount of accompanying proteins can be separated from the bioactive RNP simply by performing one of the cleaning processes mentioned once.
  • the substances contained in the fractions often adhere very strongly to one another despite their different molecular weights.
  • molecular sieve filtration for example, they are often incompletely separated according to their molecular weight due to the existence of less than ideal equilibria in protein polyelectrolytes. It is therefore advisable to carry out at least two of the separation processes mentioned in succession.
  • the protein fractions containing bioactive RNP are preferably subjected to at least three of the cleaning steps mentioned in succession.
  • Molecular sieve filtration causes the proteins to be separated according to their molecular weight. Since a predominant part of the accompanying foreign proteins have a different molecular weight than the bioactive RNP has their separation can be achieved in this way.
  • a hydrophilic, water-swelling molecular sieve is used to separate the substances according to their molecular weight. Examples of suitable molecular sieves are dextrans crosslinked with epichlorohydrin (Sephadex), agaroses crosslinked with acrylamide (Ultrogele) and crosslinked acrylamides (biogels), the exclusion limits of which are greater than the separation limits used for the separation.
  • Separation stages are used, preferably carried out as one of the first.
  • molecular sieve filtration is referred to as "preparative” or “analytical”. It is referred to as “preparative” if the chromatography is carried out on columns with a length: diameter dimension ratio of up to 10: 1 and a load of up to 1/3 of the column content or with full utilization of the total, typical matrix separation volume.
  • “Analytical” means a length-diameter ratio of more than 10: 1, preferably about 50: 1, and a load of up to 3% of the column content, even in HPLC versions.
  • gel matrices with the largest possible particle size are used in order to achieve rapid flow rates of the often somewhat viscous protein solutions at the lowest possible pressures.
  • particle size of the gel matrix is chosen to be as small as possible in order to achieve a maximum theo to achieve a reticulated bottom number of the column with technically and safety usable pressure and a flow speed of the mobile phase of 2 to 4 cm / h. These parameters depend on the gel matrix structure and differ from gel to gel.
  • the separation limit can be selected in steps.
  • An analytical molecular sieve filtration with correspondingly graduated separation limits can then be carried out.
  • the exclusion limit of the gel used must be greater than about 10,000 daltons in order to allow a volume distribution of the RNP between the stationary gel matrix phase and the mobile aqueous buffer phase.
  • the substances are applied to the molecular sieve in solution in a liquid which does not damage the substances.
  • a more specific example of a suitable solvent is 0.003 mol / l sodium potassium phosphate solution with a content of 0.3 mol / l NaCl and 0.001 mol / l cysteine and a pH of 7.4.
  • a suitable solvent is 0.003 mol / l sodium potassium phosphate solution with a content of 0.3 mol / l NaCl and 0.001 mol / l cysteine and a pH of 7.4.
  • the anion exchangers for the purification of the substances are suitable, for example, with dextran (Sephadex) crosslinked with epichlorohydrin or cellulose matrices to which functional groups with anion exchange capacity are coupled. They can be used repeatedly after regeneration.
  • a weak anion exchanger in the Cl form such as DEAE-Sephadex-A 50, which has been pre-swollen and equilibrated in a buffer solution, is preferably used, and the treatment is carried out at a pH of 8 to 10.
  • a specific example of such a buffer solution is 0.01 mol / l Tris-HCl, which contains 0.04 mol / l NaCl and 0.001 mol / l cysteine and has a pH of 8.0.
  • the substance fraction is added to such an amount of anion exchanger that is sufficient for the complete adsorption of the RNP and the positively adsorbing accompanying proteins.
  • the reaction can be designed either as a chromatography process or as a more manageable one-pot adsorption process.
  • the supernatant liquid with the negatively adsorbed proteins is separated from the anion exchanger loaded with the positively adsorbed RNP and other substances, for example by filtering (in the chromatography column), decanting or centrifuging (in the one-pot process).
  • the loaded anion exchanger is freed from adhering, negatively adsorbed compounds by washing with water or a salt solution which has a maximum ionic strength equivalent to 0.04 mol / l NaCl, preferably at most about 15 ° C.
  • a salt solution suitable for washing out is the mentioned Tris-HCl buffer solution with a pH of 8.0.
  • the anion exchanger freed from negatively adsorbed compounds and loaded with RNP and other substances, is then eluted with an aqueous salt solution which does not damage proteins and which has an ionic strength corresponding to greater than 0.04 mol / l NaCl and a pH between 4.0 and 10.
  • a high ionic strength salt solution with a pH of 5.0 to 7.0 is used.
  • a specific example of such a salt solution is a 2.0 mol / l NaCl solution which is buffered with 0.01 mol / l piperazine-HCl with a pH of 6.5 and which contains 0.001 mol / l cysteine.
  • the anion exchange reaction is designed as a chromatography process
  • the RNP and other substances can also be eluted by means of a linear NaCl concentration gradient.
  • Suitable cation exchangers for cleaning the protein fraction are, for example, dextran (Sephadex) or cellulose matrices crosslinked with epichlorohydrin, to which functional groups with a cation exchange capacity are coupled. They can be used repeatedly after regeneration.
  • a weakly acidic cation exchanger in the Na + form such as CM-Sephadex C-50, is preferably used, and the treatment at a pH of 4 performed to 6.
  • the substance fractions can be diluted with a non-protein-damaging saline solution, which has a maximum ionic strength equivalent to 0.04 mol NaCl / liter, to facilitate the adjustment of the load equilibrium before the treatment with the cation exchanger. It can also be used to adjust the pH.
  • a specific example of such a salt solution is a 0.001 mol / l potassium phosphate acetate buffer solution with a content of 0.04 mol / l NaCl and a pH of 4 to 6.
  • This cation exchange reaction can be used both as a chromatography process and as a technically easy to use one-pot process be allowed.
  • the cation exchanger is added to the substance fraction in an amount sufficient to adsorb the protein fraction. Usually about 2 parts by volume of swollen ion exchangers per part by volume of protein fraction are sufficient.
  • the supernatant liquid is then separated from the cation exchanger loaded with the substances, for example by decanting or centrifuging.
  • the loaded cation exchanger is adhered by washing with water or a salt solution which has a maximum ionic strength equivalent to 0.04 mol / l NaCl, preferably at a pH of about 4 to 6 and a temperature of preferably at most about 15 ° C , non-adsorbed compounds.
  • a specific example of a salt solution suitable for washing out is the aforementioned potassium phosphate acetate buffer solution with a pH of 5.0.
  • the cation exchanger is freed from negatively adsorbed compounds and loaded with the substances now eluted with an aqueous salt solution which does not damage proteins and nucleic acids.
  • a salt solution of high ionic strength with a pH of approximately 4 to 10 is preferably used.
  • Specific examples of such salt solutions are an aqueous 0.5 mol / l potassium phosphate solution with a pH of 6.5 to 7.5 or a 2 to 5 mol / l sodium chloride solution with the same pH.
  • salts which may be present from previous steps, e.g. Ammonium sulfate and above all phosphates, preferably by dialysis or ultrafiltration on a membrane with an exclusion limit of 500 daltons, removed before application to the hydroxyapatite.
  • the phosphaline concentration of the protein solution is critical for the success of the chromatography on hydroxyapatite.
  • the substances are eluted by means of a potassium phosphate concentration gradient, which is preferably linear.
  • the fractions containing RNP are collected and concentrated in the manner mentioned below.
  • hydroxyapatite is essential for the structure-conserving pure extraction of the metal-containing RNP. For technical and economic reasons, however, it is very difficult to chromatograph larger volumes of substances on hydroxylapatite columns. On the one hand, hydroxyapatite tends to become very constipated with larger substance volumes and is therefore unusable. On the other hand, hydroxyapatite is expensive, which prevents its use on a larger scale.
  • the majority of the accompanying foreign proteins are separated from the substance fractions in which the bioactive RNP is present as traces before the chromatography on hydroxylapatite by means of suitable process steps, thereby significantly reducing the volume of protein which has to be applied to the hydroxylapatite column.
  • the basic principle of protein separation by means of zone precipitation chromatography is different, structure-given reversible solubility properties of the proteins and RNP. They are among the most sensitive molecular separation parameters and were often used as a criterion for the detection of the molecular uniformity of a protein. Temperatures and pH, dimension of the column, type of salt, shape of the gradient and loading of the column can be varied within a relatively wide range.
  • the zone precipitation chromatography temperature can be about 0 to 40 ° C. A temperature range of approximately 0 to 10 ° C., in particular approximately 4 to 6 ° C., is preferred.
  • the pH can be between about 4 and 10; a pH in the range from 6 to 8, in particular about 7, is preferred.
  • the ratio of length: diameter of the column used should be greater than about 10: 1, a ratio of 30 to 100: 1, in particular about 50: 1, is preferred. All proteins and nucleic acids non-damaging salts can be used as salts. Examples of such salts are sodium potassium phosphate, ammonium sulfate and sodium sulfate. Ammonium sulfate is preferably used.
  • the salt concentration gradient can have any shape as long as the salting-out points of the proteins are separated on the route. Linear concentration gradients are preferred, in particular an increasing linear concentration gradient from 25 to 100% ammonium sulfate saturation.
  • the loading of the column is at most about 5%, preferably about 1%.
  • the loop or cascade molecular sieve filtration can be carried out under the conditions described above for analytical molecular sieve filtration.
  • the same molecular sieves and the same column conditions can be used.
  • Sephadex G 50 is preferred with a length-to-diameter ratio of the column of at least about 50: 1 and a loading of at most about 3% of the column content.
  • the solvents used for analytical molecular sieve filtration are preferably used as solvents and for elution.
  • the bioactive RNP-containing substance solutions obtained can be purified and concentrated from undesired salts for subsequent separations of the proteins / RNP.
  • This concentration (separation of the majority of the aqueous salt solution from the substances can be carried out in various ways.
  • the bioactive RNP and the accompanying substances can be concentrated by ultrafiltration or dewatering dialysis on a membrane with an exclusion limit of 500 Daltons or by lyophilization. This can also be done by molecular sieve filtration In the molecular sieve filtration, about 0.4 mol / l ammonium sulfate is preferably added to the substance solution.
  • ammonium sulfate in this concentration has a strong salting-in effect compared to proteins the proteins are kept in solution during molecular sieve filtration, and ammonium sulfate prevents bacterial growth and inhibits certain enzymes, thereby helping to stabilize bioactive RNP, especially if the chromatography is carried out at higher temperatures (above about 20 °) and under non-sterile conditions.
  • the temperature and pH conditions are not particularly critical when performing the cleaning steps. If it is intended to maintain the native conformation of the substances, it is advisable to maintain a temperature in the range from about 0 to 8 ° C, preferably about 0 to 4 ° C. Furthermore, the separation and purification steps must be carried out under essentially physiological pH and salt conditions. A major advantage of the method according to the invention is that it is easily possible to maintain these conditions for the first time. To prevent oxidation, the substance solution is preferably further mixed with about 0.001 mol / l cysteine.
  • the bioactive RNP obtained can be in a buffered physiological saline solution, for example in 0.0015 mol / l sodium potassium phosphate solution, which contains 0.15 mol / l (0.9%) NaCl and 0.001 mol / l cysteine and has a pH of 7. 4, after normal filter sterilization (0.2 ⁇ m pore size), be kept natively and biologically active at room temperature (for at least 200 hours) or frozen at -25 ° C (for at least 5 years). This stability of the bioactive RNP can be viewed as one of the criteria for its high purity.
  • the RNP according to the invention can also be produced using chemically or biologically synthesized partial sequences or parts and homologous sequences thereof. It is preferred that the chemically or biologically synthesized oligonucleotide or antisense nucleotide sequences in vivo or in vitro, which encode the partial sequences given according to claim 1, are used with at least 6 bases in the PCR reaction or the antisense bioprocess technique is used.
  • the examples illustrate the invention.
  • the extraction of the RNP morphogens is carried out described by leukocytes from pig blood.
  • the invention is not limited to this embodiment.
  • Cells from the reticulo-endothelial system or inflammatory, wound tissue or fluid (exudate) from other mammals can also be used.
  • 50 kg (about 10 14 ) leukocytes are isolated as a mixed cell population of physiological composition from 10,000 liters of pig blood and cultured in 20 batches of 2.5 kg (about 5 ⁇ 10 12 cells) under sterile conditions.
  • the medium listed in Table 1 is used as the culture solution.
  • 50 l of culture medium are used per batch.
  • the culture is carried out in glass vessels.
  • the cell density is initially 10 8 cells / ml.
  • the culture is maintained at 37 ° C in an atmosphere of 1 v / v% CO 2 for 40 hours.
  • the cell suspension is slowly stirred (60 rpm) and flooded with sterile, pyrogen-free, water-washed, fine (smaller than 1 mm) air bubbles (about 5 l air / hour), which have been heat-decontaminated in a quartz tube at about 500 ° C.
  • the pH value (7.1) and the D-glucose level are measured and kept constant.
  • the cells are stimulated during the culture by the polyvalent lectin (CON) in the culture medium.
  • CON polyvalent lectin
  • the number, differential and morphological viability (dye exclusion test) of the cells are continuously measured using the usual methods of hematology and cell culture technology Right.
  • the functional viability of the cells is measured based on their motility and stimulability with chemokinetic and chemotactic proteins. Mitoses are determined by chromosome counting.
  • the morphological viability of the cells at the end of the biotechnical culture is> 85%.
  • the total cell loss (mainly granulocytes) during the culture is at most 20%, which is normal for primary cell cultures.
  • the culture is terminated by separating the cells from the supernatant solution by centrifuging at 500 ⁇ g and 10 ° C. for 10 minutes.
  • the cells are washed twice with a salt solution which contains 0.15 mol / l NaCl and 0.0015 mol / l sodium potassium phosphate and has a pH of 7.1. They can be used for other purposes.
  • the culture solution is then centrifuged again at 10,000 ⁇ g for 1 hour at 4 ° C. to remove suspended particles.
  • the clear culture solution obtained (together 1000 l with a content of about 1400 g of proteins and other macromolecules) is immediately subjected to salting-out fractionation with ammonium sulfate.
  • the culture solution is mixed with 0.5 mol / l sodium potassium phosphate buffer solution to a final concentration of 0.1 mol / l.
  • Solid L-cysteine is also added to a concentration of 0.001 mol / l.
  • the culture solution is then added to an ammonium sulfate seed by adding 199 g of ammonium sulfate / l solution adjusted concentration of 35%.
  • the pH of the solution is continuously checked and kept at 6.7 by adding 2N ammonia. Some of the proteins precipitate out of the solution.
  • the protein precipitate is separated from the supernatant containing solutes by centrifugation at 10,000 ⁇ g for one hour. Protein fraction 1 is obtained as a protein slurry containing ammonium sulfate, which contains about 100 g of protein.
  • the culture solution is then adjusted to an ammonium sulfate saturation concentration of 45% by adding 60 g of ammonium sulfate / 1 solution.
  • the pH of the solution is continuously checked and kept at 6.7 by adding 2N ammonia.
  • Another part of the proteins precipitates out of the solution.
  • the protein precipitate is separated from the supernatant containing solutes by centrifugation at 10,000 ⁇ g for one hour.
  • the crude protein fraction 2 is obtained as a protein slurry containing ammonium sulfate, which contains about 60 g of protein.
  • the crude protein fraction 2 can also be separated and worked up by the process specified below to obtain its ingredients.
  • the culture solution is then adjusted to an ammonium sulfate saturation concentration of 90% by adding 323 g of ammonium sulfate / l solution.
  • the pH of the solution is continuously checked and kept at 6.7 by adding 2N ammonia.
  • the protein precipitate is separated from the supernatant containing solutes by centrifugation at 10,000 ⁇ g for one hour. It the raw protein fraction 3 is obtained as a protein slurry containing ammonium sulfate, which contains about 1080 g of protein. Most of the serum albumin is also in this fraction.
  • the crude protein fraction 3 is also worked up by the process specified below to obtain its ingredients.
  • the supernatant 4 of the crude fraction contains 160 g of protein and other macromolecules. This supernatant contains bioactive monocytic RNP.
  • the protein-containing supernatant 4 is mixed with the same volume of buffer solution A (0.15 mol / l NaCl, 0.0015 mol / l sodium potassium phosphate, 0.001 mol / L-cysteine; pH 7.4) to an ammonium sulfate degree of saturation of 45% and one Diluted phosphorus concentration of 0.05 mol / l.
  • buffer solution A 0.15 mol / l NaCl, 0.0015 mol / l sodium potassium phosphate, 0.001 mol / L-cysteine; pH 7.4
  • This solution is concentrated over a membrane with an exclusion limit of 500 daltons.
  • the substances contained in the solution are obtained as a retentate solution with a volume of 13 l (approximately 100-fold concentration).
  • the retentate solution is further cleaned separately.
  • the retentate solution is purified in the manner explained above, the preparative molecular sieve filtration being preceded by an anion exchange chromatography in the order.
  • an epichlorohydrin crosslinked dextran molecular sieve matrix (Sephadex G-50) with a particle size of 40 to 120 or 20 to 80 ⁇ m is used.
  • the separation limits are set to 7,000 to 3,000 Daltons.
  • Circulation molecular sieve filtration the eluate is recycled at a separation limit of 7,000.
  • monocytes obtained from pig blood are cultured under the conditions given in Example 1.
  • the cells are stimulated during the culture by the polyvalent lectin (CON) in the culture medium.
  • the RNPs formed in the culture solution are isolated according to the method described in Example 1 and obtained in a highly pure state. Yields similar to those in Example 1 are achieved.
  • the production of bioactive RNP from an inflammatory tissue and the separation of the RNP contained therein from the other constituents of the tissue are described.
  • the heart muscle tissue is minced at 0 - 4 ° C in a meat grinder and three times its weight is added to a 0.05 mol / 1 sodium potassium phosphate buffer solution containing 0.001 mol / 1 cysteine, pH 6.80.
  • the suspension obtained is homogenized with a homogenizer (Ultraturax).
  • the supernatant, which contains the soluble portions of the inflammatory tissue is then separated from the undissolved components by centrifugation at 10,000 ⁇ g.
  • the work is carried out at a temperature of 4 ° C.
  • the solution obtained is centrifuged at 100,000 ⁇ g for 3 hours.
  • the clear solution obtained is separated from the lipid layer floating on top.
  • the resulting RNP-containing clear solution is then subjected to a fractional precipitation with ammonium sulfate according to Example 1.
  • the concentrated retentate solution obtained is worked up to the RNP according to Example 1. About 0.03 mg of RNP morphogen is obtained.
  • Example 3 a homogenate of 500 g of leukocytes is prepared and in that specified there

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Abstract

L'invention concerne des ribonucléopolypeptides (RNP) bioactifs qui contiennent du cuivre, du zinc ou du calcium. Ces ribonucléopolypeptides sont des agents morphogénétiques non mitogènes des vaisseaux sanguins et présentent une structure primaire définie de communication intercellulaire porteuse d'informations génétiques. Les RNP de ZN/Ca/Cu peuvent hydrolyser par voie enzymatique des acides nucléiques de manière régulée (activité de nucléase régulée) et leur bioactivité peut être modulée et régulée par leur teneur en ions métalliques de Zn/Ca/Cu, qui agissent comme des "commutateurs moléculaires". Ces composés stimulent sélectivement la croissance directionnelle ou la morphogenèse des vaisseaux sanguins in vivo et in vitro et provoquent une néovascularisation des tissus. L'invention concerne également un procédé de préparation et d'extraction de RNP, ainsi que leur utilisation et des médicaments.
EP96926996A 1995-07-17 1996-07-17 Polypeptides de ribonucleotides contenant des metaux Withdrawn EP0781294A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE19525992 1995-07-17
DE19525992 1995-07-17
DE19530500 1995-08-18
DE19530500 1995-08-18
PCT/DE1996/001337 WO1997004007A2 (fr) 1995-07-17 1996-07-17 Polypeptides de ribonucleotides contenant des metaux

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EP0781294A1 true EP0781294A1 (fr) 1997-07-02

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AU (1) AU6698496A (fr)
CA (1) CA2196802A1 (fr)
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DE (1) DE19628895A1 (fr)
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Publication number Priority date Publication date Assignee Title
DE19810998C1 (de) * 1998-03-13 1999-08-26 Fraunhofer Ges Forschung Metallhaltige Ribonukleotidpolypeptide
DE19811047C1 (de) * 1998-03-13 1999-04-15 Fraunhofer Ges Forschung Metallhaltige Ribonukleotidpolypeptide
DE10109466B4 (de) * 2001-02-28 2008-01-10 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren und Mittel zur Modifikation humaner Angiogenese
UA91205C2 (ru) * 2004-12-17 2010-07-12 Венус Ремедиз Лимитед Антибиотическая комбинация амикацина и цефепима для создания общего раствора для лечения инфекций
UA91208C2 (ru) * 2005-02-14 2010-07-12 Венус Ремедиз Лимитед Фармацевтическая композиция, которая содержит антибактериальный гликопептид и цефалоспорин, для терапии резистентных инфекционных состояний
AR095196A1 (es) 2013-03-15 2015-09-30 Regeneron Pharma Medio de cultivo celular libre de suero
TW202330904A (zh) 2015-08-04 2023-08-01 美商再生元醫藥公司 補充牛磺酸之細胞培養基及用法

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Title
See references of WO9704007A2 *

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CA2196802A1 (fr) 1997-02-06
WO1997004007A3 (fr) 1997-03-13
HUP0202974A3 (en) 2005-05-30
AU6698496A (en) 1997-02-18
US6087123A (en) 2000-07-11
CZ75597A3 (en) 1997-08-13
WO1997004007A2 (fr) 1997-02-06
DE19628895A1 (de) 1997-01-23

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