Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
  • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
  • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
• • 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
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/045—Actinoplanes

Definitions

• Lipopeptide antibiotic calcium salts process for their preparation and use.
• the present invention relates to calcium salts of lipopeptide antibiotics, a process for their preparation and their use.
• EP 0 629 636 A1 discloses lipopeptide antibiotics of the general formula I in which R 1 denotes an OH or an NH 2 group and R 2 denotes a fatty acid residue (RC (O) -).
• lipopeptide antibiotics can be divided into two groups, which differ with regard to their exocyclic amino acid:
• the lipopeptide antibiotics of the amphomycin type are characterized by the exocyclic amino acid aspartic acid (Asp, where R 1 in formula I means an OH group) (RC Strong et al., Antimicrobial Agents and Chemotherapy 1970, 42-45; M. Bodanszky et al. J. Am. Chem. Soc. 95, 2352-2357 (1973)), while the asparagine-type lipopeptide antibiotics are characterized by the exocyclic amino acid Asparagine (Asn, where R 1 in formula I denotes an NH 2 group).
• the amphomycin and asparagine type lipopeptide antibiotics differ from one another by substitution 2 of the ⁇ -amino group of the exocyclic amino acid (Asp or Asn) with different fatty acid residues (R 2 in formula I).
• EP 0 688 789 A1 discloses derivatives of the lipopeptide antibiotics of the amphomycin and of the asparagine type and their pharmaceutically acceptable salts.
• salts with inorganic and organic acids for example hydrochloric acid, sulfuric acid, acetic acid, citric acid, p-toluenesulfonic acid, with inorganic and organic bases such as NaOH, KOH, are described as pharmaceutically acceptable salts of the lipopeptide antibiotics of the formula I. , Mg (OH) 2 , diethanolamine, ethylenediamine or with amino acids such as arginine, lysine and glutamic acid.
• the calcium salt of amphomycin is also known (Kirk-Othmer, Encyclopedia of Chemical Technology, fourth edition, volume 3, Antibiotics to Batteries, John Wiley & Sons, page 284). It is only sparingly soluble in water and, because of the toxicity of amphomycin due to its hemolytic activity when used systemically, was used exclusively in antibiotic ointments for topical application.
• the lipopeptide antibiotics of the asparagine type (R 1 in formula I means an NH 2 group) and their production have been described for the first time in EP 0 629 636 A1.
• the object of the present invention is to provide a salt of the lipopeptide antibiotics from asparagine type, which is characterized by a relatively high stability and good antibacterial activity and due to its high solubility in water as well as due to its low toxicity, particularly ⁇ hemolytic due to low activity, systemic (parenterally) is applicable.
• lipopeptide antibiotics of the asparagine type can have very different properties.
• the sodium salts generally have a very good antibacterial activity and are easily soluble in water. However, these have a limited shelf life, especially at elevated temperatures. Since with drugs and other commercial products, stability such. B is of great importance for the handling of the goods, stable salt forms of the lipopeptide antibiotics are required. Since the asparagine-type lipopeptide antibiotics are amphoteric compounds with an isoelectric point in the acidic range, neutral salts with numerous bases can be prepared.
• Monovalent and polyvalent ions are possible as cations, such as, for example, alkali metal, alkaline earth metal and other metal ions, but also salts with ammonia or with organic bases, such as amines. Examples of the latter are lysine and lysyllysine salts, which are very well tolerated when fully effective.
• the calcium salts of the asparagine-type lipopeptide antibiotics are not only effective and tolerable, but also readily soluble in water and, in contrast to the corresponding ones Sodium salts are particularly stable.
• R1 is a straight-chain or branched, saturated or unsaturated aliphatic acyl radical having 8 to 22 carbon atoms, which is optionally interrupted by one or more phenyl or cycloalkyl groups or linked to such groups and can also optionally be interrupted by oxygen. 5
• R1 in formula II is preferably an acyl radical which is interrupted by a phenyl or cycloalkyl group or is linked to such a group, for example
• R1 is an acyl radical interrupted by a phenyl or cycloalkyl group and by oxygen, preferably characterized in that R1 (2a)
• n is an integer from 0 to 20.
• a calcium salt of the compound of the formula II is particularly preferred, which is characterized in that R1 is a straight-chain or branched, saturated or unsaturated aliphatic acyl radical having 12 to 15 carbon atoms, R1 in formula II preferably being a fatty acid radical of the formulas shown below:
• the calcium salt of the compound of formula II can exist in two forms, the dicalcium salt or the monocalcium salt.
• the dicalcium salt can, depending on the number of anions and the fatty acid substituent (R1 in formula II)
• n is an integer from 7 to 21 and X is an anion, or (ii) for example in the case of a monounsaturated fatty acid (R1 in formula II) by the general empirical formula
• n is an integer from 7 to 21 and X is an anion
• the anion X is preferably a halide anion, CI “ , Br “ or I " , particularly preferably CiJ 10
• the monocalcium salt can furthermore, for example in the case of a saturated fatty acid (R1 in formula II), by the general empirical formula
• n is an integer from 7 to 21 in both general empirical formulas.
• the corresponding acid namely the corresponding compound of the formula II, has, for example, the empirical formula C 59 H 9 N 1 Oi 9 .
• the acid A 1437-D which corresponds to the preferred calcium salt (3d) mentioned above (the corresponding compound of the general formula II), has the structure shown below (FIG. 1): 11
• Fig. 1 Structural formula of A 1437-D (acid form of 3d)
• Table 1 shows the assignment of the 1 H and 13 C NMR signals using the example of the A 1437 D sodium salt.
• the amino acids preferably have the following configuration: Pip-3: D; Me-Asp-4: L-threo; Asp-5, -7: L; Dab-2: L-threo; Dab-9: D-erythro; Val-10: L; Pro 11: L; Asn-1: L
• the process for the preparation of the calcium salt of the compound of the formula II described above is characterized in that a sodium or ammonium salt of the compound of the formula II is dissolved in a suitable organic solvent, a calcium salt dissolved in ethanol is added to this solution and the calcium salt the compound of formula II isolated as a precipitate.
• a suitable organic solvent is ethanol.
• the calcium salt to be added dissolved in ethanol is preferably a calcium halide, CaCl 2 , CaBr 2 , Cal 2 or the corresponding hydrates.
• the dicalcium salts of the compound of the formula II are preferably formed in this process.
• the A 1437 D calcium salt which is sparingly soluble in the organic solvent, precipitates in the form of a precipitate.
• Calcium salts which are soluble in ethanol and are suitable for the precipitation are, for example, CaCl 2 , CaBr 2 Cal 2 and their hydrates.
• This precipitation process leads to salts that may contain in addition to the calcium cation or anion of the precipitant salt, for example the halides Ch, Br, and K, the empirical formulas of the precipitated calcium salts may be, for example: C 59 H 92 N ⁇ O ⁇ 9 Ca 2 halide 2 such.
• Crystallization is another way of obtaining or purifying calcium salts of the compound of formula II.
• the property of the antibiotic calcium salt is used to dissolve in pure water, in dimethyl sulfoxide, in pure methanol and in other polar solvents.
• the process for producing a calcium salt, preferably the monocaicium salt, of the compound of the formula II is distinguished by the fact that a dicalcium salt of the compound of the formula II, which can be obtained, for example, by the method described above, is dissolved in a polar solvent, then the solution obtained is mixed with a less polar solvent or a mixture of less polar solvents and the calcium salt, preferably the monocalcium salt, is isolated as a precipitate.
• Another process for the preparation of a calcium salt, preferably the monocaicium salt, of the compound of the formula II is characterized in that a sodium or ammonium salt of the compound of the formula II is dissolved in a polar solvent, and a calcium salt dissolved in the same solvent is added to this solution , then the solution obtained is mixed with a less polar solvent or a less polar solvent mixture and the calcium salt, preferably the monocalcium salt, is isolated as a precipitate.
• the sodium or ammonium salt or the dicalcium salt of the compound of the formula II is preferably dissolved in a polar solvent which is selected from the group consisting of water, dimethyl sulfoxide and methanol.
• the less polar solvent with which the solution obtained is added is preferably selected from the group consisting of alcohols, acetone and acetonitrile.
• the sodium or ammonium salt or the dicalcium salt of the compound of formula II is dissolved in water and the less polar solvent is methanol.
• the calcium salt, preferably the monocalcium salt, of the compound of formula II can be prepared by dissolving a sodium or ammonium salt of the compound of formula II in methanol, adding a calcium salt dissolved in the same solvent to this solution, then the resulting solution with Water or a mixture of water and butanol are added and the calcium salt, preferably the monocalcium salt, is isolated as a precipitate.
• a calcium halide is preferably added as the dissolved calcium salt, which is selected from the group consisting of CaCl 2 , CaBr 2 , Cal 2 and their hydrates.
• the A 1437-D-dicalcium salt can be concentrated in a readily dissolving solvent and then mixed with a miscible agent which is less solvent for the antibiotic salt.
• a miscible agent which is less solvent for the antibiotic salt.
• examples of the latter are less polar, organic solvents such as alcohols, acetone, acetonitrile and others.
• the mixture of water and methanol forms a special case. While these pure solvents easily dissolve A 1437-D-Ca salts, the mixtures of both solvents have significantly weaker solvent properties.
• the A 1437-D monocalcium salts (3d / iv) can thus be precipitated and crystallized from water (from methanol) with addition of methanol (water). Calcium salts can be produced or purified in this way.
• the A 1437-D monocalcium salt (3d / iv) easily forms gels in water-methanol mixtures. This gel formation is unfavorable for the crystallization because the rate of crystallization is greatly slowed down. Measures must therefore be taken for the crystallization in order to suppress the formation.
• Such a measure can e.g. B. the addition of small amounts of a suitable substance, such as butanol.
• Another process for the preparation of the calcium salt of the compound of formula II, preferably its monocaicium salt, for example the A 1437-D monocalcium salt (3d / iv). consists in the use of a carrier, for example adsorption resins, reverse phase carriers, molecular sieves and ion exchangers, which with an aqueous solution of a sodium or ammonium salt of the compound of formula II (for example the sodium or ammonium salt of compound A 1437-D), which with a calcium halide has been added, or optionally an aqueous solution of a dicalcium salt of the compound of formula II (for example the dicalcium salts (3d / iia), (3d / iib) or (3d / iic)) is loaded, after which the carrier is optionally provided with a suitable one Washed solvent and finally the calcium salt of the compound of formula II, preferably the monocalcium salt, for example the A1437-D-monocalcium salt (3d / iv
• adsorbent resins for use as an adsorbent resins are, for example Amberlite XAD 7 (Rohm & Haas), DIAION ® HP20SS (Mitsubishi Chem. Corp.), Porous '' 20 R2 or polyamide 6 (Riedel-deHaen), with the use of reversed-phase carriers are, for example LiChrosorb , n RP-select B (E. Merck), but carriers can also be used 19 that are commonly used in hydrophobicity interaction chromatography (HIC) for example for protein purification.
• Such carriers are for example, Phenyl Sepharose ® or TSKgel Phenyl ToyopearP.
• Molecular sieves such as those used for "size exclusion chromatography” or gel filtration chromatography are also suitable.
• the basis of this process is the tendency of the compound of formula II (e.g. A1437-D) to bind calcium ions.
• a mixture of ammonium or sodium salts of the compound of the formula II with calcium salts in water is prepared and this mixture is separated on the carriers in a manner known per se.
• an aqueous solution of a corresponding dicalcium salt can also be introduced.
• an aqueous solution of A1437-D-sodium salt and calcium chloride to an adsorption resin such as applied for example to DIAION ® HP 20SS, washing the loaded resin with water to remove excess salts, remove, and then the calcium salt is the lipopeptide with hydrous or anhydrous solvents, preferably eluted from the support with methanol.
• the A1437 calcium-containing fractions are dried.
• a product obtained in this way has, for example, the elemental composition C 59 H 92 N 14 O 19 Ca (monocalcium salt of the compound of the formula II (3d / iv)).
• ion exchangers preferably anion exchangers
• any aqueous, low-ion solution of the compound of formula II is bound to an anion exchanger at pH values between pH 5 and pH 9, and after washing the loaded carrier with water, the monocalcium salt of the compound of formula II is washed with a increasing concentration of a water-soluble calcium salt eluted.
• the column eluate, which contains the monocalcium salt, is desalted and dried, for example, by reverse osmosis.
• the monocalcium salt can be isolated by other methods such as crystallization. 20th
• the compounds of formula II, the precursors of the calcium salts according to the invention can advantageously be prepared, as is known from EP 0 629 636 A1, by fermentation of Actinoplanes sp., Preferably Actinoplanes friulensis (DSM 7358), and optionally, as in EP 0 688 789 A1 (US Pat. No. 5,629,288), derivatized by replacing the acid residue R1 in formula II with a non-naturally occurring acid residue.
• the compounds of the formula II obtained in this way can be converted to their calcium salts as described above.
• the object stated at the outset to provide an improved process for the fermentative preparation of the compound of the formula II, the acid precursor of the calcium salts according to the invention, in which the compound of the formula II is preferably produced by the microorganism, is achieved in that the fermentation of actinoplanes spe ⁇ , preferably from Actinoplanes friulensis DSM 7358, the fermentation solution with one or more complexing agents, preferably chelating agents, and supplemented with the amino acid asparagine.
• Citric acid or ethylene diamine tetraacetate (EDTA) is preferably used as the complexing agent.
• the fermentation solution can also advantageously be supplemented with EDTA and citric acid.
• the fermentation solution can additionally be supplemented with the amino acid L-leucine.
• the fermentation solution can also be supplemented with the amino acid L-valine.
• the present invention also relates to a process for the preparation of a calcium salt of the compound of the formula II, which is characterized in that, in a first step, the compound of the formula II, as described above, by fermentation of Actinoplanes spec, preferably Actinoplanes friulensis (DSM 7358), wherein the fermentation solution is supplemented with one or more complexing agents, preferably chelating agents, and with the amino acid 7 ⁇ asparagine, and in a subsequent step the calcium salt of the compound of the formula II, as explained above, by precipitation, crystallization or treatment wins on a carrier.
• Actinoplanes spec preferably Actinoplanes friulensis
• the calcium salt of the compound of the formula II as explained above, by precipitation, crystallization or treatment wins on a carrier.
• the present invention also relates to a calcium salt of the compound of formula II for use as a medicament.
• the calcium salts of the compound of formula II are preferably suitable for the preparation of a medicament against bacterial infections, the calcium salts (3c) and in particular (3d) described above or the dicalcium salts (3c / iia), (3c / iib) and (3c / iic) ) and in particular the dicalcium salts (3d / iia), (3d / iib) or (3d / iic) are particularly suitable, the dicalcium salt (3d / iia) being particularly preferred.
• the monocaicium salts of the compound of the formula II are also particularly suitable for the preparation of a medicament against bacterial infections, the monocaicium salts (3c) and in particular (3d) with the empirical formula (3c, 3d / iv) C59H 92 Ni 4 O 9 Ca being preferred.
• the abovementioned calcium salts of the compound of the formula II are preferably suitable for the preparation of a medicament against bacterial infections which are caused by gram-positive bacteria, preferably by glycopeptide-resistant bacteria. 22
• the medicaments containing at least one calcium salt of the compound of the formula II can furthermore contain the customary pharmaceutical auxiliaries.
• the dicalcium salt of the compound of the formula II preferably the chloride 3d / iia
• the dicalcium salt of the compound of the formula II can be dissolved in water to prepare a medicament with equal proportions by weight of mannitol and then lyophilized.
• the calcium salts of the compound of the formula II are particularly suitable for parenteral administration in the form of an injectable solution. Accordingly, the present invention also relates to injectable solutions containing one or more calcium salts of the compound of the formula II, preferably the calcium salt (3d / iv) with the empirical formula Cs 9 H 92 Ni 4 O 9 Ca or particularly preferably the calcium salt 3d / iia with the Molecular formula C59H 92 N- ⁇ Oi 9 Ca 2 CI 2 .
• the lyophilisate which consists of equal proportions by weight of mannitol and calcium salt, can be dissolved in appropriately treated water.
• the acid form of the compounds of the formula I is designated A1437.
• FIG. 2 gives an overview of the lipopeptides produced or used in the examples.
• Actinoplanes spec in particular Actinoplanes friulensis DSM7358, forms at least eight antibiotically active ones by fermentation 24 compounds of the formula I of both the amphomycin and the asparagine type.
• the lipopeptides A1437-C and A1437-D both compounds of the formula II
• A1437-C and A1437-D are cyclic decapeptides with an exocyclic asparagine which is acylated in the case of the C peptide with a C13 and in the case of the D peptide with a C14 fatty acid (Fig. 2).
• the fermentation can be controlled in the direction of C or D peptide formation.
• a peptide (A1437-A) is always formed simultaneously with the C peptide and the B peptide (A1437-B) is formed with the D peptide, which only differs from the C or D peptide in the exocyclic position (aspartic acid instead of asparagine) differs.
• the titers of these peptides are even higher than those of the C and D peptides; in the best case the ratio is approx. 1: 1.
• the yields in the shake culture ranged between 40-150 mg / l D-peptide at 50-250 mg / l B-peptide (Example 1).
• In 30 I and 200 I steel fermenters typically 800-1100 mg / l B-peptide could be achieved with 20-40 mg / l D-peptide (Example 2).
• the maximum yields were, for example, 1.2 g / l A1437-D at 280 mg / l A1437-B (example 2).
• the selective shift and increase in yield towards A1437-D was also observed in shake culture To be 25.
• the yield of A1437-D could also be increased to 1 g / l in shake culture with the same average yield of A1437-B (Example 1).
• the pH of the fermentation broth is 7.0 to 7.8 at the end of the fermentation.
• the yields were between 20 and 155 mg / l A1437-D at 60-250 mg / l A1437-B.
• a 300 ml Erlenmeyer flask with 100 ml of nutrient solution 1 is inoculated with the contents of an ampoule stored at -190 ° C and shaken for 5 days at 28 ° C and 240 rpm.
• Inoculated Erlenmeyer flask which is also shaken for 5 days at 28 ° C and 120 rpm.
• Nutrient solution 2 is filled and for 150 - 250 h at 28 ° C, 0.7 vvm and one
• Fermentation broth is 7.0 to 7.8 at the end of fermentation.
• the yields are typically between 20 and 40 mg / l A1437-D at an A1437-B concentration of 800 - 1000 mg / l. 28
• the A1437-D yields could be increased up to 1.2 g / l with a considerable reduction in the A1437-B concentration.
• a 300 ml Erlenmeyer flask with 100 ml of nutrient solution 1 is inoculated with the contents of an ampoule stored at -190 ° C and shaken for 5 days at 28 ° C and 240 rpm.
• the pH of the fermentation broth is 7.0 to 7.8 at the end of the fermentation.
• the yields are typically:
• the antibacterial activity of the A 1437 D - Ca salt is summarized in Table 3.
• the value of the compound is based on its effectiveness against the resistant and multidrug-resistant pathogens: the spectrum of action makes the antibiotic - in addition to the good tolerability listed below - an enrichment of the pharmaceutical treasure.
• a 1437 D-calcium bromide salt prepared according to Example 7 ' are dissolved in water and applied to a prepared, 16 ml column filled with MCI Gel CHP20P, 75-150 ⁇ . Elute as quickly as possible with a solvent gradient from 10% methanol in water (solution A) to 90% methanol and 10% butanol (solution B). First the impurities are washed off the column, then the pure A 1437 D-monocalcium salt with pure solution B. Concentration in vacuo gives 0.8 g of A 1437 D-monocalcium salt with 96% A 1437 D (HPLC) and 3% calcium, corresponding to the composition C 59 H 92 N ⁇ 4 O ⁇ 9 Ca.
• a 1437 D calcium bromide salt obtained as described in Example 7, is dissolved in 100 ml of water and mixed with 36 ml of a mixture of methanol-butanol (9: 1). The first clear solution gradually becomes cloudy, and is left to stand for 12 hours to complete. The precipitate formed is collected by centrifugation, washed with 50 ml of cold 40% methanol in water and dried in vacuo. 720 mg of monocalcium salt, C 59 H 92 N 1 O 9 Ca, obtained with 3J% calcium, ⁇ 1% bromine and 95% A 1437 D-monocalcium salt. 33
• a 1437 D sodium salt 1 g is dissolved in 30 ml of water and a solution of 200 mg of CaCl 2 in 10 ml of water is added. 14 ml of a mixture of 90% methanol and 10% butanol are added to the clear aqueous solution and centrifuged off at 4 ° C. after one hour. The precipitate, washed twice with 30 ml of aqueous methanol (40%), gives 810 mg of A 1437 D-monocalcium salt with 3.2% calcium, ⁇ 1% chloride and 94.5% A 1437 D-monocalcium salt, calculated as the free acid.
• Freshly drawn venous blood from humans, rhesus monkeys or beagle dogs is used to measure hemolytic activity.
• the blood is collected in heparinized tubes and divided into 12 ployethylene tubes in 200 ⁇ l aliquots.
• One aliquot is mixed with 200 ul distilled water and serves as 100% standard, another is mixed with 200 ul physiological saline (0.9% NaCl) (0% standard).
• 200 ⁇ l of substance dilutions in physiological saline at 1600, 800, 400, 200, 100, 50, 25, 12.5, 6.25 and 3.125 mg / l are distributed to the other tubes. All tubes are gently swirled and then incubated for 3 hours at 37 ° C.
• the 100% standard is then filled with 5 ml of distilled water, the remaining tubes are each filled with 5 ml of physiological saline and centrifuged at 700 g for 5 minutes.
• Hemolysis is determined by measuring the absorption of the supernatant in a spectrophotometer at a wavelength of 540 nm. The absorption of the standard with complete hemolysis is set as 100%. The absorption of the 34
• Test preparation dilutions and the 0% standard are measured and stated as a percentage of the maximum inducible hemolysis.
• Table 4 shows the result of the hemolysis test with A 1437-B, A 1437-G in comparison to A1437-D Ca salt, carried out with monkey blood.
• Table 4 shows an example of the in vitro hemolysis of monkey blood as a function of the antibiotic concentrations.
• Antibiotic concentrations (mg / 1): 12.5 25 50 100 200 400 800 1600
• the end product contains 80.5% A 1437 D-lipopeptide, calculated as free acid, 4.5% water, 5.1% calcium, 7.5% chloride and 2.1% sodium according to the empirical formula C 5 9H 92 N 1 O 19 Ca 2 Cl2 with an admixture of approx 4.6% NaCI.
• a 1437 D-dicalcium dichloride and 100 mg of apyrogenic mannitol are dissolved in 2 ml of sterile water and lyophilized.
• the entire lyophilisate, which is in the form of a powder, is dissolved in 2 ml of water for injection solutions, filled into a sterilized ampoule and the ampoule sealed with a septum.

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