DE1296143B - Process for the production of water-soluble sodium salts of the polyene antibiotics nystatin and levorin - Google Patents

Description

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The invention relates to a process for producing satisfactory yields and preparations with water-soluble sodium salts of polyene antibiotics favorable pharmacological application possible-nystatin and Levorin for the purpose of obtaining solubles.

antifungal preparations. In contrast to the good the method according to the invention for production

water-soluble polyene antibiotic trichomycin, 5 water-soluble sodium salts of polyene antibiotics which is an exception in this, are nystatin and nystatin and levorin is now characterized by Levorin practically insoluble in water. that one nystatin or levorin in anhydrous

The semi-synthetic production of water-methanol is known, with an anhydrous, methanol-soluble one antifungal antibiotics by acylic solution of sodium butylate until reached lation of polyenes, e.g. B. in the form of succinates, io a pH of at most 10.1 potentiometrically However, only the derivatives of the heptaenen have anti-titration, insoluble impurities are separated off, then biotika have a biological activity, but compared to the solution obtained in a nitrogen stream to the starting heptane, z. B. perimycin. on reduced! Pressure concentrates, filtered, from the filtrate drops 2 to 16 times. the inactive ones by adding ethyl acetate

Also the production of water-soluble salts ampho- 15 accompanying substances and the sodium salt formed tereric polyenic antibiotics, e.g. B. of nystatin, after nystatin or levorin falls and Blocking of the nitrogen by acetylation is then the separated sodium salt with acetone known. The N-acetyl derivatives of the tetraene anti or methylene chloride washes and under reduced However, biotics are dried out at increased pH a pressure. According to a preferred embodiment, they are subjected to hydrolysis and lose in the cleavage process of the method according to the invention of the macrolid lactone ring their antibiotic ac- the dry sodium salt of nystatin or des activity, while the N-acetyl derivatives of Levorin are dissolved in water and then in high-volume Heptaenes, e.g. B. of the candidate, subjected to freeze-drying in the acetyl vacuum. The new lation at low temperature in the anhydrous process enables the sodium-methanol to be obtained maintain their reduced activity. 25 salts of nystatin for external use with one It has also been proposed to use nystatin by means of a yield not less than 60% (based on the methanolic hydrogen chloride in the water-soluble activity and the weight of the nystatin used) pharmacologically active hydrochloride form over and with a yield of not less than 38 bis lead, however, only in the presence of a considerable 45% (based on the activity) and a weight amount weak organic bases such as Isonico- 30 yield more than 50% of the freeze-dried tinylhydrazide and / or hydroquinone, which is the sterile preparation for inhalation application and for virological examinations of the preparation, which is very difficult to preserve. The weight gain make pharmacologically inapplicable, with the sodium salt of levorine contributes more than 61%, sufficient yield is feasible. and the yield, based on the activity of the

It is also known to bring Nystatin 35 into solution, which is up to 97% in water by means of anionic emulsifiers, of which, in addition to reducing the yield when freezing

however, e.g. sodium lauryl sulphate on liver dry toxic up to 38 to 45% additional losses occur works. of the sodium nystatin salt when dissolved in water

Further, it is known that the solution (approximately calculated J ^e 75 mg solution into a solution of the sodium salt convert dry matter) by bacterial filter tericin A using methanolic sodium hydroxide 40 during filling into vials under aseptic Leaves crystalline Ampho- sterile filtration, which can be precipitated with acetone or ethyl acetate; conditions, with repeated freeze-drying, with the sodium salt of amphotericin B, on the other hand, a part of the finished product that is not formed so quickly and is difficult to use for external use and due to aqueous solution is excreted in control. 45 insufficient sealing of the rubber stoppers and

The manufacture of sodium salts of the soft aluminum caps, which sometimes rotate Most amphoteric tetraene and heptaene carboxyl leave on. However, these measures are sustainable Antibiotics in the usual way in the aqueous position of a sterile and apyrogenic preparation that However, a solution is ruled out, as the pH value corresponds to the relevant drug regulations of solutions of tetraene salts above 10.50 speaks, indispensable.

that of heptaene salts is above 11, d. i.e., this as a result of partial inactivation during the

Salts are neither neutral nor stable and therefore intra- evaporation of the sodium nystatin salt solution can cause the venous not applicable. Due to the lability of the yield drop by up to 60%, based on the active macrolides Laktonring occurs whose complete hydrolysis did of the nystatin used. The yield of the already in a 0.0In NaOH, z. B. at pH 12 55 sodium nystatin salt depends, as in Examples 1 in 3 hours, while in a salt formation reaction and 2 it can also be seen from the quality of the einin anhydrous medium the hydrolysis then occurs, the nystatin is deposited and the higher the higher, the higher it is if water of reaction is formed in the process. is the biological activity of the latter, the

The object on which the invention is based has requirements placed on it in different countries consists in the development of a technically and economically 60 different (from 2000 to 3000 U / mg), Economically simple method of making water The method preferably becomes more soluble as follows Salts of the polyene antibiotics nystatin and led: A 0.8 to 1% nystatin levorin is presented in anhydrous medium, which has the disadvantages or Levorin solution in anhydrous methanol. as the known method, in particular the option of titrant is a 0.1 n sodium butylate solution in the formation and accumulation of water of reaction, 65 used dry methanol and the titration with that in an alkaline medium hydrolysis of the macro of a glass electrode up to a pH value of 10.1 liden lactone rings and, as a result, the loss of carried out. After filtering off the insoluble antibiotics Activity causes, excludes, and the mixtures are evaporated, the filtrate in a stream of nitrogen

to one eighth to one tenth of the original volume, the excreted inactive admixtures are filtered falls from the concentrate by fractionation first the admixtures and then from the filtrate the sodium salt of the polyene by adding 5 times the amount of ethyl acetate, washes the Polyene sodium salt on the filter with acetone or methylene chloride and dry in the vacuum drying cabinet at a temperature not exceeding 25 ° C and at a pressure of 50 mm of mercury.

The nystatin sodium salt is a light yellow, crystalline powder and forms completely transparent, watery ones Solutions in concentrations up to 100 mg / ml. If left in air at room temperature for a long time the more than 25 mg / ml concentrated solutions gel - due to the intrinsic viscosity of the colloid solution.

The solutions of the nystatin sodium salt used for inhalation purposes in a concentration of 5 to 10 mg / ml have a pH value of 9.1 to 9.4, stay at room temperature for 7 to 10 hours Storage in the refrigerator for several days, transparent. The stability of the salt is during the Storage at 4 ° C due to a 1 to 2% loss of activity per month, as for nystatin itself, marked. By freeze-drying the concentrated solutions, it is increased so that the Loss of activity is only 5% within one year. The milligram activity of the salt is at least 1900 to 3400 U / mg, determined by the diffusion method in agar in comparison with the international nystatin standard from the National Institute for Medical Research in London.

The levorin sodium salt is a yellow amorphous powder which gives completely transparent aqueous solutions up to a concentration of 50 mg / ml and slightly opalescent solutions up to 100 mg / ml. Compared to solutions of the nystatin salt, levorine salt solutions are distinguished by the fact that they do not gel. The salt is at + 4 ⁰ C after many years of storage

ίο stable. By freeze-drying, the stability of the salt is also increased so that it is stable at +4 ⁰ C to 1,5 years.

The milligram activity of the levorine salt amounts to at least 25,000 U / mg according to the diffusion test in agar against a Levorin standard kept in the Tarasevich Control Institute in Moscow will. The attempts at inhalation administration were carried out first on rats, then on dogs and finally carried out on volunteers. The healing dose

ao of the salt in clinical use when inhaling the solution is a concentration of 40,000 U / ml 1 to 2 times a day, 200,000 units (the medicinal levorin dose is 1,500,000 units). Of the The term "healing dose" results from the minimum, average and maximum partial and daily doses of the Antibiotic, which are provided in the instructions for use of these preparations (see for example M. D. Maschkowski, "Medicines" Moscow, Verlag Medizina, 1967).

The doses in the clinical trial are in detail:

preparation

Nystatin

Sodium nystatin salt

Levorin

Sodium levorin salt.

This means that the healing effect of the sodium nystatin salt is greater than that of the sodium nystatin salt by a factor of 13 Nystatins. The healing effect of the sodium levorin salt is 4 to 6 times greater than that of levorin.

The test dose for white mice with intravenous introduction is 200 U / mouse or 0.4 mg / kg for levorin sodium salt, and 80 U / mouse or 2 mg / kg for nystatin sodium salt. The term “test dose” indicates the tolerable dose (LD ₀ ) for white mice when introduced into the tail vein (intravenous application) of the selected dose of the preparation, calculated in units per mouse, administered in 0.5 ml of water, and is as a test to understand the harmlessness of the preparation.

The biological activity of the sodium salts is determined according to the international standard method using microbiological diffusion in agar-agar as determined by the London National Institute of Medicinal Research for nystatin is established (see Pharmacopoea Britannica, 1963, and Pharmacopoea Internationalis, 1967, under "Nystatin"); the yeast Torula utilis serves as the test organism. As a Levorin standard, which is used for comparison is that from the Actinomyces levoris Krass strain. No. 879 received Series 115-66 (deposited in the scientific Allunion Research Institute for Antibiotics).

Partial dose Daily dose in units of activity min. Max. 2,000,000 4,000,000 min. 150,000 300,000 500,000 1200,000 1,500,000 150,000 200,000 400,000 400,000 100,000 Max. 1,000,000 150,000 500,000 200,000

By converting nystatin and levorin into their sodium salts, the toxicity is reduced. During the production of the nystatin salt, a 40% detoxification takes place (taking into account the milligram activity). The lethal dose LD ₅₀ for this salt when administered intraperitoneally in white mice is 35.7 mg / kg - for nystatin 18 mg / kg based on the standard activity.

This is due to the removal of toxic ballast by filtration after salt formation, evaporation, fractionated precipitation and washing out of the inactive admixture of the albonurinsin formed by Actinomyces noursei No. 153 from the preparation with the aid of acetone or methylene chloride. In the corresponding production of the levorin sodium salt, the toxic impurities are also removed and the actinomyces levoris crass is washed out. No. 879 antibiotic levoristatin achieved by means of acetone or methylene chloride. The LD _{50 of} the salt in white mice is 60,000 U / kg or 2.4 mg / kg (intravenous) or 1200 mg / kg (perorally); with Levorin, on the other hand, 110,000 U / kg or 4.4 mg / kg (intraperitoenal) or 1000 mg / kg (perorally). The apparently greater tolerance of Levorine is due to its practicality

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Insolubility in water and the associated electrode equipped potentiometer is titrated. To insufficient absorbability. Throwing off the undissolved sediment is steamed

the lightened filtrate in vacuo at 50 mm Hg im

Pharmacological application nitrogen flow to one eighth to one tenth of one.

While nystatin and levorin are administered orally, the precipitate is separated off and cation because of its poor solubility and lack of transparent concentrate for the purpose of precipitating the If resorption from the gastrointestinal canal adhere to little, little active, thrown-off admixtures are effective or remain ineffective, their 1.7 times the volume of anhydrous ethyl acetate are up to Sodium salts with advantage z. B. added to the turbidity in candidiasis.

Mucous membranes of the oral cavity, the nasopharynx, ethyl acetate, as well as aspergillosis and coccidiosis, can be used from the filtrate by adding 3.3 times the volume of dry ethyl acetate for the lungs, the airways and the genitals. Nystatin sodium salt precipitated. The absorption of the sodium salts can be achieved with rectal cream-colored sediment for washing out or peroral application in the form of enteric albonursin with a small amount of dry tablets or when fed with milk. 15 acetone or methylene chloride is then washed and then washed. The sodium salts in aerosol therapy can be used for the abovementioned erosion in a vacuum at 50 mm Hg and 25 ^{0 C, diseases.} Salt yield 1.6 g, activity 3337 U / mg, activity

On inhalation of the aqueous solution, in particular 75% related yield. The sodium salt of nystatin becomes in the blood one that is fungi-. ,.

static concentration exceeding 10 times 20 Example /

achieved. Inhalation of 100 g of nystatin with an activity of 2500 U / mg has proven to be effective

Dose of 150,000 U against pulmonary aspergillosis, which are dissolved in 121 dry methanol and which cannot be influenced by other means. This dose of solution with vigorous stirring with 14.7 g of sodium is 20 times less than the usual daily dose of 3 bis butylate in the form of its 0.1 n-methanol solution with 4 million units of nystatin, which is ineffective in such cases. 25 room temperature with vigorous mixing, with the help of the advantage of using the sodium levorin- a potentiometer up to the pH-value of 10.1 salt consists in the lowering of the necessary titrations. The activity yield is 70%. The un-healing dose by a factor of 5 in comparison with the insoluble admixtures are separated on the pressure filter absorbable Levorin, which therefore does not cure by pressing on nitrogen. In candidiasis of the mucous membranes and the pulmonary tract, the filtrate is steamed onto a ninth of the original and the genitalia. In addition, the volume exerts in a rotary evaporator in embroidery Natriumlevorinsalz its healing effect in cases of the material flow at 20 mm Hg and a water bath Clinical ineffectiveness of Natriumnystatinsalzes temperature not exceeding 35 ⁰ C a. The inactive admixtures observed in clinical practice, which have failed, are separated out on the print as a result of the increasing nystatin resistance of the pathofilters. The yield (based on the genetic candidate strains. With solutions of the original sodium activity) is 60% of the rinses used in a concentration of nystatin.

of 1000 U / ml remove wound after a few days. The concentrate becomes the less active

sponges of the oral cavity and nasopharynx admixtures by adding 1.7 to 2.5 volume space. 40 parts dry ethyl acetate precipitated and precipitation Sodium salts, in particular nystatin sodium, secreted on the pressure filter. The salt of the filtrate can also be used for the prevention of yeast - the sodium salt of nystatin is precipitated by the addition and fungal infection of pure tissue cultures for the purpose of 3.3 to 2.5 parts by volume of ethyl acetate; Precipitation on the pressure filter washes away with measles, whooping cough, flu and other virus 45 1.51 dry acetone or methylene chloride and diseases as well as in the preparation of the nutrient medium for drying in a vacuum drying cabinet during a microbiological and virological examinations. temperature of at most 25 ⁰ C and a pressure of The treatment of cattle and poultry 50 Torr. Immediately before the widespread coccidiosis disease, the powder is dissolved by rectal freeze-drying in water to form a 50 mg / l turn of the nystatin sodium salt in combination with 50 solution.

Oral application, causes complete healing. The solution is in medical glass vials

within 5 to 6 days, while it was previously bottled at 150,000 units (about 1.5 ml), lyophilized Treatment of coccidiosis is not sufficiently effective and for the purpose of complete sealing with rubber stoppers Funds gave. and aluminum caps closed. Yield, loading

Furthermore, nystatin and levorine sodium salts are also attracted to the original activity, 38 to 45% of the 55 applicable to inhibiting the fermentation process of the nystatin used and at least in terms of weight Wine must in the production of dessert wines, 50%.

also for the preservation of fruit, vegetables and examples 3

other agricultural products as well as for

Combat the pathogen Verticillium Dahliae of the 60 Ig Levorin raw material, which occurs in the fermentation of verticillic wilting of the cotton bush. Actinomyces levoris Krass, with an activity of

. -I ₁ 6400 U / mg was obtained, is in 150 ml of water

Oil free methanol dissolved and with a methanolic

2.5 g nystatin with an activity of 2884 U / mg 0.1 n sodium butylate solution up to a pH of are dissolved in 300 ml of anhydrous methanol and titrated 65 10.1. After filtering off the undissolved by-products vigorous stirring at room temperature with a mixture of the filtrate in vacuo in the stick-0.1 n-Sodium butylate solution in methanol evaporated to the tenth part of the stream, the exhaust pH value of 10.1 with the help of a sediment that fell with a glass and separated from the

the sodium salt was precipitated with 5 times the volume of ethyl acetate. The remaining levonstatin is washed out of the precipitated precipitate with anhydrous acetone or methylene chloride and dried in a vacuum oven. Salt yield 610 mg, activity 10 240 U / mg, activity yield 97%. Shelf life at 4 ⁰ C about 1 year.

Claims (2)

Claims: IO 1. Process for the preparation of water-soluble sodium salts of the polyene antibiotics nystatin and Levorin, characterized in that you nystatin or levorin in anhydrous Methanol dissolves with an anhydrous, methanolic solution of sodium butoxide up to Reaching a pH value of at most 10.1 potentiometrically titrated, insoluble additions separated off, then the resulting solution is concentrated in a stream of nitrogen under reduced pressure, filtered, the inactive accompanying substances from the filtrate by adding ethyl acetate and the sodium salt of nystatin or levorin formed is fractionated, precipitated and then the separated sodium salt was washed with acetone or methylene chloride and reduced under reduced pressure Print dries. 2. The method according to claim 1, characterized in that the dry sodium salt of Dissolve nystatin or levorine in water and then freeze-drying in a high vacuum subject. 909522/479