DD209392A5 - PROCESS FOR PRODUCING STERILE PREPARATIONS OF HIGH VISIC SOLUTIONS AND SUBSTANCES - Google Patents

Abstract

A new process is described for the preparation of sterile preparations of substances which are either inaccessible to conventional methods of sterilization or are altered by them undesirably. The process is characterized in that a sterilizing agent is allowed to act under mild conditions, this then almost quantitatively removed again together with the solvent in a subsequent lyophilization or spray drying and the product optionally by additions in a pharmaceutically acceptable bzw.zur application to isolated animal or human cells or cell cultures suitable form.

Description

2 * 3 * 7 / / Q Π -'f- Berlin, 2.7.1982 O / 4 ^ ö U APA 61 K / 237 448/0

(60 379/11)

Process for the preparation of sterile preparations of highly viscous solutions

Application of the invention

The present invention relates to processes for the preparation of sterile preparations of substances which are either inaccessible to conventional methods of sterilization or are undesirably altered by them.

Characteristic of the known technical solutions

For germination of substances and solutions of substances, the following methods are common:

- addition of preservatives,

- Heat sterilization and

- Sterile filtration.

In the production of sterile solutions of highly viscous substances, problems arise in particular when

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- there is an interference between the preservative and the active ingredient by adding preservatives or the allergenic component of the preservative has negative side effects.

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- The active ingredient is changed unfavorably by heat sterilization or

- Due to its molecular size and / or viscosity, the substrate to be sterilized can no longer be filter-sterilized through membrane filters.

Particularly difficult has the recovery of sterile solutions of blood plasma or sterile fractions of ^T """'' bloodpTäsmas, ^ various high molecular weight nucleic acids such as deoxyribonucleic acid, ribonucleic acid as well as long-chain polynucleotide homopolymers, such as polyinosinic-Polycytidyisäurekompiexen '(Poly I: C) or polyadenylic acid-polyuridinyl acid complexes (poly A: U).

Poly I: C refers to a two-stranded complex of polyinosinic acid and polycytidylic acid, which was determined as a trigger for the formation of interferon and is therefore referred to as interferon inucer (see, for example, US Patent 4,124,702 and DE-OS 20 10 734). ,

Such complexes are obtained by combining mutually complementary homopolynucleotides in solution under certain conditions (e.g., enzyme catalysis, ultrasound, etc.). Here, for example, a polyinosinic acid strand (poly I) and a polycytidic acid strand (poly C) combine to form hydrogen bonds between the bases to a duplex and these in turn due to "stacking forces" to a double-helical tertiary structure of poly I: C together , ·

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If the temperature of a solution of poly I: C is increased, one finally reaches a temperature at which the structure of the double strand breaks apart. The solution now contains the separate ("denatured") single strands. It is said that the double strand has melted and the temperature at which this takes place is called the melting temperature. If the solution, which now contains the molten individual strands, is cooled slowly, double-strand formation ("renaturation") occurs again.

The melting process also involves the repulsive charges of the phosphate groups. High salt concentrations can shield this repulsion. Therefore, earlier solutions of Poly I: C were prepared by reacting solutions of Poly I and Poly C in the presence of a saline solution, e.g. 0.1 mol talcium chloride solution at a pH of about 7 at room temperature or with little heating mixed.

This solution was allowed to stand for 1-2 weeks. During this time, a gel - like non - sterile viscous mass formed.

Sterile poly I: C solutions are first obtained according to DE-OS 20 35 7 90, by dissolving poly I and poly C separately in the presence of a buffered saline solution, the separate solutions to form gelatinous, turbid complexes, the complexes in Autoclave '. let cool.

heated to 135 C in an autoclave and then slowly

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Investigations have shown that sterile poly I: C solutions prepared by the above process no longer have the original chain length due to the high thermal load and have a lower interferon-inducing effect.

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" ⁴ " 2.7.1982

ΑΡΑ61 Κ / 237 448/0 (60 379/11)

The results required for this are obtained in the following way: If a double strand of poly I · poly G is melted, an increase in the absorbance at 260 nm occurs. A melting curve is obtained, the abscissa being the temperature and the ordinate the ordinate is applied. The melting process is cooperative · It follows from the theory of such cooperative processes that at high MoIe-V_x kulargewicht of the double strands a steep melting curve is obtained · The smaller the molecules, the sooner the melting process starts and the flatter the melting curve · A quantitative idea, the average molecular weight is obtained by determining the sedimentation constant in the analytical ultracentrifuge

Biese sedimentation constants are given in S (= Svedberg-Sinheiten) and are for inventively sterilized poly I: G 12-15 »while material that is sterilized according to DE-OS 2,035,790, results in values below 10.

Neither the individual polynucleotides nor the long-chain polynucleotide complexes can be sterile-filtered by conventional membrane filters because of their secondary and tertiary structure.

Object of the invention

The aim of the invention is to provide an improved process for the preparation of sterile preparations of highly viscous solutions, which is carried out with extremely gentle sterilization.

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A? A 61 K / 237 443/0 (60 379/11)

sation the blood plasma and the ο. G. Nucleic acids not decomposed, as long as possible to obtain chains of homopolymers, thus providing a higher interferon-inducing 'effect of the poly I: C complex.

Explanation of the essence of the invention;

The invention has for its object to leave a sterilizing agent to act under mild conditions, this then approximately quantitatively to remove together with the solvent in a subsequent lyophilization or spray drying and the product optionally by additives in.eine pharmaceutically acceptable or for use in isolated animal or human cells or cell cultures suitable form ·

This object is achieved surprisingly simply by killing the germs under relatively mild conditions with a preservative and followed by a lyophilization process or spray-drying process, in the course of which, as well as the solvent, the preservative is largely sublimated or evaporated.

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The inventive principle is of course not limited to the above-described special case of sterilization of poly I: C solutions, but can be used in many ways for the sterilization of highly viscous solutions and substances.

The invention therefore relates to processes for the preparation of sterile preparations of highly viscous solutions and substances, such as blood plasma or fractions of blood plasma, of various high molecular weight nucleic acids, such as deoxyribonucleic acid, ribonucleic acid as well as long-chain polynucleotide homopolymers and complexes of such polymers, which characterized in that one kills the germs with a preservative, the preservative largely wegsublimiert or evaporates along with the solvent in the course of a subsequent lyophilization or spray drying process and optionally produces a pharmaceutically acceptable form by adding suitable conventional adjuvant.

For the purposes of the invention, preparations are understood as meaning all preparation forms of substances which are suitable directly or indirectly for pharmaceutical use or for use on isolated animal or human cells or cell cultures. These are, for example, solutions, suspensions, ointments, granules, powders or tablets.

As preservatives are all substances that show the corresponding bactericidal activity in the treatment of blood plasma, nucleic acids and polynucleotide homopolymers according to our method, the structure of the active ingredients and the activity of the polynucleotides during the sterilization process not

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interfere with the lyophilization or spray-drying from the solution or vaporize. The preservatives must be eliminated during lyophilization or spray drying since these - e.g. may interfere with interferon-inducing activity at poly I: C or another desirable property. Such preservatives are, for example, chloroetone, benzyl alcohol, p-chloro-m-cresol or pyrocarbonic acid diethyl ester. Preference is given to chloroetone or pyrocarbonic acid diethyl ester.

The concentration of the components to be sterilized in the polynucleotide complexes before lyophilization max. 0.3%, preferably 0.1%, in the deoxyribonucleic acid and ribonucleic acid max. 2%, preferred. 0.1%.

The concentration of the various preservatives depends on the concentration of the active ingredients and can be determined on a case-by-case basis by methods familiar to those skilled in the art.

A. Embodiment of the method according to the invention consists in dissolving blood plasma, nucleic acids or polynucleotide homopolymers or complexes of such polymers, optionally in the presence of buffer substances, adding the required amounts of preservative to these solutions and, if necessary, heating. The preservative or disinfectant is allowed to act at 25-100 ° C. (preferably 4O-8O ° C.) for 10 minutes - 4 hours (preferably 30 minutes 2 hours). Thereafter, the drug solution is lyophilized or spray dried with the preservative sublimated or evaporated during lyophilization or during spray drying. The lyophilisates and spray

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dried granules are readily soluble in water and give a sterile solution.

A preferred embodiment of the method according to the invention is the preparation of sterile preparations of polyinosinic-polycytidylic acid complexes (poly I: C), polyadenylic acid-polyuridic acid complexes (poly A: U), polyadenylic acid-cytidylic acid (poly A: C), polyadenylic acid-guanylic acid Uridylic acid (poly A: G: U), • thionicotinamide adenine dinucleotide phosphate (thio-NADP) or 2-deoxyadenosine 5'-triphosphate (dATP) (disodium salt) and sterile concentrates of t-ribonucleic acid or blood plasma ,

Surprisingly, it has been found that the concentrated sterile polynucleotide homopolymer complexes prepared by the method of the present invention produce a higher interferon-inducing effect.

Amazing here is the fact that after a very short exposure time of the preservative complete killing of the germs occurs , so that in a further operation (lyophilization or spray drying) the no longer required preservative can be almost completely removed, because preservatives are usually used to to inhibit the growth of germs, but not to completely kill the germs.

As subsequently stated, e.g. at poly I: C much longer chains of homopolymers and thus a higher interferon-inducing effect. The homopolymer complexes obtained by our process are largely freed from the volatile preservative by the lyophilization process or by azeotropic drying. This is an important factor as the preservatives limit the interferon-inducing effect of the polynucleotides.

Q 7 / L P Π "~ ^S ~ 2.7.1982

J / .4 H O U AP A 61 K / 237 443/0.

(60 379/11)

A further subject of the invention are therefore pharmaceutical preparations or preparations suitable for use on isolated animal or human cells or cell cultures which contain the preparations of highly viscous solutions and substances sterilized according to the invention,

Ausfuhrangabeispiel

The invention is explained in more detail below with reference to some examples.

The following examples are intended to explain the subject of the invention in more detail, but in no way limit it:

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example 1

Sterile poly I: C lyophilisate

All glassware containing the Poly I: C solution

are thoroughly cleaned with chromic acid and then rinsed well with water for injections.

In a purified 10-12-1 glass flask with reflux condenser and stirrer motor, as described above, 9 water are introduced for injection purposes and dissolved therein with heating to 50-60 C 100 g of poly I: C with so-called interferon buffer.

In this 100 g of the above mixture are 10 g of poly I: C and 90 g of interferon buffer (consisting of 0.006 M Na ₃ PO ₄ · 12 H ₂ O and 0.15 M NaCl, adjusted to pH 7.0). After this. Dissolution of the solid is made up to the final volume of TO 1 with water. In the finished solution is made up to the final volume of 10 1 with water. In the final solution, 50 g of chloroethene are added and stirred at 80 C at reflux for 1 hour. The solution is cooled to room temperature. Now the solution

spray-dried or filled to 5 ml in sterile ampoule bottles under aseptic conditions and lyophilized under conditions previously considered optimal.

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Example 2

Sterile Poly A: U-Lyophili sat

As described in Example 1, all equipment necessary for the production are cleaned. Thereafter, 31.2 g of poly A: ü are dissolved in 2.9 1 of water for injections.

There are added 14.5 g of chloroetone and heated for 1 hr. At 80 C under reflux and stirring. After cooling to room temperature, the solution is spray-dried. You can also fill the cooled solution to 5.1 ml in the sterilized vial bottles and lyophilize under the previously determined as optimal conditions.

Example 3 Sterile blood plasma

500 ml of blood plasma are mixed with 2.5 g of chloroetone and heated to 35 C for 15 min. The solution is cooled to room temperature and lyophilized under conditions previously considered optimal.

Example 4 Sterile t-RNA lyophilisate or spray granules

500 mg of transfer ribonucleic acid (t-RNA) are dissolved in 500 ml of 1.1% interferon buffer solution and heated to 60 ° C. with addition of 5.0 g of diethyl pyrocarbonate. Thereafter, the solution is lyophilized or spray dried under determined conditions.

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Ex iel 5

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Sterile polyadenylcytidic acid (Poly A: .C)

500 mg of polyadenyl-Cytidylsäure (poly A: C) are dissolved in 500 ml of 1,1 "iiger interferon buffer solution and heated with the addition of 5.0 g of chloroethenone at 60 ⁰ C for 1 hour.

Subsequently, the solution is lyophilized or spray-dried under determined conditions.

Example 6 15

Sterile polyadenyl-guanyl-uridylic acid (poly A: G: U)

500 mg of polyadenyl-guanyl-uridylic acid (poly A: G: U) are dissolved in SOO ml of 1- iger Interferonpufferlösung and heated with the addition of 5.0 g of chlororetone for 1 hr. At 60 ⁰ C.

Thereafter, the solution is lyophilized or spray dried under determined conditions.

Example 7

Sterile thionicotinamide adenine dinucleotide phosphate (thio-NADP) 3o

1.0 g thionicotinamide adenine dinucleotidphoshat (thio-NADP) are dissolved in 500 ml of water and heated with the addition of 5.0 g of diethyl pyrocarbonate for 1 hr. At 60 ⁰ C. Subsequently, the solution is lyophilized or spray-dried under the determined conditions.

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example Sterile 2-deoxy-adenosine 5'-triphosphate (dATP) (disodium salt)

1.0 g of 2-deoxy-adenosine 5'-triphosphate (dATP) (disodium salt) are dissolved in 500 ml of water and heated with the addition of 5.0 g of diethyl pyrocarbonate for 1 hr. At 60 ⁰ C. Subsequently, the solution is lyophilized or spray-dried under the determined conditions. 1o

In all cases, a low-germ, highly viscous solution with high interferon-inducing effect resulted after redissolution of the lyophilisates obtained in this way (the latter does not apply to Example 3).

Claims (3)

Erfindunffsanspruch ή ' Verfahren zar production of sterile preparations of. Blood plasma, t-ribonucleic acid, polyinosinic-polycytidylic acid complexes (poly I: C-), polyadenylic acid-polyuridic acid complexes (poly A: U), polyadenylic acid-cytidylic acid (poly A: C), polyadenylic acid-guanilic acid-uridylic acid (poly A: G: U), ^ hionicotinamid adenine dinucleotide phosphate (thio-NADP) or 2-deoxy-adenosine 5 'triphosphate · (dATP) (disodium salt), characterized in that d n ate egg preservative or disinfectant can act amittel , then lyophilized or spray-dried and, if appropriate, subsequently converted by addition of customary sterile auxiliaries to a pharmaceutically acceptable form or suitable for use on isolated animal or human cells or cell cultures. 2 /, procedures after. Item 1, characterized dadureh, .daß that the preservative or disinfectant is selected from the group of chloroetone, benzyl alcohol, p-chloro-m-cresol and pyrocarbonic acid diethyl ester. 3., Method according to point ^, characterized in that. That the preservative or disinfectant is chloroetone or pyrocarbonic acid diethyl ester. ty. Method according to one of the items 1 to 3; characterized dadureh that the preservative or disinfectant 10 min - 4 hours at 25 to 100 ⁰ C acts. Method according to point H _t characterized dadureh that the preservative or disinfectant 30 minutes to 2 hours at 40 to 80 ⁰ C acts.