DE2201513A1 - New modification of natural double-stranded ribonucleic acids with antiviral activity and reduced acute toxic effect, processes for their production and medicinal preparations containing such a modification - Google Patents

Description

"New modification of natural double-stranded ribonucleic acids with antiviral activity and reduced acute toxic effect, processes for their preparation and a Medicinal preparations containing such modification "

Priority: January 14, 1971, Great Britain, No. 1940/71

It is known per se that double-stranded ribonucleic acids and their salts, which are hereinafter abbreviated as RNA, represent highly effective interferon inducers and are therefore of great importance for the broad-spectrum prophylaxis of viral infections and to a lesser extent can also be used to treat such infections. These Activity as interferon inducers are shown by both the synthetic ones as well as the RNA of natural origin. Double-stranded ribonucleic acids with antiviral activity can e.g. Virus particles are extracted, which are found, among other things, in certain infected fungi, such as in some strains of Penicillium chrysogenum, Penicillium funiculosum, Penicillium stoloniferum. In addition, such ribonucleic acids come in cytoplasmic polyhedrosis viruses, in the Reovirus 3 virion, as well

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in the replicative phage form of MS2 from E. coli and in the replicative phage form of MU9 of a mutant E. coli.

However, there is evidence that double-stranded ribonucleic acids Of natural origin in humans and animals exert too high a toxic side effect and therefore their application limited in human and veterinary medicine.

Based on these facts, there is a need to assess the toxicity of such natural double-stranded ribonucleic acids to reduce, but at the same time maintain their favorable activity as an interferon inducer and thus their antiviral activity th.

Surprisingly, it was found that this important problem by a new modification of natural double-stranded Ribonucleic acids can be dissolved.

Accordingly, the invention relates to a new modification of natural double-stranded ribonucleic acids with antiviral ones Activity and reduced acute toxic effect, which is characterized in that it

a) reduced hyperchromicity,

b) more strongly degraded molecules _;

c) a lower viscosity than solution as well

d) has a higher LDc _Q value when administered intraperitoneally in mice compared to unmodified ribonucleic acid of the same natural origin,

e) the same composition with regard to the base building blocks and

f) the same sedimentation behavior on a sucrose gradient

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served

like an unmodified ribonucleic acid of the same

of natural origin shows

g) in the event of degradation up to the complete irreversible separation of the molecular strands under conditions which per se are not hydrolysis of the phosphate diester bonds cause single polynucleotide strands to form, which are of lower molecular weight have the same as single strands of the unmodified ribonucleic acid formed under the same conditions of natural origin and

H). in mice infected with encephalomyocarditis virus in vivo Shows antiviral activity 24 hours after dosing.

The invention also relates to salts of such modified ones Ribonucleoic acids, "where ammonium salts, amine salts and metal salts are particularly suitable.

About the differences between the modification according to the invention and the normal natural double-stranded ribonucleic acids To understand correctly, the characteristic properties of such a normal form of double-stranded ribonucleic acids must first be understood explained. Such a normal form is understood to mean that form in which the relevant Ribonucleic acids by extraction from the corresponding naturally occurring substances are obtained. By means of such extraction measures, all substances are separated off, which are not double-stranded ribonucleic acids. This extraction is carried out under conditions in which the powert is kept in the range of 2 to 10 and where no degradation of the double-stranded molecules of the ribonucleic acids in

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shorter nucleotide fragments or polynucleotide fragments occur, which contain less than about 25 nucleotide building blocks and which also have no change in composition of the base units of the ribonucleic acid in question occurs. The above extraction conditions seem to be very sharp, but the applicant is not aware of any literature which would describe extraction measures, which are not carried out in compliance with the aforementioned conditions. Accordingly, it really is under the conditions listed above, a mode of execution which is the customary or normal form of the double-stranded Supplies ribonucleic acids. Examples of such extraction methods for obtaining double-stranded ribonucleic acids from natural starting material are described in British patents 1,170,929 and 1,211,142 as well as in Belgian patents No. 739 424 and 739 425.

There is a very general view that the normal form of double-stranded ribonucleic acids obtained in this way is made up of double-stranded molecules a high degree of order exist, with between the facing base building blocks of the individual strands there is a strong hydrogen bond. The two single strands of each molecule of such a normal Double-stranded ribonucleic acid consists of single uninterrupted polynucleotide chains and when examined by means of the electron microscope, the molecules of such a normal double-stranded ribonucleic acid are presented as long thread-like structures, which are only slightly curved.

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In contrast to this, the molecules of the new modification according to the invention are naturally occurring double-stranded ribonucleic acids more degraded. The term "more strongly degraded" ("aggregated") is understood in the context of the invention, that the total amount of double-stranded ribonucleic acid when treated on a column of a gel swollen with water from agarose (agarose is a linear polysaccharide made up of alternating units of D-galactose and 3,6-anhydrc-L-galactose) with a molecular weight limit for proteins of 20 to 30 χ 10 completely into the void volume of the gel is eluted. (Such an agarose gel can be produced from commercially available agarose, for example from the commercial product "Sepharose 2B").

An indication of the greater degradation of the molecules of the invention Modification of double-stranded ribonucleic acid results from the behavior during gel electrophoresis Use of polyacrylamide gels. It turns out that the new modification has a lower mobility in such Gel electrophoresis has compared to the normal form of double-stranded RNA.

Qualitative evidence for the existence of such a degradation is obtained from observation under an electron microscope the non-branched, only slightly curved thread-like molecules of the normal form of RNA differ very well from the shorter ones Molecules of the new modification can be distinguished, which show a rougher or more angular plain crack shape.

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The present invention also relates to a method of extraction this new modification of natural double-stranded Ribonucleic acids, which ^ is characterized in that a double-stranded ribonucleic acid of natural origin is used a p n value of 11.0 to about 12.5 in an aqueous electrolyte solution with an alkali metal hydroxide under such conditions as the temperature, the treatment time, the Electrolyte concentration and the p "value treated that the both polynucleotide molecular strands begin to separate from one another immediately, but no more than a total of 25 percent each Strand of the phosphate diester bonds are hydrolyzed, and that the treatment solution is then neutralized.

If desired, the new modification of the double-stranded ribonucleic acids can be obtained in solid form from the treatment solution by precipitation with ethanol or by freeze-drying. Of course, the reaction parameters, namely the temperature, the duration of treatment, the electrolyte concentration and the Pu value, can be varied practically continuously. For severe conditions, that is taking place too high a _pH value, too high a temperature, however, are too long reaction times and high electrolyte concentrations are avoided, so not a real degradation of ribonucleic acids. The choice of the individual treatment conditions cannot be prescribed, but depends on the nature of the ribonucleic acid concerned, that is to say on the starting material from which it has been obtained. In general, however, it is expedient, treatment at room temperature, that is in the range of 20 to 25 ^0C and ₁ in an electrolytic solution to carry out,

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which has a molarity of 0.1 to 0.8. The p ^ value is expediently chosen under these conditions in the range from 11.0 to 11.8 and the duration of treatment is then in general about 5 to 30 minutes. In this way, the new modification of the double-stranded RUS will certainly be in general educated.

If desired, the method of treatment defined above can also be carried out with the extraction of the double-stranded ribonucleic acid be combined from the relevant natural starting material, so that you can then immediately the new modification according to the invention receives.

The invention also relates to medicinal preparations which have such Contains modification of a natural double-stranded ribonucleic acid as an active ingredient, optionally in combination with. one or more pharmacologically acceptable carriers and / or auxiliaries. Such medicinal preparations can be formulated for oral, parenteral or topical administration in particular they can be prepared in the form of a nasal spray, particularly expediently in the form of an aerosol.

The invention is illustrated in more detail by the following examples.

example 1

400 mg of a double-stranded RNA extracted from virus particles of Penicillium chrysogenum (ATCC 10002) are dissolved in 80 ml dissolved in a 0.15 molar sodium chloride solution. From this solution three portions of 26 ml each are taken and placed in the

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treated in the following way.

1st part: Dilute with 0.15 molar sodium chloride solution

up to 130 ml. This preparation is hereinafter referred to as preparation INT 218.

2nd part: It is treated with 6.5 ml of a 0.1 η sodium hydroxide solution. The pji value of the solution is 11.8. This solution is left to ^{stand for 10 minutes at 2O 0} C, then neutralized by adding 32.5 ml of 0.02 η hydrochloric acid and finally diluted by adding 65 ml of 0.23 iuular sodium chloride solution to a total volume of 130 ml The solution obtained contains 1 mg / ml of the modified double-stranded RNA in a 0.15 molar sodium chloride solution with a Ρτ, value of 7. This preparation is hereinafter referred to as preparation INT 219.

3rd part: You treat in the same way as the second

Portion, but carries out the alkali treatment for a total of 20 minutes. That way you get a preparation, hereinafter referred to as preparation INT 220.

Physico-chemical properties
1 · Tm value and hyperchromicity

These two parameters are determined by recording the UV absorption of the test material at a certain wavelength, the temperature of which is gradually increased. The UV absorption a double-stranded RNA at 258 mu is always less than the absorption of the same material in single-stranded

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ger shape at the same wavelength. As the temperature of the double-stranded material rises, the hydrogen bond between the strands becomes somewhat weaker, until the strands finally separate. It follows that the UV absorption of a double-stranded material decreases with increasing temperature. The difference between the two limit values of the absorption, expressed as a percentage of the absorption of the double-stranded material, is referred to as the "hyperchromicity" of this material (see 1. P. Doty, H. Boedtker, JR Fresco, R. Haselkorn and M Litt, "Proc. Nat. Acad. Sci." Wash., 45, -482 (1959).
2. J. Marmur and P. Doty, "J. Mol. Biol.", 5., 109 (1962))

So if the UV absorptiori at 258 mu against the temperature is ~ worn, it is found that the absorption is greater at high temperatures than at low temperatures. The increase in absorption goes on gradually and it is possible to determine a temperature at which the absorption is in the middle lies between the absorption of the double-stranded material and the single-stranded material. This temperature is called the Denotes Tm value.

The hyperchromicity and the Tm value are measured on solutions in 0.15 molar sodium chloride solution in 1 cm cuvettes using a Unicara spectrometer of the SP 800B type. The temperature of the test solutions is increased ^{by 1/2 ° C. every minute.} The measured values obtained are summarized in the table below.

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- 10 - Gel electrophoresis 2201513 preparation Hyperchromicity. %
(258 mu) Tm value INT 218 38.2 92 INT 219 30.0 90 INT 220 29.4 89

The electrophoresis is carried out using a 4prozentigcn acrylamide gel and one with acetic acid to a ρύ value of 7.8 adjusted 0.04 molar Tris buffer, which is 0.02 mol Sodium acetate and 2 nano-molars of the sodium salt of ethylenediaminetetraacetic acid is carried out. The electrophoresis is carried out in a tube 0.6 cm in internal diameter and about 10 cm

_o man

Length performed at 20 C with M for up to 3 hours Voltage gradient of 10 volts / cm at 5 mA / gel applies. The gel will then removed and stained with a methylene blue solution. The bands that appear are evaluated.

The modified ribonucleic acid of the preparations INT 219 and INT 220 appears in the form of a single band on the gel. the However, unmodified double-stranded RNA of the preparation INT 218 shows a higher mobility and therefore splits into three gangs up.

3. Chromatography on a column made of "Sepharose 2B" The chromatographic treatment is carried out on a column 90 cm in length, an internal diameter of 2.5 cm and a volume of about 500 ml. The column is eluted in the upward direction with a solution which contains 0.15 mol of sodium chloride, 0.05 mol of Tris buffer and 0.05 mol of magnesium chloride and has a Pu value of 7.5. It gets with a flow rate

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worked from 0.4 ml / minute. There are fractions of 7.2 ml collected. The nucleic acid content of the individual fractions is determined by measuring the UV absorption at 260 μm and recorded.

The elution diagram for the preparation INT 218 indicates that the nucleic acid with the highest molecular weight has been eluted into fractions nos. 16 to 21 (maximum content at fraction No. 18, Kav = 0.05).

Kav ⁺ ) = distribution coefficient between liquid phase and gel phase In the case of the preparations INT 219 and 220, on the other hand, the main component is eluted into fractions no. 16 to 19 (maximum content in fraction no. 17, Kav = 0).

4. Sedimentation analysis

A 100 ug ribonucleic acid-containing solution is a preformed 5 to 25 percent sucrose gradient in 10 mM Tris-HCl, 150 mM NaCl with a p _H ~ ¥ ert of 7.0 overlaid. It is then centrifuged for 5 hours at a temperature of 20 ° C. in a Beckmann centrifuge, type Spinco SW50, at an acceleration of 100,000 g. A UV analyzer (type Iscq model 222) is used to determine the material distribution at a wavelength of 254 mju.

Under these conditions, the sedimentation speed of the preparations INT 219 and 220 does not differ noticeably from that of the preparation INT 218, which contains the unmodified form of double-stranded RNA.

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Acute toxicity and activity of the preparations INT 218, INT and INT 220 against the encephalomyocarditis virus (EMC) Test methods;
Toxicity:

Groups of 8 test animals each (mice of strain CD1, Live weight 18 Ms 22 g) the preparations concerned are administered intraperitoneally in an amount of 5, 10, 20, 40, 60, 80, 110, 125 or 150 mg / kg are injected. The test animals are then observed for 8 days and the deaths that occur are recorded recorded (see FIG. 2). The dose of each preparation which is fatal for 50 percent of the examined mice, was determined according to the method of Reed and Muench (see Reed, L.J. and Muench, H. (1938) "American Journal of Hygiene", Vol. 27, p. 493).

Protection against the EMC virus

24 hours before infection with the 160-fold or 16-fold LDcQ dose of EMC virus will be groups of 10 mice each injected intraperitoneally with doses of the preparations INT 218, INT 219 and INT 220. One uses doses of the preparations in question of 0.5, 5, 50, 250 jig / kg or of 0.5, 2.5, 5 and 10 mg / kg. During the following 10 days, the Deaths are registered and the total mortality is shown graphically in FIG. 1 below.

■ Results

The acute toxicity LDc ₀ for intraperitoneal administration is derived from the following summary:

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INT 218 40 mg / kg INT 219 140 mg / kg INT 220 150 mg / kg

From Figure 1 it can be seen that the level of the antiviral protective effect at the higher virus dose in the sequence INT 218, INT 219 and INT 220 corresponds. At the lower virus dose, however, all three preparations practically show that same level of antiviral protective effect.

In appreciating these results, however, it must be borne in mind that the method used for evaluating antiviral activity does not show particularly good reproducibility when using groups of only 10 test animals. For example, lie the results of the preparations INT 218 and 219 with both virus doses within the range of variation, which experience has shown also occurs if the two identical tests are carried out on the same day with only the preparation INT 218.

Electron microscopic examination

For this investigation, preparations of the natural double-stranded RIiS as well as of the modifications according to the invention (preparation INT 219 and INT-220) according to the microdiffusion technique of Mayer and Jordan (1968) produced.

Stock solutions of the natural double-stranded RNA and the modifications in a concentration of 1 jig / ml in 0.2 molar Ammonium acetate solution are microdiffused into a Layer of denatured Cytochrome C (starting concentration 10 fng / ml each stored. Contrast vapor deposition is then carried out by means of a platinum-palladium alloy (80:20), the pre-

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is held ready at an angle of 8 °. The preparations made in this way are then in a Phillips electron microscope, type EM 300, at a high voltage of 40 KV and a magnification of 11,000 examined. This is followed by a photo-optical re-enlargement of up to 30,600.

At the concentration of 1 jig / ml shows the untreated double-stranded RNA a variety of unbranched filamentous Molecules of various lengths, which, however, have only a slight curvature over the entire length (cf. Figure 3).

The preparations INT 219 and INT 220, on the other hand, show molecules that are shorter and have a sharper outline (see Figures 4 and 5). The molecule length is shorter in the transition from * preparation 219 to preparation 220 and the angularity of the molecules decreases to. In contrast, no change is observed in the width of the molecules.

Example 2

This example illustrates the structural changes that occur in double-stranded RNA with different alkaline pjj values .

To get a closer look at those conditions, which are necessary to the modifications according to the invention with degraded molecules of double-stranded RNA according to the example 1 (preparations INT 219 and INT 220) and improve the physico-chemical properties of these modifications to recognize, experiments are carried out in which the

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Immediately developing hyperchromicity is measured and for which this parameter is also measured after several days in one 0.15 molar sodium chloride solution with different p ^ values is determined.

Test conditions

1 ml of a solution of double-stranded RNA in 0.75 molar sodium chloride solution, which contains 250 µg of the RNA in question per ml, is added to each 4 ml of sodium hydroxide solutions with alternating normality in the range from 0.001 η to 0.1 η. Immediately after adding the RNA solution, mix well and then determine the UV spectrum. The pjj value of the solution is then measured. After 4 days of standing at 20 ° C the UV spectrum and the p _H is - ¥ ert investigating solutions in turn determined. In Figure 6 is the immediate hyperchromicity as well as the terminal hyperchromicity. each determined at a wavelength of 258 mp, plotted against the p ^ value.

Experimental results and discussion of the same

When evaluating the test results, various aspects of particular interest emerge. First, it is apparent that the double-stranded RNA at 20 ⁰ C in 0.15 molar sodium chloride solution to a PJR value of 11.0 is completely stable. In a p ~ value of 11.4 _H is an immediate Hyperchromicität starts to become noticeable and this sharply increases, up to a _pH value of 12.1. A further increase of the p _H -value has no further effect on the immediate Hyperchromicität which in this final state has a magnitude of 29 percent. This immediate or immediate hyperchromicity is due to the removal of protons from the hydrogen bonds.

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fertilize between complementary base units in the two molecular strands, the protons being removed by the hydroxyl ions present in the test medium. This results in a loosening of the two strands of the double helix and with p ^ values of 12.1 or above the nucleic acid in question therefore exists in single strand form. When the double-stranded RNA p _H values is exposed to over 11.0 a long time, then finds a hydrolysis of Phosphatesterbindungen in Nucleotidmolekülen the single strands instead, WO · is further increased by the Hyperchromicität. After 4 days at 20 ⁰ C at a _pH value of 12.3 and above this hydrolysis is complete. The final value of hyperchromicity is then 53 percent.

Under the conditions under which the formation of the modifications according to the invention (preparations INT 219 and 220) is observed, namely at a p ^ value of 11.8 _; Separation of the molecular strands up to about 50 percent takes place immediately and, in addition, to some extent hydrolysis of the phosphate diester bonds is also observed.

It is possible that the detached single strand parts then in turn form hydrogen bonds with other segments of the molecular chain enter, but then a different structure is formed than was present in the original double-stranded RNA Has. That means that a very significant change in the structure of the molecule occurs and this change could well explain the physico-chemical properties observed.

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■ Example 3

This example illustrates the influence of the concentration of the sodium chloride solution on the training of the various modifications. In particular, the influence of the sodium chloride concentration becomes investigated for the hyperchromaticity of the double-stranded RNA that sets in immediately at alkaline pjr values.

Test conditions

For each 4 ml of sodium hydroxide solutions of varying concentration In the range from 0.001 η to 0.1 η, 1 ml of an RNA solution is added in each case, which contains 250 ^ g / ml of the RNA in question and which has been dialyzed against a solution containing 5 times the desired final sodium chloride concentration. Immediately after adding the RNA solution, mix carefully and then determine the UV spectrum as well as afterwards the pu value. The results achieved in water, in one 0.15 molar sodium chloride solution and in a 0.75 molar sodium chloride solution are shown graphically in FIG.

Experimental results and discussion of the same

With decreasing salt concentration v / ground higher p _H values needed to modify the double-stranded RNA. Only minor differences are observed between the 0.15 molar and the 0.75 molar sodium chloride solution, but a significantly higher p ^ value is required in pure water in order to separate the two molecular strands from one another. In addition, this separation takes place only in a narrower Pu value range. Corresponding results have also been observed with deoxyribonucleic acids (cf. FW Studier, "J. Mol. Biol." (1965), Vol. 11, p. 373) and these results are based on the fact

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attributed that in the absence of positively charged counterions the approach of the hydroxyl ions is prevented by the negative charge from the nucleic acid.

Example4

This example explains the residual hyperchromicity, i.e. the irreversible residual hyperchromicity that occurs after neutralization of solutions of double-stranded RNA in 0.15 molar sodium chloride solution and is still present in water after these solutions have previously been exposed to an alkaline p "value.

Test conditions

^{1 ml of a solution of double-stranded Rl 1} JS in 0.75 molar sodium chloride solution or in V / water is added to each 4 ml of sodium hydroxide solutions of varying concentration in the range from 0.001 η to 0.1 η, these solutions having a concentration of 250 yg RNA per ml. Immediately after adding the RNA solution, mix thoroughly, then leave to stand for 10 minutes at 20 ° C. and finally neutralize with 1 ml of a hydrochloric acid solution, the normality of which corresponds to four times the normality of the sodium hydroxide solution used. The UV spectra of the examined solutions are then determined. For the investigation solutions of the RNA in pure water, an identical volume of 0.3 molar sodium chloride solution is added before the UV spectrum is measured. In Figure 8,

thus determined Resthyperchromicität which the dilution factor was corrected for, plotted against the _pH-value.

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Test result and discussion of the same

In 0.15 molar sodium chloride solution, the Hyperchromicität lOminütigen after a treatment at 20 ⁰ C at pu values below 11.7 is completely reversible. From the course of Hyperchromicität skurve of Figure "1 it can be seen that when the _pH value of 11.7 to about 50 percent occurs a separation of the two strands of the molecule. A sharp increase in the irreversible Hyperchromicität occurs in the range of ^ p 11.7 to 12. At even higher p ^ values, the increase in hyperchromicity increases more slowly. The sharp increase in the hyperchromicity curve reflects the constantly growing large entropy factor, which is associated with a complete reunification of the two molecular strands as soon as the first Separation has become practically complete. But even after this treatment at the high p n value of 12, 50 percent reunification to a double strand appears to take place as a result of the neutralization hydrolysis takes place and this hydrolysis is likely to lead to the further development of irreversible hyperchrorai city is responsible for ρττ values above 12 and could also play a role in the pjr value range from 11.7 to 12.

Even with the test solution in pure water, the strong increase in irreversible hyperchromicity occurs in that one PtT value range which is responsible for a separation of the molecular strands (see FIG. 7). A reunion of the However, single strands to form a double strand are not as effective at a low ion concentration and this is a reflection

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again in the high residual hyperchromicity, after which the Solution pjj-V / erten was exposed to which values above those which are necessary for a separation of the individual strands. When the ion concentration of the neutralized solution is adjusted to a value of 0.15 molar with sodium chloride, then there is a further union of the single strands instead of.

Under those conditions in which the preparation INT 219 was produced (p n value 11.8) is just the beginning of one The development of irreversible hyperchromicity can be observed. this is in agreement with the observation that the preparation INT 219 exhibits reduced hyperchromicity after thermal modification compared to that of unmodified double-stranded RNA.

Example 5

This example illustrates the rate of hydrolysis. The observation, that a higher p ^ value is required for the separation of the individual strands from one another in water than for a solution in 0.15 molar sodium chloride appeared to make it possible to double-strand the internucleotide phosphate bonds To hydrolyze RNA in water at lower p ^ values than are necessary for a separation of the molecular strands. Accordingly, a study of the rates of hydrolysis was carried out at a PjT value of 11.8 in water and in sodium chloride solution, respectively.

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Test conditions

1 ml was added to each 4 ml of a 0.03 η sodium hydroxide solution a double-stranded RNA solution in 0.75 molar sodium chloride or added in v / ater, which contains 235 µg RNA per ml contained. The ρττ value of the test solution was always 11.8. The hourly UV absorption of the two solutions was then measured at a wavelength of 258 μm for a total of 16 hours determined. In FIG. 9, the optical density is plotted against the duration of the experiment.

Experimental results and discussion of the same

After the hyperchromicity initially assumes a corresponding value as a result of the separation of the double strand into single strands, a reduced increase in optical density is observed in the test solution with 0.15 molar sodium chloride, which then linear with respect to time. This linear increase in hyperchromicity is due to the hydrolysis of the. Phosphate bonds in the nucleotide molecule are returned.

In the case of the test solution in pure water, on the other hand, there is no immediate separation of the molecular strands and the subsequent hyperchromicity increases to a much less pronounced extent. Since the total hyperchromicity is low anyway (about 6 percent after 16 hours) it is not possible to decide whether this hyperchromicity is a result of a separation of the single strands or a result of hydrolysis, but it is evident from the experimental results that with a small one ion concentration of both the Strarigtrennung than required for the hydrolysis _pH value is much higher than at a higher ion concentration.

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These speed investigations can also be used as an approximation calculate the number of continued fractions that occurred during the formation of the modification according to the preparation INT 219 occur from natural double-stranded RNA.

It has been shown above that the complete separation the hyperchromicity based on molecular strands is a value of 29 percent and that the total hyperchromicity, which is caused by the separation of the molecular strands and the hydrolysis stems from, is 53 percent. The hyperchromicity based on the total hydrolysis therefore has a value of 24 percent. From FIG. 4, the increase in hyperchromicity in 0.15 molar sodium chloride solution at a p ^ value of 11.8 after the Separation of the individual strands can be calculated as follows: '

= 0.05 units of optical density within 10 hours.

χ 100 = 5 »3 percent hyperchromicity within 10 hours

= ■ ■ ·· = 0.0883 percent hyperchromicity in .10 minutes. 60

If, therefore, a molecular weight of 2 χ 10 is assumed for double-stranded RNA and an average molecular weight of 340 is fixed for the nucleotide structural unit, then the number of chain breaks per molecule of double-stranded RNA results

_ 0.0883 " 2 χ 10 ⁶ _ ₉₉

Accordingly, the number of continued fractions per single strand is 11,

A corresponding calculation for the preparation INT 220 shows that there are 22 continued fractions per single strand.

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Example 6

This example illustrates the influence of the cations on the formation of the modified double-stranded RNA.

The result of a treatment of double-stranded RNA with cesium hydroxide in 0.15 molar cesium chloride solution is compared with the Effect of sodium hydroxide in 0.15 molar sodium chloride solution compared.

Test conditions

1 ml of a solution of double-stranded RNA in 0.75 molar cesium chloride solution, which contains 250 pg of the RNA per ml, is added to each 4 ml of solutions of cesium hydroxide of different concentrations in the range from 0.001 η to 0.1 η. Immediately after adding the RNA solution, mix carefully and measure the UV spectrum. In addition, the PjT value of the solution is then measured.

In FIG. 10, that is determined at a wavelength of 258 μm Hyperchromicity plotted against the ρττ value and with those Data have been compared, which can be obtained in a corresponding manner using sodium hydroxide in a 0.15 molar sodium chloride solution receives.

Experimental results and discussion of the same

In the presence of cesium ions, a separation of the single strands occurs at a lower pjj value than in the presence of sodium ions. Similarly, it has been reported for deoxyribonucleic acids that a separation of the single strands occurs in the lower the p ^ value, the larger the ionic

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radius is the relevant counterion (cf. M. Ageno,

E. Don and C. Frontali, "Biophys. J." (1969), Vol. 9, p. 1281).

Example7

This example explains the modification of double-stranded RNA using sodium carbonate solutions, with the increase in particular the hyperchromicity is measured.

Test conditions

Solutions of double-stranded RNA in water (162 μg RNA / 1.5 ml) are freeze-dried and 3 ml of n-sodium carbonate solution or 0.1 η sodium carbonate solution or water are added to the solids thus obtained. The UV spectra of these solutions are measured immediately after their preparation and then every half hour for a total of 18 hours at a wavelength of 258 rough. The optical density determined in this way is plotted against the duration of the experiment in FIG.

Experimental results and discussion of the same

In 0.1 η sodium carbonate solution, with a p ^ value of 11.3, neither a separation of the individual strands nor hydrolysis takes place. In an n-sodium carbonate solution (p _H 11.6), although the Hyperchromicität immediately entering relatively low (9.2 percent) a separation of the single strands will However, quite rapidly and held and is completed in about 2 hours. The further increase in hyperchromicity, which can be attributed to the hydrolysis reaction, takes place somewhat faster (by 6 percent within 10 hours) than with a p ^ value of 11.8 in 0.15 molar sodium chloride solution. This behavior can be attributed to the somewhat higher concentration of sodium ions.

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The difference in the relative rates of increase with regard to the separation of the single strands and the hydrolysis in n-sodium carbonate (p ^ 11.6) and in 0.15 molar sodium chloride solution with an addition of 0.02 n-sodium hydroxide (ρ _Η 11.8) shows indicates that the action of n-sodium carbonate on double-stranded RNA does not result in the same products as those in the preparations INT 219 or INT 220. This fact is confirmed by an experiment in which double-stranded RNA is treated with n-sodium carbonate solution for 10 minutes and 20 minutes at 20.degree. The thermal modification properties of these products, measured in 0.15 molar sodium chloride solution, are identical to those of the double-stranded RNA originally used. Although these products show good antiviral protective action, an increase in the PD (-Q values is observed with the increase in the treatment time with sodium carbonate and therefore sodium carbonate is not considered to be a suitable reagent for preparing the novel modifications of double-stranded RNA according to the invention.

Example 8

Physico-chemical properties of the preparations INT 219 and INT 220

In addition to the physical properties of these preparations given in Example 1, there are also the following Properties have been investigated:

1. A comparison of the thermal modification of INT 218 and INT 219 examined in citrate-buffered saline solution (see FIG. 12).

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In a medium composed of 0.15 molar sodium chloride and 0.015 molar trisodium citrate with a p ^ value of 7, these correspond to Curves for the hyperchromicity are mutually exclusive, although those with the preparation INT 219 in unbuffered 0.15 molar sodium chloride solution the overall hyperchromicity is somewhat lower, and the Tm value is also 2 ° lower. On the other hand will be in a medium of 0.015 molar sodium chloride solution and 0.0015 molar trisodium citrate with a ρττ value of 7 however, large differences were observed. With the preparation INT 219, denaturation begins at very low temperatures and the corresponding curve appears to be in three phases. However, the overall hyperchromicity for the preparation INT 219 is again significantly less than for preparation 218 and also the Tm value is lower. *

2. Gel permeation chromatography (spherical agarose particles

74 to 149 μ) The chromatographic treatment is carried out on a column of

26 cm in length, an inner diameter of 2.5 cm and a volume of about 130 ml. The column is upstream. eluted with a solution which contains 0.15 mol of sodium chloride, 0.05 mol of Tris buffer and 0.005 mol of magnesium chloride and has a Pjr value of 7.5. A flow rate of 0.08 ml per minute is used. In each case, fractions of 3> 3 ml are removed. The ribonucleic acid content of the fractions is determined by measuring the UV absorption at a wavelength of 260 μm. The elution scheme of the preparation INT 218 indicates that the nucleic acid with the highest molecular weight is eluted into fractions nos. 16-27. (Maximum content in fraction No. 21, Kav = 0.2).

209831/1091.

INT
In the case of preparation ¹ '219, the main component is eluted into fractions no. 13 to 18 (maximum content in fraction no. 15, Kav = O).

3. Viscosity

The viscosity measurements are carried out in a micro-capillary viscometer, which is a photoelectric timer. Preliminary investigations have shown that the size ^ specific / c hardly depends on the extent of the shear force as soon as the absolute size is> 10 dl / g. The viscosities were ^{measured at 25 °} C. in a 0.15 molar sodium chloride solution. The results obtained are shown in FIG.

4. Stability

INT
The stability of the preparation '' 219 when precipitated by means of ethanol and freeze-drying was investigated because these are measures which are usually used in the isolation of nucleic acids. After precipitation from a solution in 0.15 molar sodium chloride containing 1 mg / ml of ribonucleic acid, with 2 parts by volume of ethanol being used, the total content of ribonucleic acid in the preparation INT 219 was recovered. In addition, a corresponding solution was freeze-dried. In both cases, the solid product obtained was again worked up to a solution in 0.15 molar sodium chloride which contained 1 mg of the ribonucleic acid in question per ml. These solutions showed identical physicochemical properties (hyperchromicity, Tm value, gel electrophoresis and chromatography with Sepharose 2B) as the solutions of the original preparation INT 219.

20983171091

5. Denaturation with Formald ehy d

Test conditions were worked out under which an irreversible separation of the two single strands of naturally occurring double-stranded RNA and the alkali-modified product (preparation INT 219) occurs. For this purpose, 4 mg of the nucleic acid in question in 4 ml of a 0.15 molar solution of sodium chloride and treated at 50 ⁰ C with 1 ml 40prozentigem aqueous formaldehyde and 15 ml of dimethyl sulfoxide for 30 minutes. Previous studies had shown that unmodified double stranded RNA is denatured at 50 ⁰ C in 75prozentigem dimethyl sulfoxide, and it was therefore applied these relatively low reaction temperature to avoid a crosslinking reaction with the formaldehyde. The reaction solutions in question were then cooled, dialyzed at a _pH value of 7.5 0,5prozentige against 2 liters of formaldehyde solution, and then chromatographed by chromatography on a column of Sepharose 2B. A column 35 cm long with an internal diameter of 1.5 cm and a volume of about 60 ml was used for this. The column was eluted upstream with a solution which contained 0.5 percent formaldehyde and 0.15 mol of sodium chloride and had a p ^ value of 7.5. The flow rate was 0.08 ml / minute. Fractions of 3.3 ml each were removed. The nucleic acid content of the individual fractions was determined by measuring the UV absorption at a wavelength of -260 mu.

The results obtained are shown in FIG.

209831/1091

Although under these conditions, according to earlier observations regarding the degradation of the molecular chain, the preparation INT 219 occurs one fraction earlier than the original double-stranded RNA (preparation INT 218), the preparation INT 219, which has been denatured by means of formaldehyde and dimethyl sulfoxide, becomes two fractions
eluted earlier than the corresponding denatured original RNA in preparation 218. These results are in agreement
with the conclusions from the studies of hyperchromicity, according to which in the manufacture of the modification from
Preparation INT 219 from the original double-stranded RNA, about 11 chain breaks per molecular strand occur.

Example 9

Results of biological investigations A. Protective effect against the EMC virus .

The results of earlier investigations with regard to the antiviral protective effect of the preparations INT 218 and INT 219 are compiled in Table I and FIGS. 15 and 16. As already described in Example 1, the preparations in question
Administered 24 hours before viral infection. It can be seen from the results that the day-to-day observations
fluctuate and therefore it can be concluded that the difference in the protective effect between the preparations INT 218 and INT 219 is only insignificant.

209831/1091

NAL to
O Table I. - 30 - preparation Stanm: CDI) PräOarat INT 218, mortality 1 I. 2/11/70 28/1/71 9/2/71 3/30 ι
j 17/30 7/6/71 • I. 0/10 j Total percent 1 co. Protective effect of the preparations INT 218 and INT 219 against the EMC virus in mice ( dose
(mg / kp) 13/10/70 9/10 30/30 2/10! 6/30 14/29 9/10 I. 48/50 96 fa" Virus CEMC) 0.0005 9/10 29/30 I. 2/30 8/10 j 46/50 92 "** Dose (intra-
■ peritonealj 0.005 5/10 9/10 • 14/20 70 O 0.05 6/10 5/10
j ! 6 / 3O 3/30 27/50 54 «Ρ 0.25 7/10 2/10 13/30 0/10 5/10 32/70 46 10 ~ ⁴ 0.5 5/10 0/10 φ ο 0/10 [ 3/30 0/10 0 (16OxLD ₅₀ ) 2.5 0/10 2/10 I ο / ι ο 5/10 10/60 17th 1/10 ' 9/50 18 ' 10 7/10 24/40 <
60 0.005 4/10 18/39 46 I. ■ 0.005 2/10 4/40 10 y 0.05 1/10 ; 1/20 5 y 0.25 4/10 i 7/60 12: 0.5 1/10 ^; 1/10 io I 10 ~ ⁵ 2.5 0/10 3/50 6 y (16xLD ₅₀ ) 5 0/10 0/20 : 0 N | 10 O cn co

Table I - continued -

Virus (EMC; * fm preparation l1 3/10/70 Preparation INT 219, 28/1/71 mortality i 24/30 7/6/71 i Total percent peritoneal) ίο "- ⁷ (ms / kff) 2/11/70 9/2/71! 25/30 8/10 i 45/50 90 (16XLD ₅₀ ) 0.0005 9/10 28/30 y 6/10 42/50 84 ■ * 0.005 8/10 28/30 10/30 9/10 17/20 85 10 ~ ⁴ 0.05 8/10 8/10 8/30 39/50 78 (16OXLD ₅₀ ) 0.25 8/10 7/10 6/10 17/30 6/10 48/70 69 0.5 3/10 9/30 3/10 30th 2.5 3/10 0/10 8/30 5/10 18/60 30 ' 5 3/10 2/10 13/50 26th 10 3/10 5/30 21/40 53 * 0.005 4/10 11/40 28 0.005 2/10 • L / 30 12/40 30th 0.05 4/10 3/10 5/20, j 25 0.25 2/10 0/10 1/10 7/60 12th 0.5 1/10 I 1/10 j 10 2.5 1/10 0/10 I 5/50 10 5 1/10 0/10 '1/20 5 10 ! 1/10

B. Intraperitoneal Toxicity Further studies were also carried out on the relative toxicities of the INT 218 and INT 219 preparations when administered intraperitoneally. The procedure was the same as in Example 1. The results obtained are shown in Table II below and in FIG.

It must again be pointed out that the observations vary from day to day, but the toxicity of the preparation is INT 219 considerably lower than that of the preparation INT 218.

209831/1091

Tabel

Intraperitoneal toxicity of the ING preparations I. 14/10/70 Preparation INT 218, mortality 20/1/71 15/2/71 j Total : 2ΐε 100 'and INT 219 at Mice i 0/8 j 20/1/71 3/10 10/10 15/2/71 Total % preparation 0/8 2/11/70 ι O / 16 I. 0/8 ■ 6/10 i 10/10 0/16 0 dose 0/8 0/8 0/20 0/20 0/56 % 88 • 0/8 10/10 j
t 0/16 t
0 5 5/8 0/8 6/20 7/20 " j - 18/56 0 Preparation INT 219, mortality i 10/10 0/20 0/36 0 i 10 6/8 0/8 '9/20 15/28 0 14/10/701 2/11/70 0/8 I 10/10 0/8 0! 20th 4/4 9/20 12/20 31/52 32 0/8 I.
i 2/20 2/32 6 I. 30th 4/4 6/8 4/4 54 0/8 1/8 1/4 25 y 40 20/20 12/20 37/48 60 0/8 0/8 • 4/20 8/38 21 i 50 5/8 20/20 15/20 43/48 100 0/8 8/20 14/38 ³⁷ h 60 8/8 77 0/4 0/8 20/20 30/30 icoj 80 8/8 89 1/4 0/8 ! * ί 100 ' 8/8 1/8 17/20 27/30 90 I. 110 8/8 100 1/8 13 \ 120 8/8 • 8/8 9/20 19/30 | 63 y 125 7/8 18/18 lOOJ 140 7/8 20/20 20/20 1001 150 10/10 1100; 160 K) 200 ro 0 cn co

C. Toxicity in the event of intravenous administration The intravenous toxicity of the preparations INT 218, INT 219 and INT 220 is determined in mice of the CDI strain with a live weight of 18 to 22 g. The test animals are observed for a total of 10 days and the deaths are determined (see Table III). The toxicity with intravenous administration is somewhat lower with the preparations INT 219 and INT 220 compared to that of the preparation INT 218. However, the differences are not as great as with intraperitoneal administration.

Table III
Intravenous toxicity of the preparations INT 218, INT 219 and INT

preparation dose
(mg / kg) mortality LD ₅₀ (mg / kg) INT 218
INT 218 10
20th
40 0/20
8/20
20/20 21 «
INT 219 10
20th
AO
80 0/20
5/20
12/20
10/10 32 INT 220 10
20th
• AO
80 0/20
3/20
8/20
7/10 A6

D. Intranasal protective effect against mouse influenza Mice of the PR8 strain were infected with the influenza virus in question for 1 hour by means of an aerosol preparation, virus particles with a size of about 8 yes being used.

- 201831/1091

30 hours or 6 hours before infection and 18 hours after

INT of the infection, the mice were administered either the preparation * ¹ 218 or the preparation INT 219, namely by means of an aerosol preparation, which was exposed for 30 minutes, a mist being generated by means of a solution containing 1 mg of the RInFS in question each ml in 0.15 molar sodium chloride.

From the numerical values given it can be deduced that each mouse has about 1.2 μg of the active ingredient within 30 minutes recorded. 10 days after the virus infection, the mice were examined and the extent of the hepatization of the lungs was determined determined, based on the following assessment of the lung condition. '

0 no injuries

1 0 to 14 percent injuries.

2 15 to 37 percent injuries

3 37 to 62 percent injuries

4 62 to 85 percent injuries

5 85 to 10% injuries

The evaluation in each test group with 20 test animals was totaled and this overall result became an average value educated. In addition, the number of mice with lung injuries in each group was determined and derived from it Percentage, based on the size of the study group, formed and this percentage below as the frequency of injuries given in percent.

. 209831/1091

Average assessment of the injured skin lung condition, of the percent

Negative control 2.0 85 INT 218 0.4 20th INT 219 0.5 30th

From these test results it can be concluded that the preparations INT 218 'and INT 219 do not differ significantly in terms of their activity.

E. Antitumor protective activity in tumor system L517OY For this study, mice at least 8 weeks old, DBA 2 / j, were used, and a tumor in an ascites form was injected into the abdominal cavity in an amount of 10 cells per week. For the experiment in question, an infectious dose of the tumor was applied to the shaved tissue of the test animal in an amount of 10 cells in 0.1 ml of the substance

diums 199 applied. The dosage of the active ingredient took place after one week and a total of 3 doses were given. The protective effect is given as the number of regressions per number of test animals in a group.

Experiment No. 1

The preparations INT 218, INT 219 and INT 220 v / ground each tested in an amount of 100 pg per dose, with the relevant Single doses are administered 7 days, 10 days and 12 days after tumor infection. The results are in the table below IV summarized.

⁺ ^ Composition: see Appendix

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Table IV

Days after the
infection INT 218 INT 219 INT 220 control
PBS 7th o / ₅ Ve Ve ^O / 1O 10 ^5/5 Ve Ve ■ ^O / 1O 12th Vs Ve ^O / 1O 14th Vs Ve Ve ° / io

From these results it can be concluded that the preparation INT 219 is just as active as the preparation INT 218 and in of the dose used gives rise to regression of the tumor in each case. However, the preparation INT 220 is less active.

Experiment number 2

In this case, the activity of different single doses of the preparations INT 218 and INT 219 compared with each other. In this case, too, the single doses are again 7 days, Administered 10 days and 12 days after tumor infection. The results are summarized in Table V below.

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- 38 Table V

Days after
the In 50 INT 218
Dose (μβ) 5 INT 219
Dose (ns) 50 15th 5 control
PBS fection ° / 5 15th 7th 7s 75 7s 7th ^5/5 ° / 5 ° / 5 Vs ■ ° / 5 7s 78 10 ^5/5 7s 75 Vs ° / 5 7s 78 12th ⁵ A 75 7s Vs 75 7s ° / e 14th Vs 7s

The preparations INT 218 and INT 219 again show the same activity, both being somewhat more active after 3 doses of 15 mg / mouse each and causing complete regression at a dose of 50 mg / mouse. ^%

F. Serum Interferon Concentration

Test groups of 6 mice each are administered intraperitoneally 10 μg of the preparations in question. The blood is then drawn by cardiac puncture and the blood samples are pooled. The concentration of interferon is measured on plates of L-929 (mouse fibroplast) cells, the cells in question having previously been treated with dilute solutions of the blood sera and then the reduction in the number of areas with dissolution caused by EMC virus is determined. The PDD ^ _Q value is the reciprocal amount of the serum dilution which is sufficient to reduce the number of dissolution areas by 50 percent compared to the control sample. The results obtained are summarized in the table below.

209831/1091

Q at -specific times after injection of the preparations

Preparation 2 hours 4 hours 24 hours INT 218 385> 1OOO 165 INT 219> 1OOO 500 <4

The preparation INT 219 gives a maximum of the interferon concentration at an earlier point in time than the preparation INT 218.

209831/1091

Claims (6)

Claims d) a higher LD ^ Q value when administered intraperitoneally in mice compared to unmodified ribonucleic acid of the same natural origin, e) the same composition with regard to the base building blocks and f) the same sedimentation behavior on a sucrose gradient as an unmodified ribonucleic acid of the same natural origin shows, g) in the event of degradation up to the complete irreversible separation of the molecular strands under conditions which per se are not hydrolysis of the phosphate diester bonds cause single polynucleotide strands to form, which are of lower molecular weight have the same as single strands of the unmodified ribonucleic acid formed under the same conditions of natural origin, h) in mice infected with encephalomyocarditis virus in vivo Shows antiviral activity 24 hours after dosing. 209831/1091 2. Modification according to claim 1, characterized in that that they are in 0.15 molar sodium chloride solution at ρ ,, 7 and one Wavelength of 258 mp has a hyperchromicity of 29 to 55 percent and in particular of 30 percent. 3. Modification according to claim 1 and 2, characterized in that that there are 5 to 20 phosphate diester bonds per molecular strand in the hydrolyzed state. 4 »Method of obtaining a new modification more natural Double-stranded ribonucleic acids according to Claims 1 to 3 ^, characterized in that a double-stranded ribonucleic acid of natural origin with a ρττ value of 11.0 to about 12.5 in an aqueous electrolyte solution with a Alkali metal hydroxide under such conditions of temperature, treatment time, electrolyte concentration and the p ^ value treats that the two polynucleotide molecular strands begin to separate from one another immediately, but not more than a total of 25 percent per strand of the phosphate diester bonds are hydrolyzed, and that the treatment solution is then neutralized. 5. The method according to claim 4, characterized in that the treatment by means of sodium hydroxide is carried out at a Pu value of about 11.8 in a 0.15 molar aqueous sodium chloride solution at a temperature of about 20 ^° C. for about 10 minutes. 6. Medicinal preparations, characterized by a content of a modified double-stranded ribonucleic acid according to claim 1 to 5 as an active ingredient. . ·.