Complexs Of Polyriboinosinic Acid And Polyribo-2-thiocytidylic Acid

Abstract

Two-component complexes consisting of a polyriboinosinic acid of the formula ##SPC1## Wherein k is an integer from 1 to 3, inclusive; and b is an integer from 1 to 2,000, inclusive; and the metal and ammonium salts thereof, and a polyribo-2-thiocytidylic acid of the formula ##SPC2## Wherein m is an integer from 1 to 3, inclusive; and n is an integer from 1 to 2,000, inclusive; and the metal and ammonium salts thereof, exhibit antimicrobial, in particular antiviral, activity.

Description

BACKGROUND OF THE INVENTION

This invention relates to novel polynucleotides. It further relates to a method for the production of interferons. It is known that interferons which in the literature are also referred to as "virus inhibiting factors" are involved in the body defenses of vertebrates against viral infections. They are the only broad spectrum antiviral agents known in the prior art. Interferons are proteins of cellular origin having different molecular weights which can be destructed, and thereby made ineffective, by protein destructing enzymes, e.g., trypsin. They do not inactivate the viruses directly but rather inhibit the virus reproduction by an intracellular mechanism wherein the ribonucleic acid and protein synthesis is involved. Interferons are species-specific, i.e., they are effective only in cells of the same or a nearly related species from which they were obtained. They are, however, virus non-specific, i.e., they are effective against different, non-related virus species.

Thus, a compound which stimulates the production of interferon can be used as a broad spectrum antiviral agent.

It is an object of this invention to provide novel antiviral polynucleotides, a process for their preparation, and a method for the treatment and prevention of viral, bacterial and protozoal infections by administering these polynucleotides. Another object is to provide a process for the preparation of interferons which comprises treating cell cultures with the polynucleotides according to the invention and isolating the interferon thus produced. Other objects will be apparent to those skilled in the art to which this invention pertains.

SUMMARY OF THE INVENTION

The novel polynucleotides of this invention are two-component complexes (hereinafter referred to as AB) consisting of a polyriboinosinic acid (hereinafter referred to as A) of the Formula I ##SPC3##

wherein k is an integer from 1 to 3, inclusive; and b is an integer from 1 to 2,000, inclusive; and a polyribo-2-thiocytidylic acid (hereinafter referred to as B) of the Formula II ##SPC4##

wherein m is an integer from 1 to 3, inclusive; and n is an integer from 1 to 2,000, inclusive; and the metal and ammonium salts thereof, i.e., of the A component, of the B component and of both.

In its process aspect, this invention relates to a process for the preparation of the two-component complexes AB according to the invention which comprises (a) admixing an aqueous solution of the homopolynucleotide A and an aqueous solution of the homopolynucleotide B at a temperature of from 10.degree. to 95.degree. C., or (b) producing one of the homopolynucleotides A or B in a reaction mixture containing the other homopolynucleotide, or (c) treating an aqueous solution containing one of A and B in the homopolynucleotide form, and the other in the form of their monomeric nucleoside diphosphate or triphosphates, with a polymerizing enzyme.

In a method of use aspect, this invention relates to a method of inducing the formation of interferons, thereby inhibiting the growth of viruses, both in vertebrates and in cell cultures of vertebrates, by administering to these organisms or cultures, respectively, a two-component complex AB according to this invention. Thus, the two-component complexes of this invention are useful for protecting cells from virus infections both in vitro and in vivo. Further, administering AB or a metal or ammonium salt thereof protects against infections of other disease germs, e.g., bacteria and protozoa. It is believed this effect is due to an increase of the specific as well as the non-specific infection defense mechanisms of the organism. The increase of the specific, i.e., immunological defense mechanisms is due, for example, to an increased production of antibodies. This effect can be used to improve the results of vaccination with vaccines which do not contain reproducible germs or viruses. The production of antibodies triggered by a vaccine can be magnified by simultaneous administration of AB or a metal or ammonium salt thereof. Thus, a satisfactory vaccination can be achieved with a smaller than usual amount of vaccine. Vaccination with the usual amount of vaccine will result in an increased production of antibodies.

DETAILED DISCUSSION

Of the two-component complexes AB of this invention, including the metal and ammonium salts thereof, preferred are those wherein:

a. k is 1;

b. m is 1, preferably wherein both k and m are 1;

c. b is greater than 2, preferably greater than 10, most preferably greater than 100;

d. n is greater than 2, preferably greater than 10, most preferably greater than 100;

e. both b and n are greater than 10, more preferably both are greater than 100, especially those of (a) or (b).

Preferred salts of A and B and thus of AB are the physiologically acceptable salts, including the alkali metal salts, e.g., lithium, sodium and potassium, the alkaline earth metal salts, e.g., magnesium and calcium, the heavy metal salts, e.g., manganese, zinc and iron, and the unsubstituted and substituted ammonium salts, including the alkyl, dialkyl and trialkyl ammonium salts, wherein alkyl contains one to eight, preferably one to four carbon atoms, and salts of the corresponding amines wherein 1, 2, or 3 of the alkyl groups is substituted by a .beta.- or .gamma.-hydroxy group, e.g., trimethyl-, triethyl-, monoethanol-, diethanol-, triethanol- and tri-n-butylammonium salts. Other salts can be employed for isolation, purification and characterization purposes.

The two-component complexes according to this invention are molecular aggregates built up from polyriboinosinic acids A of Formula I and polyribo-2-thiocytidylic acids B of Formula II, or of a salt of one or both of them. The molecular proportions of A and B in these novel complexes may vary considerably, as well as the molecular shape of the complexes. An AB complex can be in the form of a double helix, with the two homopolynucleotide strands therein of substantially equal length, i.e., both homopolynucleotides contain substantially the same number of bases. However, because the homopolynucleotides A and B are polymers themselves, consisting of molecules with varying molecular weights, which contain different numbers of bases and are of different length, the complexes AB can, of course, be of largely different shapes. Thus, one helix of the two-component complex AB may even contain odd numbers of the complementary bases. One A strand can be combined with two or more shorter or even longer B strands or vice versa. This may even be the case if the overall concentrations of the polyriboinosinic acid and the polyribo-2-thiocytidylic acid are equal. Triple helices may represent other molecular structures of the two-component complexes AB according to the invention. As the stoichiometric composition of the active complex molecule is not precisely known, it may well be that the reaction mixture contains an excess of one or the other homopolynucleotide which cannot be separated easily from the complex. The antimicrobial activity of the complex, however, is not adversely affected thereby.

A particular advantage of the novel two-component complexes according to the invention is their increased stability against the action of heat and body fluids. Thus, no T.sub.m (i.e., the temperature where 50 percent of a given amount of the complex AB is dissociated into the components A and B) of aqueous solutions of AB could be determined at temperatures up to 100.degree. C. Further, the two-component complexes AB remain effective after a 24 hour in vitro treatment with human serum.

In the preparation of the new two-component complexes usually at least one of the homopolynucleotides A and B is used as starting material. The homopolynucleotides A and B possess a pentose phosphate backbone, the pentose therein being ribose. The nucleic bases contained are hypoxanthin and 2-thiocytosin, respectively. A and B are most frequently prepared using biochemical standard procedures, e.g., treating the corresponding nucleoside di- or triphosphates with a polymerizing enzyme.

The preferred method of preparing the new two-component complexes AB comprises combining separate aqueous solutions of the homopolynucleotides A and B at a temperature of from 10.degree. to 95.degree. C. This reaction is usually carried out in the presence of one or more inorganic and/or organic salts, preferably an alkali metal salt, e.g., sodium chloride, in order to maintain a definite ionic strength of preferably from 0.001 to 1. The pH value of the solutions of A and B must be from 5.0 to 11.5, preferably from 6.5 to 11.0. In order to maintain a constant pH value, the homopolynucleotide solutions usually are buffered. Suitable buffer materials are, e.g., organic or inorganic alkali metal salts, preferably sodium salts, e.g., sodium acetate, potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium hydrogen tartrate, sodium citrate and, most preferably, sodium cacodylate. Organic buffer materials can also be used, e.g., tris-(hydroxymethyl)-aminomethane/hydrochloric acid.

Optionally, an organic solvent miscible with water can be added, e.g., a mono- or polyvalent alcohol, e.g., methanol, propanol, ethylene glycol and glycerin, or an aprotic dipolar solvent, e.g., dimethyl sulfoxide, formamide and dimethyl formamide.

A reaction mixture with the desired ionic strength favorable for the complex formation is preferably obtained by combining equal volumes of equally concentrated solutions of the components A and B (calculated on the base content thereof), both of which are adjusted to the desired ionic strength. Of course, it is also possible to combine odd volumes of solutions which are different in the concentrations of A and B, and in their ionic strengths, in such manner that the reaction mixture eventually has the desired ionic strength. In this case, it is also preferred that the reaction mixture contains euqimolar amounts of the homopolynucleotides A and B (with respect to the bases). It is, however, also possible to use different molar amounts of A and B in the preparation of the two-component complexes according to the invention. The type of the complex formed in a given reaction mixture is nearly independent from the molar proportion of its components A and B present during its formation, the type of the complex AB formed depending mainly on the ionic strength and the pH value of the reaction mixture wherein it is formed.

Another process for the preparation of AB or its metal or ammonium salts comprises the production of one of A or B in a reaction mixture which already contains the other homopolynucleotide. Thus, in an aqueous solution containing A and, e.g., polyribo-2,4-dithiouridylic acid, the latter homopolynucleotide can be converted to polyribo-4-sulfo-2-thiouridylic acid by treating the polyribo-2,4-dithiouridylic acid with sulfite and/or bisulfite ions, preferably with an alkali metal sulfite and/or bisulfite, e.g., sodium bisulfite, in the presence of an oxidizing agent, e.g., molecular oxygen, at a pH value of 4.5 to 9, preferably about 7. The polyribo-4-sulfo-2-thiouridylic acid thus obtained is then reacted with ammonia and/or ammonium ions, which preferably are provided in the form of an ammonium halide, e.g., ammonium chloride, at a pH of 7 to 10, preferably about pH 8.5 to produce B, which, with the homopolynucleotide A already present, will form the two-component complex AB according to the invention immediately or after adjusting the pH and the ionic strength of the reaction medium to suitable values.

A variant of the above-described process comprises treating an aqueous solution containing one of the homopolynucleotides A and B, and the monomeric nucleoside di- or triphosphate corresponding to the other homopolynucleotide, with a polymerizing enzyme. In this reaction, too, the second component of the complex according to the invention is prepared in situ. It is not important, however, whether or not the monomeric nucleoside phosphates prior to the polymerization are associated with the homopolynucleotide already present, e.g., by hydrogen bonding. Suitable enzymes for the enzymatic synthesis of homopolynucleotides A or B from the corresponding nucleoside phosphates are polynucleotide phosphorylases, e.g., the polynucleotide phosphorylase E.C.2.7.7.8, obtainable, for example, from Escherichia coli. The enzyme can be utilized in the polymerization in the form of crude extracts or in purified form. The polymerization is ordinarily carried out at a pH of from about 5.5 to 9.5, preferably 8 to 9, at a temperature of from 0.degree. to 80.degree. C., preferably 20.degree. to 45.degree. C., especially at about 37.degree. C. Buffer substances, e.g., tris(hydroxymethyl)-aminomethane ("tris"), ammonium carbonate, or sodium cacodylate, are advantageously added. It is also advantageous to add an inorganic salt during the polymerization, for example, magnesium chloride, calcium chloride or manganese (II) chloride. The polymerization is normally terminated after about 1-72 hours.

Homopolynucleotides A or B with quite narrow mol weight ranges are obtained e.g., by gel filtration. By selecting appropriate fractions of the filtrate the polymers with b or n greater than 100 are separated from those with smaller b or n values.

Within a wide range, the antimicrobial and interferon inducing activity of a two-component complex according to the invention is not dependent upon the conditions by which it is produced. The desired activity generally is due to that structure of the complex which is the most stable under the physiological conditions in the organism of the host, i.e., the buffer capacity and the salt content of the physiological system of the particular vertebrate treated, which determine a definite pH value and ionic strength. An AB complex produced under non-physiological conditions may possibly be rearranged to form the active complex in the host organism, or in the cell culture grown under physiological conditions. Thus, it is theoretically possible to bring about a protective effect by separate administration of A and B, if the two components are administered in a manner whereby the active complex AB is formed in situ in the organism or cell culture, respectively.

The complexes AB according to the invention can be characterized by physical methods, for example, by the determination of the hyperchromic shift in the ultraviolet absorption spectrum. This hyperchromic shift is determined by comparing the UV spectrum of the complex with a spectrum obtained by the addition of the UV spectra of the components A and B registered at a concentration of 50 percent of that of the complex. The presence of AB is preferably proven by the determination of the hyperchromic shift at some different wavelengths. Other physical methods for the characterization of AB include the determination of the ORD spectra; of the Svedberg values s.sub.20,w ; of the T.sub.m values, i.e., the temperatures at which one-half of the complex molecules originally present in a given solution, has been dissociated into A and B; saccharose density-gradient fractionation; and chromatography.

It is, however, preferred to establish the presence of the new complexes AB of this invention by biological methods, e.g., by their ability to induce the production of interferon. This is the most important method of characterization of the new complexes because its sensitivity is greater than the physical methods mentioned above. It has been established that neither of the homopolynucleotides A and B separately exhibit interferon inducing activity under the test conditions described in the examples.

The two-component complexes according to the invention are particularly well characterized by a determination of their protective effect against a virus infection in a cell culture. Thus, confluent monolayers of secondary rabbit kidney cells are treated with a dilution series of the complex in the cell culture maintenance medium, and are then incubated at 35.degree. to 37.degree. C. for 18 to 24 hours. Thereafter, the liquid is removed from the cell culture vessels, and the cultures are infected with a suitable virus, for example, Herpes Simplex virus. The infected cultures are covered with agar and incubated at 35.degree. to 37.degree. C. until untreated controls exhibit visible holes in the cell layer (hereinafter referred to as plaques) resulting from the destruction of the cells by the virus infection. At this state, the cells are stained, and the number of the plaques is determined. From the figures thus obtained, the concentration of the complex AB is determined which brings about 50 percent decrease of the plaques number in the complex treated culture compared with the untreated control.

In analogously conducted experiments using different types of cells of different animal species, and also other viruses, it was proven that the protective effect of the novel complex is neither associated exclusively with a particular kind of cell nor limited to a particular species of virus.

In order to confirm the interferon induction by the complex AB, confluent monolayers of, for example, rabbit kidney cells or mouse embryo cells are covered with a solution of AB in the cell culture maintenance medium. Thereby, the cells are stimulated to produce interferon which according to biochemical standard methods, can be isolated from the supernatant liquid and can be characterized as such.

In order to confirm the virus inhibiting activity of the thus produced interferon, cell layers of rabbit kidney cells are incubated overnight in the presence of rabbit interferon, and are then infected with a virus. The further conduction and evaluation of these experiments are the same as in the plaque reduction test described above. The interferon content of the liquids tested can thus be determined. By using different species of virus, e.g., Herpes Simplex, Vaccinia, or Vesicular Stomatitis virus, the virus non-specificity of the interferon is shown. The specificity of the interferon with respect to the animal species is, for example, proven by treating mouse embryo cells with rabbit interferon, which does not inhibit virus reproduction therein. It can also be shown that the interferon after being treated with trypsin no longer has a protective effect. The dependence of the interferon effect on the protein and RNA synthesis of the cells is shown, for example, by the fact that interferon exhibits no protective effect against vesicular stomatitis virus in such cells which have been treated with actinomycin D.

The production of interferon in vertebrates, following the administration of AB, can be shown by injecting a solution of the complex in a physiologically acceptable solvent, for example, Hanks' salt solution, intravenously, collecting some blood from the patient 2 to 6 hours later, and isolating the interferon from the serum and characterizing it as described above.

The protective effect of AB against virus infections can also be shown directly by animal tests. Thus, for example, mice are treated intraperitoneally with a solution of AB in a physiologically acceptable solvent, and one day thereafter are infected with a pathogenic virus, e.g., Herpes Simplex. Depending on the dose of AB administered, the treatment results in an increased survival time or even full survival of the infection, while control mice which have not been treated with AB, are killed by the infection.

By similar experiments the protective effect of the complexes according to the invention against a great variety of viruses including Vaccinia virus, Vesicular-Stomatitis virus, and Influenza virus can be shown.

Analogously, the protective effect of AB against other infections, e.g., infections by yeasts, for example, Cryptococcus Neoformans, by bacteria, for example, Pneumococcus, and by protozoae, for example, Plasmodium Berghei or Eperythrozoon Coccoides, can be shown.

A particular advantage of the novel complexes AB is their ability to increase the specific defense mechanisms of the host organism. If, for example, a number of guinea pigs are vaccinated with anti-influenza vaccine, and one-half thereof additionally with AB, antibodies are earlier detectable in the serum of the animals to which AB is administered. Further, the antibody titer, as determined by the hemagglutination inhibition test or the complement fixation test, becomes higher in the AB-treated than in the AB-untreated vaccinated guinea pigs.

The increase of the vaccination protection by additional administration of AB can also be shown directly, for example, by vaccinating mice with an anti-influenza vaccine and also treating one-half of the vaccinated mice with AB and, 14 days after the vaccination, infecting all of the animals with influenza virus. A greater percentage of those animals treated with AB in addition to the vaccination, survived the infection than those which were not treated with AB.

When the novel complexes are used in the in vitro production of interferons, they are usually added to cell cultures of the vertebrate species to be treated with the thus-produced interferon, preferably in the form of a solution or suspension of AB, in free acid form or as a metal or ammonium salt thereof, in the cell culture maintenance medium. Sterilants or antibiotics can also be added to prevent bacterial contamination of the culture during the incubation. The incubation usually takes place at a temperature near the normal body temperature of the respective vertebrate species. Temperatures of 33.degree. to 40.degree. C. are preferred. The culturing time necessary for optimal yields of interferon varies from one species to another. Good results are obtained after culturing times of 5 to 48 hours. The interferons thus produced by the cell cultures are isolated using standard procedures for the isolation of proteins, comprising the steps of filtration, precipitation with, for example, ammonium sulfate, and purification by, for example, re-precipitation and/or dialysis.

Living organisms can also be used in the production of interferons. For that purpose, a suitable amount of AB is administered to the organism, and after a time necessary for building up at least a considerable interferon level in the serum, blood is collected from the organism and the interferon isolated therefrom in the usual way.

In experiments conducted on laboratory animals, AB has displayed anti-tumor activity, in mice which were inoculated with Ehrlich ascites tumor cells, the mice which were treated with AB survived considerably longer than untreated ones.

The novel complexes of this invention can be employed in mixture with solid, semi-solid, liquid or gaseous excipients as drugs. The AB complexes, including their metal and ammonium salts, can be administered enternally, parenterally or topically. A preferred mode of enteral application is oral, e.g., in the form of capsules, syrups or elixiers. The complexes can also be administered rectally, e.g., in the form of suppositories. Parenteral administration can be by the injection of sterile solutions thereof in physiologically acceptable solvents subcutaneously, intramuscularly or intravenously. A particularly well suited mode of application of the novel complexes is the topical administration in the form of solutions, lotions, ointments, creams, powders or aerosol sprays. Preferably, solutions or sprays are applied to mucous membranes, e.g., intraorally, intranasally or conjunctivally.

Suitable excipients for pharmaceutical preparations containing the two-component complexes of this invention, including their metal or ammonium salts, are those organic or inorganic substances adapted for enteral, parenteral or topical application and which do not react with the novel complexes, such as, for example, water, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, vaseline, cholesterol, as well as conventional propellants for aerosols, e.g., fluorochlorohydrocarbons. The pharmaceutical compositions according to this invention can optionally be sterilized or mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, or wetting agents, emulsifiers, salts for influencing the ionic strength, buffers, coloring, flavoring and/or aromatous substances.

Pharmaceutical compositions according to this invention, including those which contain AB as the only active ingredient and those which also comprise one or more other active substances, usually contain 0.001 to 200 milligrams of the active ingredient per dosage unit, and preferably 0.01 to 50 milligrams per dosage unit. In combination preparations, preferably at least 10 percent of the active ingredients consists of AB.

The theoretically effective doses of the novel two-component complexes vary widely from species to species, and depend further on the virus species against which they are used. In mice, the threshold dose is about 0.5 milligrams per kilogram, while in rabbits, it is only about 0.05 micrograms per kilogram.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever.

In the examples set forth below, the temperatures are indicated in degrees Centigrade. The s.sub.20,w -values were obtained by sedimentation velocity analyses by means of an analytical ultracentrifuge. The figures given for the concentrations of the holopolynucleotides refer always to the monomeric nucleotides which build up the polymeric ones, the molecular weights of which can be calculated from that of the nucleoside monophosphate minus a molecule of water. The slight deviation from actual molecular weight because the end groups are not included is negligible and can be ignored. In place of the cell culture medium used in the experiments described below, other conventional media can also be used. The type of the cell culture medium is only important for the cell culture and does not influence the effect of the two-component complexes according to the invention.

The polyriboinosinic acid (A) used throughout the following examples is a commercial product of C. F. Boehringer & Soehne G.m.b.H., Mannheim-Waldhof, having a s.sub.20,w -value of 5.3.

PREPARATION

The starting polyribo-2-thiocytidylic acid B was prepared employing the method described in Example 1(b) of the copending application filed Aug. 20, 1971, Ser. No. 173,656, as follows:

4 ml. of an aqueous mixture (pH 8.3) containing 0.4 mmol of tris-(hydroxymethyl)-aminomethane hydrochloride (hereinafter referred to as tris.HCl), 0.008 mmol of magnesium chloride, 0.04 mmol of the disodium salt of 2-thiocytidine-5'-diphosphate, 0.04 mmol of dithiothreitol, and 10 enzyme units of polynucleotide phosphorylase (specific activity 0.165 mmol UDP/hour .times. mg. protein at 37.degree.) are incubated for 4 hours at 37.degree.. After removal of the protein, and a 48 hour dialysis of the aqueous phase concentrated to 1.5 ml. at 3.degree. against 0.01 M tris.HCl.(pH 7.0), B having a s.sub.20,w -value of 8.3 is obtained.

Polynucleotides B having other s.sub.20,w -values can also be used with similar success. The same holds for polynucleotides A.

EXAMPLE 1

To 0.53 ml of a 0.01 M tris.HCl buffer solution (pH 7.0) containing 0.19 micromol of the polyribo-2-thiocytidylic acid described in the Preparation above per ml., 1.37 ml. of Hanks' salt solution (cf. J. M. Hoskins, Virological Procedures, London 1967, page 313) is added, and the combined solution is mixed with 0.10 ml. of a Hanks' salt solution containing 1 micromol of the commercially available polyriboinosinic acid A per ml. This mixture, thus containing 0.1 micromol of each of A and B, is set aside for 1 hour to form a solution of AB exhibiting the following properties:

Hyperchromicity of the UV spectrum in comparison with a spectrum obtained by the addition of the UV-spectra of each of A and B alone, as shown in the following table: Wavelength Hyperchromicity (nm) (%) ______________________________________ 250 18 260 18.2 270 20.8 280 17 ______________________________________

The UV spectrum shows .lambda..sub.max 247.5 nm; .lambda..sub.min 226 nm;

[M].sub.340 = -1.2 10.sup.3, [M].sub.320 = 0, [M].sub.303 = 1.4 10.sup.3,

[m].sub.297 = 0, [m].sub.262 = 21.8 10.sup.3, [m].sub.242 = 0, [m].sub.232 = 18.8 10.sup.3 ;

apparent pK value 11.5; isobestic point 230 nm.

In aqueous solution, no T.sub.m of AB can be determined between 0.degree. and 100.degree. C. In an aqueous mixture containing 30 percent of ethylene glycol and 0.05 M Na.sup.+ ions, a sharp transition is shown with a T.sub.m -value of 77.degree..

The average s.sub.20,w -value of AB is 12.4; the integral distribution of the s.sub.20,w -values of AB is shown in the following table (the values were determined in a 0.1 M phosphate buffer at pH 7.0, after 97 minutes at 20,000 rpm):

s.sub.20,w C'/C. ______________________________________ 5.5 0.02 6.5 0.04 7.5 0.08 8.5 0.13 9.7 0.20 10.5 0.28 11.5 0.39 12.5 0.50 13.5 0.83 14.5 0.93 15.5 1.00 ______________________________________

Contrary to the free polyribo-2-thiocytidylic acid (B), B contained in the two-component complex AB is not attacked by polynucleotide phosphorylases.

EXAMPLE 2

0.32 mg of A are dissolved in 10 ml. of a 0.001 M solution of sodium cacodylate (pH 7.0) containing 59 mg. of NaCl. 1 ml. of a 0.001 molar solution of B is added, and the mixture is left standing at room temperature. The complex formation is followed spectrometrically. A solution of AB is obtained whose properties resemble those of the complex AB described in Example 1.

EXAMPLE 3

To 5 ml. of an aqueous mixture (pH 8.3) containing 0.5 mmol of tris.HCl buffer, 0.01 mmol of MgCl.sub.2, 0.05 mmol of A and 0.16 mg. of disodium 2-thiocytidine-5'-diphosphate, 5 enzyme units of polynucleotide phosphorylase (specific activity 0.165 mmol UDP/hour .times. mg protein at 37.degree.) are added, and the mixture is incubated for 4 hours at 37.degree.. After removal of the protein by repeated extraction with chloroform-isoamyl alcohol (25:2 parts by volume), the aqueous phase is concentrated to a volume of 2 ml. at 15.degree., and is dialyzed for 48 hours at 3.degree. against 0.01 M tris.HCl buffer. By this procedure, a solution of the complex AB is obtained which exhibits the same properties as that described in Example 1.

EXAMPLE 4

To 4 ml. of a 0.001 M sodium cacodylate solution containing 24 mg. of NaCl, 0.1 mmol of polyribo-2,4-dithiouridylic acid, and 0.1 mmol of A, 20 microliter of a sulfite reagent solution consisting of 3 parts by volume of an aqueous 1M Na.sub.2 SO.sub.3 solution and 1 part by volume of an aqueous 1M NaHSO.sub.3 solution, are added, and air is sucked through the reaction mixture. After 1 hour, again 20 microliter of the sulfite reagent solution are added, and the air is bubbled through for another hour. Thereafter, 0.5 ml. of an aqueous 0.2M NH.sub.4 Cl solution are added and the mixture is adjusted to pH 8.5 by adding aqueous ammonia. After standing for 1 hour at room temperature, the reaction mixture is concentrated to 2 ml., and is dialyzed for 60 hours at 3.degree. against 0.01 M tris.HCl buffer. A solution of AB having the same properties as that described in Example 1 is thus obtained.

EXAMPLE A

From an AB solution obtained according to Example 1, a standard solution is prepared by adding 18 ml. of a cell culture maintenance medium. This medium is prepared from a commercial tissue culture medium (TCM 199 manufactured by Wellcome Reagents Limited, Beckenham) by adding a 0.168 percent NAHCO.sub.3 solution as well as 100 IU of penicillin and 100 microgram of streptomycin per ml. By repeated diluting this standard solution with the above-described cell culture maintenance medium in a ratio of 1:10, a dilution series is prepared. 10 Ml. of each of these solutions are used to layer 7 days old primary rabbit kidney cell cultures grown in square bottles. Three bottles are used for each concentration of AB, and three similar bottles are layered with the above-described cell culture preserving medium alone. All cultures are incubated overnight at 35.degree., and then infected with Herpes Simplex virus. The infected cultures are layered with agar, incubated for 48 hours at 35.degree., and are then coated with a second agar layer containing a dye for staining the cells. After an additional incubation for 24 hours at 35.degree., the then macrosopically visible virus plaques are counted macroscopically a magnifying lens. From the three values obtained for each concentration of AB, the average value is calculated and is brought in relation to the average value calculated from the untreated controls. From these figures, the 50 percent Plaque Reduction Dose (PRD.sub.50), i.e., the dose of AB reducing the plaque member to 50 percent of the untreated control, is determined graphically.

In further experiments with the same AB standard solution, dilution series in a ratio of 1:2 and 1:4 were tested. Moreover, in other experiments secondary instead of primary rabbit kidney cells were used, as well as Vaccinia virus instead of Herpes Simplex virus. The results are shown in the following table:

Test Cell Culture Used Virus PRD.sub.50 No. (microgram/ml) ______________________________________ 1 prim. rabbit kidney Herpes Simplex 0.0035 2 prim. rabbit kidney Herpes Simplex 0.0065 3 sec. rabbit kidney Vaccinia 0.0030 4 sec. rabbit kidney Herpes Simplex 0.0061 ______________________________________

The same tests were also conducted with A and B alone. Under no circumstances was a reduction of the number of plaques observed at concentrations of up to 0.61 microgram/ml.

EXAMPLE B

In order to determine the biological activity of the complex AB obtained according to Example 2, plaque reduction tests were conducted as described in the foregoing Example A. Unlike Example A, in this test secondary rabbit kidney cells were used in all experiments, and further, during the experiments referred to as No. 3 and No. 4 in the following table, no streptomycin was added to the cell culture maintenance medium.

______________________________________ Experiment PRD.sub.50 No. Virus microgram/ml. ______________________________________ 1 Herpes Simplex 0.013 2 Vaccinia 0.00067 3 Herpes Simplex 0.0015 4 Herpes Simplex 0.0057 ______________________________________

EXAMPLE C

According to Example 1, a solution was preared which contained 325 micrograms AB/ml. To demonstrate the interferon production in vivo, 23 ml of this solution were applicated intravenously to each of two rabbits of conventional breed weighing 2.3 kg, i.e., 325 micrograms AB/kg. After 2 hours blood was taken from the rabbits by heart puncture. The serum was separated and frozen to -70.degree. C. In order to measure the interferon titer, the sera were separated and diluted in 10-fold steps with cell culture maintenance medium. These dilutions were applied to confluent monolayers of sec. rabbit kidney cells in square bottles (10 ml per bottle; three bottles per dilution). The cells were incubated overnight at 35.degree. and then challenged in conventional manner with Herpes Simplex virus. The further procedure and evaluation was as in example A.

A 50 percent reduction of Herpes Simplex virus plaques was obtained by the following serum dilutions:

Serum Dilution ______________________________________ I 1:1800 II 1:1050 ______________________________________

(Sera which were taken from the rabbits before treating them with AB had no effect under the test conditions).

In a second experiment 0,1 ml of serum I were diluted with 2.7 ml of Hanks' salt solution without Ca.sup.2.sup.+ and Mg.sup.2.sup.+ (pH 7.4) and 0.4 ml of a solution containing 1 mg of twice crystallized bovine trypsin (obtained from SERVA Feinbiochemica GmbH & Co, Heidelberg) per ml Hanks' salt solution were added. The mixture was incubated for 3 hours at 37.degree., then 0.8 ml of a solution containing 1 mg soybean trypsin inhibitor (obtained from SERVA Feinbiochemica GmbH & Co, Heidelberg) per ml Hanks' salt solution and the mixture set aside for 1 hour at 22.degree..

A control was run with the respective solvents only, i.e., without trypsin and inhibitor.

The interferon titer was measured as described above. A 50 percent reduction of Herpes Simplex virus plaques was obtained with the following dilutions:

Solution Dilution ______________________________________ Serum I + trypsin 1:180 Serum I (as control 1:1450 without trypsin) ______________________________________

From this it can be seen that the residual interferon activity of serum I after treatment with trypsin was only 12 percent.

EXAMPLE D

A 45.5 percent reduction of plaques Vaccinia virus was obtained by serum I in a dilution of 1:1000 under test conditions analogous to those described in Example C.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims

What is claimed is:

1. A two-component complex of a polyriboinosinic acid of the formula ##SPC5##

wherein k is an integer from 1 to 3, inclusive; and b is an integer from 1 to 2,000, inclusive; and of a polyribo-2-thiocytidylic acid of the formula ##SPC6##

wherein m is an integer from 1 to 3, inclusive; and n is an integer from 1 to 2,000, inclusive; and the physiologically acceptable metal and ammonium salts thereof.

2. A two-component complex according to claim 1 wherein k is 1.

3. A two-component complex according to claim 1 wherein m is 1.

4. A two-component complex according to claim 1 wherein both k and m are 1.

5. A two-component complex according to claim 1 wherein b is greater than 10.

6. A two-component complex according to claim 1 wherein n is greater than 10.

7. A two-component complex according to claim 1 wherein b is greater tha 100.

8. A two-component complex according to claim 1 wherein n is greater than 100.

9. A two-component complex according to claim 1 wherein both b and n are greater than 100.

10. A two-component complex according to claim 4 wherein both b and n are greater than 10.

11. A two-component complex according to claim 4 wherein both of b and n are greater than 100.