IE42170B1 - Antiviral agentss - Google Patents

Description

PATENT APPLICATION BY (71) BEECHAM GROUP LIMITED, A BRITISH COMPANY OF BEECHAM HOUSE, GREAT WEST ROAD, BRENTFORD, MIDDLESEX, ENGLAND.

Price 12lp This invention relates to antiviral substances, to a method for their preparation, and to pharmaceutical compositions comprising them.

It is now generally recognised that double-stranded 5 ribonucleic acids are potent inducers of interferons and thus are of value in the broad spectrum prophylaxis of viral infections, and, to a lesser extent, in the treatment of such infections. Double-stranded ribonucleic acids of both natural and synthetic origin have been shown to possess interferon-inducing and antiviral activity in tissue-culture and in whole animals. Among the specific sources of interferon-inducing double-stranded ribonucleic acid which have been reported are the virus particles found in some strains of the genus Penicillium such as P.chrysogenum, P.funiculosum, P.stoloniferum, and of the genus Aspergillus such as A.niger and A.foetidus; cytoplasmic polyhedrosis virus} reovirus 3 virion; and the replicative form of MS2 coliphage and of MU9 mutant coliphage.

However, there is some evidence which suggests that double-stranded ribonucleic acid of natural origin may be unacceptably toxic in mammals and its medical and verterinary use may be limited. There is thus a need for an antiviral agent which is less toxic than double-stranded ribonucleic acid alone, and which has comparable or better'antiviral activity. .

This invention is based on the discovery that complexes of double-stranded-ribonucleic acids (d.s. RNA) of natural - 2 42170 origin with polyarginine are less toxic (as evidenced by higher LD^qS in small mammals) than the parent d.s. RNA., and their complexes give a longer duration of antiviral protection than the parent d.s. RNA..

In order to put this invention into its proper perspective, it is necessary to consider the prior art in some depth.

It is of course well know that nucleic acids form ionic complexes with many basic polypeptides such as pro10 tamines and polylysine, although the vast majority of the published papers on the subject have been concerned with complexes between polybases and deoxyribonucleic acid (DNA) and single-stranded ribonucleic acid (ss. RNA). Such complexes of polybases with nucleic acids are known to be more stable to nuclease degradation and thermal denaturation than the parent nucleic acids. When it was discovered that double-stranded ribonucleic acids (d.s. RNA) were inducers of interferons, many workers believed that treatment of the d.s. RNA with polybases would result in stabilisation against 2.0 degradation by x-ibonuclease, and this in improved or prolonged antiviral activity. The results published in support of this theory were not particularly encouraging. Billiau and co-workers (Ann. N.Y, Acad. Set., 173 (1), 657 (1970)) reported that the stimulation of the interferon mechanism in tissue cultures by d.s. RNA can be enhanced up to 100 fold by addition of polybasic substances such as neomycin, streptomycin, diethvlaminoethyl dextran, methylated albumin, - 3 42170 protamine, histone and colistin, these substances having been previously reported to inhibit the action of ribonuclease or to augment cellular uptake of RNA. These workers speculate that Conceivably some of the basic substances could be useful in the therapy of viral disease by interferon inducers but qualify this by adding that it was found that the induction of interferon by Poly I:Poly C in vivo was enhanced by diethylaminoethyl dextran but hot by neomycin or protamine.

High molecular weight poly-D-lysine has been shown (Rice et al: Appl. Microbiol. 19 (5), 867, (1970)) to enhance interferon induction by Poly I:Poly C in mice, and it is reported to be superior to diethylaminoethyl-dextran in this respect. The animals treated with the combination of Poly I: Poly C and poly-D-lysine did not show any immediate signs of toxicity, although the dose of material was well below that for which the toxic effects of Poly I:Poly C itself would become evident.

French Patent No. 178,955 discloses a formulation of d.s. RNA containing d.s. RNA 1 mg.; polyornithine 200 μg; and vitamin Bg 50 mg., the polyomithine being said to enhance the uptake of RNA by the cells.

In our view the prior art on this topic indicated that there were good grounds to suppose that complexes of certain polybases with d.s. RNA would show enhanced and/or prolonged interferon levels in vitro and in vivo. As is well known, however, interferon levels do not strictly - 4 42170 parallel antiviral activity; a compound which is only a moderate interferon inducer may be a very good antiviral agent and vice versa. In addition, in the prior art there is no general agreement about what type of polybase is necessary to give the enhanced interferon levels in vivo. Combinations of protamine and d.s. RNA are said by some workers to be less satisfactory than d.s. RNA, while combinations of d.s. RNA and D5A.E dextran are said to be superior. These erratic results are, in our opinion, confirmation that there is no general trend supported by the prior art; it is not possible to say that the combination of d.s. RNA with any particular polybase which forms an ionic complex with the d.s. RNA will always be superior in interferon-inducing activity (much less in antiviral activity) than the d.s. RNA itself.

The utility of an antiviral agent depends principally on two factors - the duration of protection conferred by a dose of the agent and the toxicity of the agent. The first of these factors may or may not be connected with its capacity to induce interferon production and thus, according to the prior art, may or may not he influenced by the addition of a polybase, while the second of these factors the toxicity - is something upon which the prior art is completely silent.

According to the present invention there is provided an antiviral substance which is a principally ionic complex, the cationic component being a poly cation of a linear poly arginine molecule having at least 5 arginine units and the anionic component being either (a) a doiible5 42170 stranded ribonucleic acid polyanion, said double-stranded ribonucleic acid being of natural origin or (b) a poly anion of a double-stranded derivative of a double-stranded ribonucleic acid of natural origin, The term double-stranded" used in connection with ribonucleic acid refers to the characteristic whereby two ribonucleic acid molecules are associated by hydrogen bonding between complementary bases' in each molecule. Ribonucleic acids may vary in the degrees of double-strandedness.

The term double-stranded ribonucleic acid of natural origin means any double-stranded ribonucleic acid which is isolatable from a naturally-occurring source (e.g. these sources listed earlier in this specification), and excludes synthetic double-stranded ribonucleic acids such as Poly I: Poly C, Poly A:Poly U and Poly G:Poly C.

The term double-stranded derivative of a doublestranded ribonucleic acid of natural origin means any doublestranded ribonucleic acid of natural origin which has been subjected to a chemical or biochemical (e.g. enzymatic) reaction which alters the primary and/or secondary and/or tertiary structure (e.g. the N-oxides described in our British Patent Specification no. 1,284,150» or the alkalimodified double-stranded ribonucleic acids described in our British Patent Specification No. 35980), provided that the resultant derivative retains a substantial degree of basepairing between complementary strands.

The double-strandedness of a double-stranded ribonucleic - 6 42170 acid, or a derivative of a double-stranded ribonucleic acid can be measured by two parameters known as the hyperchromicity and Tm. These parameters are obtained by recording the ultra violet absorption of the material at 260 nm while gradually raising the temperature of the material. The u.v. absorption value of a double-stranded material at this frequency increases with increasing temperature until a constant value is reached, corresponding to the absorption of the thermally denatured (i.e. single-stranded) ribonucleic acid. The difference between the two extremes of absorption expressed as a percentage of the absorption of the doublestranded material is termed the hyperchromicity of that material.

When the u.v. absorption at 260 nm of a double-stranded material is plotted against temperature, it is found that the absorption is greater at high than at low temperatures. The temperature at which the absorption is mid-way between the absorption of the double-stranded material and that of the thermally-denatured (i.e. single stranded material) is called the Tm of the material.

The cationic component present in the complex of this invention is a linear polyarginine molecule having at least 5 polyargdnine units. Suitably its molecular weight is in the range 1,500 to 1 million, preferably about 6,500. By the word linear when used in this specification in connection with the pclyarginine molecule is meant that there is substantially no cross-linking between the basic amino groups of the arginine - 7 43170 units in the polyarginine molecule.

Suitably, the polyarginine is a poly-D-, a poly-L-, or a poly-D,L-arginine but preferably the polyarginine is a poly-L-arginine.

The polyanions present in the complex of this invention are (a) double-stranded ribonucleic acid polyanions, said double-stranded ribonucleic acid being of natural origin or (b) polyanions of a double-stranded derivative of a doublestranded ribonucleic, acid of natural origin. Preferred sources of double-stranded ribonucleic acid include the virus-like particles found in certain of the Penicillia, •e.g. P.chrysogenum (British Patent No, 1,170,929), P.stolonlferum (Banks et al. Nature 225, 135 (1968)), and in certain of the Aspergllli, e.g. A.niger and A.foetldus (British Patent Specification no. 1,300,259). Pi'eferably also the component (a) or (b) should be capable of inducing interferon production in live mammals, · (This can be confirmed by the method of Lampson et al. G.P, Lampson, A.A. Tytell, A.K. Field, M.M. Nemes and M.R. Hilleman Proc. Nat, Acad. Sci., £8 (1967), 782).

The antiviral substance of this invention has been described as a principally ionic complex. The complex is characterised by a strong electrostatic interaction between the polyarginine cationic moiety and the ribonucleic acid anionic moiety. However, other types of interaction may vzell operate, although the precise nature of such bonding is not yet understood. - 8 42170 In the complexes of this invention, substantially all the anionic sites of the d.s. RNA are paired with corresponding cationic sites on the polyarginine component, the cationic sites being the basic amino groups of the argininemonomers. In order to obtain this pairing between cationic and anionic sites, the polyarginine component may be made up of either a single molecule having substantially the same number of cationic sites as the d.s. RNA has anionic sites, or number of smaller polyarginine molecules so that the total number of cation sites provided by the sum of the smaller molecules is substantially the same as the anionic sites of the d.s. RNA. These 1:1 complexes confer long duration of antiviral protection and low toxicity.

The complexes of this invention may be prepared by a process which comprises contacting, in solution, polyarginine or a salt thereof with either (a) double-stranded ribonucleic acid polyanicns said double-stranded ribonucleic acid being of natural origin, or (b) polyanions of a double-stranded derivative of a double-stranded ribonucleic acid of natural origin.

The polyarginine polycation component can be contacted with the ribonucleic acid polyanion in aqueous solution.

For example a solution of polyarginine hydrochloride can be made up in an aqueous solution of an inorganic salt, eg. NaCl. A similar solution of the ribonucleic acid component can be made up in an aqueous solution containing an inorganic salt, and the two solutions mixed together. If the molarity of the resultant salt solution is not too high, the desired complex - 9 42170 will precipitate directly. If it does not precipitate, the ' solution can be diluted to reduce the molarity, and this will generally precipitate the desired complex.

Alternatively, a physical mixture of the neutral polyarginine and the ribonucleic acid or ribonucleic acid derivative can be added to a salt solution and, if necessary, the resultant solution can be diluted to precipitate the desired complex.

Preferably a small excess of the polyarginine polycation is contacted with the ribonucleic acid polynucleic acid polyanion (the excess being calculated on the basis of the number of basic amino sites capable of reacting with the phosphoric acid sites on the RNA polyanion).

The complexes of the present invention are antiviral in activity, having a wide spectrum of activity against a variety of RNA and DNA viruses, e.g. EMC virus, Seml.iki Forest virus, Foot and Mouth Disease'virus, and Herpes Simplex virus. It· is believed that their mode of action is at least partially by induction of interferon in host cells, thereby conferring protection against virus attack.

One surprising property of these complexes is that the toxicity (measured as LD^0 in mice) of the complex is less than that of the RNA component alone.

Thus, in another of its aspects, the present invention provides a pharmaceutical or veternary composition compris- 10 42170 ing a complex in accordance with this invention and a pharmaceutical or vetemary carrier.

The choice of the pharmaceutical carrier is determined by the preferred mode of administration and standard pharmaceutical practice. The mode of administration may be by injection, e.g. subcutaneously or intramuscularly, in which case the carrier will be an injectable liquid in which the complex may be.dissolved ox· suspended as a fine suspension. In the case of topical application, the carrier may be a liquid for topical application to the mucous membrane of the nose or throat.

The following Examples illustrate the present invention. In these Examples, the abbreviation d.s. RNA stands for double-stranded ribonucleic acid.

EXAMPLE 1 Preparation of the complex from ds-RNA isolated from P.chrysogenum, ATCC 10,002 virus-like particles and Polyarginine To a stirred solution of the ds-RNA sodium salt (100 mg) in 0.15M sodium chloride (200 ml) at room temperature was added a solution of poly-L-arginine (M.W. 6,500 200 mg) in 0.15M sodium chloride (200 ml). A precipitate was obtained. The reaction mixture was stirred for 16 hrs. at room temperature and then centrifuged. Measurement of the u.v. spectrum of the supernatant solution indicated that it contained no nucleic acid. The precipitate was washed with water (200 ml) and then with methanol (200 ml) and dried at room temperature in vacuo giving the product as a white solid (162 mg). The precipitate was insoluble in 3M NaOl and was formulated' as a suspension for biological evaluation.

The toxicity and biological activity of this product are given in the following Tables.

Biological Activity A. Toxicity in mice The toxicity of the ds-RNA starting material and of the complexes prepared in Example 1 was compared in 16-20g mice of the strain CD1. The compound was administered by intraneritoncal injection and the animals observed for 10 days after.

Ds-RNA-polyarginlne Complex : Toxicity . Compound DossS (mg/kg) Mortality LD50 (mg/kg) ds-RNA-polyarginine 25 °/5 >100 complex in 1.5M NaCl (suspension) 50 % 100 °/5 ds-RNA 12.5 V10 25 in 0.15M NaCl. 25 '’/io 509/io Spose refers to amount of ds-RNA present in each case mortality = number of deaths size of group B. Antiviral Activity An intraperitoneal injection of the test compound was administered to lS-20g. mice of the strain CD1, Either 24 hours or 72 hours later these mice were challenged with one of two dilutions of encephalomyocarditis (EMC) virus also administered by the intraperitoneal route. The animals were observed for 12 days and mortality rate of treated mice v;as compared with that of untreated control mice. - 15 42170 .ort Term Antiviral Activity of ds-HNA - Sasic Polyarginine Complex o v §* 0 hO Λ O d φ fi Ή r4 (ϋ μ ο μ I Ό CO φ Ρ • % Ο £ Ν-Ζ < § 2 Η δ e 5! ί Compound administered 1 days prior to virus infection Φ W o Q in· to o δ •H > Φ W C «5 to o 3 r£< •H > faO λ b0 S Φ to a 'd § ft a 0 o 0.05 CM Ch in I o V ΦΟ WCM Q .in 6 CM O v- I in o in in o ό \o σ> «4 I o 5- ΦΟ W CM OS PO CM H δ > in O m o T" in «4* O ν’ Compound administered 3 days prior to virus infection 0) to o pm co o r •H > Φ (0 O to 0 δ •H in o • o IS co Untreated Controls Mortality in • Λ p r- in m oo in o ό IS o v- . in o' cn o V m σ> o V τ3 . Ο Ο ο H O !§ ί (Ur r4 'H 0 U C-H CtJ ·Η s ban S O S| 3 £3 i a to to o •d _ -ί/» _ EXAMPLE 2 I Preparation of the complex from ds-RNA isolated from P.chrysogenum, ATCC 10,002 virus-like particles and Polyarginine To a stirred solution of the ds-RNA sodium salt (500 mg) in 0.15M sodium chloride (500 ml) at room temperature was added a solution of poly-L~argininc hydrochloride (Miles code number 71-103A) (318 mg) in 0.15M sodium chloride (100 ml). A precipitate was obtained. The reaction mixture was stirred for 3 hours, at room temperature and then left at /+°c overnight. The precipitate was then centifuged. Measurement of the u.v. spectrum of the supernatant solution indicated that it contained no nucleic acid. The precipitate was resuspended in 0.15M sodium chloi‘ide (50 ml), recentrifuged and the precipitate was resuspended in 0.15M sodium chloride. The precipitate was micronised to give a fine suspension of the complex (ds-RNA content 20 mg/ml).

' The precipitate was insoluble in 3M NaCl and was used as a suspension for biological evaluation. - 15 42170 Duration of Activity The duration of the protection offered by the complex against low and high challenge levels of EMC virus'(8 and SO x LD^q) was evaluated at discrete time intervals up to 5 weeks. A subcutaneous (S.C.) injection of the complex was administered to l6-20g mice of the strain CD1. On day -35, -21, -7 or -1 on day 0, these mice were challenged with one of two dilutions of encephalomyocarditis (EMC) virus administered by the intraperitoneal route. The animals were observed for 12 days, and the mortality rate of mice treated with complex was compared with that of control mice to which an equivalent dose of d.s. RNA had been administered by . either S.C. or intraperatoneal (i.p.) Injection, and challenged in an identical manner. The time intervals, dose-rates and routes are shown in Tables 3 and 4. Because of the unusually large size of the mice at the end of the pretreatment period a trial LD^g of BMC was determined at Day -7· Appropriate challenge levels could then be chosen.

EMC LD50 : trial: 10-6,91' 6 6’ at termination: 10" 2.0 LDcjq is the dose of virus required to kill 50% of test animals.

Duration of Protection of Ds-RNA Complexes Table 3 Pretreatment Time (days) Test Compound Dose mg/kg Route 1 7 21 35 ds RNA 5 i.p. °/io3/io - - o.s RNA 5 s.c. °/io7/io - - Complex 100 s.c. %o3/io1/92/io Control - - 10/ '10 - - - • (Deaths/Group Size) Table A 80 x LD50 Pretreatment Time (days) Drug Dose mg/kg Route 1 7 21 35 ds RNA 5 i.p. °/ '10 -10/ '10 - - ds RNA 5 s.c. 2/ z10 10, '10 - - Complex 100 s.c. 10/ '10 5/ '10 5./ '10 9/ '10 Controls - - 10/ '10 - - - (Deaths/Group Size) - 17 43170

Claims (13)

1. CLAIMS A

1. An antiviral substance which is a principally ionic complex, the cationic component being a polycation of a linear polyarginine molecule having at least 5 arginine units, and the anionic component being either, (a) double-stranded ribonucleic acid polyanion, said double-stranded ribonucleic acid being of natural origin; or (b) a polyanion of a double-stranded derivative of a double-stranded, ribonucleic acid of natural origin.

2. An antiviral substance as claimed in claim 1 wherein the cationic component is a poly-L-arginine cation.

3. · An antiviral substance as claimed in either of the preceeding claims wherein the polyanicn is the polyanion of the ribonucleic acid of the virus particles found in infected strains of Penicillium chrysogenum, Penicillium stoloniferum. Aspergillus nlger or Aspergillus foet-idus.

4. An antiviral substance as claimed in either claim 1 or claim 2 wherein the polyanion is the polyanion of the ribonucleic acid of the replicative form of a phage.

5. An antiviral substance as claimed in claim 1 or 2 wherein the polyanion is the polyanion of the ribonucleic acid of the replicative form of the phage mutant MO—9 of MS2 coliphage.

6. An antiviral substance as claimed in any one of the preceeding claims wherein substantially all the anionic sites on the ribonucleic acid component are involved in ionic - 18 X'- * bonding to the cationic sites on the polyarginine component.

7. An antiviral substance as claimed in claim 1, substantially as hereinbefore described in Example 1 or Example 2. 5

8. A process for the preparation of an antiviral substance as claimed in any one of claims 1-7 which process comprises contacting in solution, linear polyarginine polycations with either, (a) double-stranded ribonucleic acid polyanions, said double-stranded ribonucleic acid being of natural 10 origin; or (b) polyanions of a double-stranded derivative of a double-stranded ribonucleic acid of natural origin.

9. A process as claimed in claim 8 wherein a solution of a polyarginine in an aqueous electrolyte solution, is mixed 15 with an aqueous electrolyte solution of the double-stranded ribonucleic acid or double-stranded derivative of a doublestranded ribonucleic acid.

10. A process as claimed in claim 8 substantially as hereinbefore described in Example 1 or Example 2. 20

11. An antiviral substance whenever prepared by a process as claimed in any one of claims 8 to 10.

12. A pharmaceutical or veterinary composition comprising an antiviral substance as claimed in any one of claims 1 to 7 together with one or more pharmaceutical or veterinary 25 carriers. 13. ·· A pharmaceutical or veterinary composition as claimed in claim 12 which includes a live or killed virus. - 19 > USKBrSr' ii-·· ' « « 14. Apharmaceutical or veterinary composition as claimed in claim 12 or 13 which is in a form suitable for injection.

13. A pharmaceutical or veterinary composition as claimed in claim 12 or 13 which is in a form suitable for application 5 to the mucous membrane of the nose or throat.