Field of the Invention

The present invention relates to a pharmaceutical composition for the prevention and treatment of viral infections and optionally inflammations accompanying viral infections. The invention relates more specifically to pharmaceutical compositions comprising /Mupeol as the antivir ally active substance. The invention relates furthermore to a method of preventing and treating viral infections and optionally inflammations by oral administration of the pharmaceutical composition to a person with a need thereof.

Background Art

Until now, it has been impossible to provide an efficient composition for preventing and/or treating viral infections caused by cold virus etc, such as influenza virus, Rhino virus, Corona virus etc. or other viruses in the upper respiratory passages. Practically all humans suffer from infections in the upper respiratory passages from time to time, such as cold and flu. The symptoms of these infections include a sore throat and earache (otitis), a runny nose, itchy eyes, and a pain in the muscles and the joints. The infections are caused by a variety of different viruses which together are referred to as "cold virus". Although vaccines are available for a restricted number of influenza strains, no efficient methods are known for preventing or treating most of the infections in the upper respiratory passages. Such viral infections, e.g. caused by Rhino virus, which is responsible for

- approximately 50% of all viral infections in the upper respiratory passages, are wide-spread and can cause ill health or be directly potentially lethal for susceptible groups, such as children, elderly people, and persons suffering

from a deficient immunity, such as AIDS-patients, cancer patients etc. A method of treating these symptoms and the underlying infections would be of immense importance.

GB Patent Application No. 2, 1 98,041 A discloses compositions which i.a. contain lupeol. The compositions are stated to have an effect on alcohol addiction, but it does not appear that this effect can be ascribed to lupeol.

EP-A-0 287 000 discloses a process for the preparation of plant extracts, which i.a. may contain lupeol. These extracts are stated to be applicable by the treatment of prostatic hypertrophy, but it does not appear whether the effect can be ascribed to lupeol.

WO 90/14764 discloses a number of terpenozonides having an antiviral effect. These compounds differ, however, essentially from Mupeol, as they contain three oxygen atoms to form a trioxycyclopentane ring. The antiviral effect is ascribed to this trioxycyclopentane ring system.

Aqueous, unpurified extracts of bitter ginseng orally administered have been used for many years in China against chronic hepatitis. The chemical compound or compounds active by the above treatment are, however, not known. Thus it could not be foreseen that a specific fraction can be extracted from bitter ginseng, viz. β- lupeol, which has the unexpected useful effect described in the present specification.

Brief Description of the Invention.

The invention relates according to a first aspect to a pharmaceutical composition for the prevention and/or treatment of viral infections, said composition being characterised by comprising

one or more ? -lupeol derivatives of the formula

where R represents a hydrogen atom, a straight-chained or branched aliphatic C.,_ ₆ -hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C.,. ₆ -acyl group, which may be straight-chained or branched and may contain one or more unsaturated bonds selected from double and triple bonds, or a group, which is easily decomposed under the conditions prevailing in the human or animal body to release the β- lupeol derivative, as well as conventional pharmaceutically acceptable adjuvants, additives, and carriers.

The aliphatic C.,_ ₆ -hydrocarbyl group includes methyl, ethyl, branched and unbranched propyl, butyl, pentyl and hexyl, ethenyl, branched and unbranched propenyl, butenyl, pentenyl and hexenyl, ethynyl, branched and unbranched propynyl, butynyl and hexynyl and corresponding compounds containing two or more double or triple bonds.

The C- _j . _β -acyl group includes methanoyl, ethanoyi, branched and unbranched propanoyl, butanoyl, pentanoyl and hexanoyl, ethenoyl, branched and unbranched propenoyl, butenoyl, pentenoyl and hexenoyl, butynoyl, branched and unbranched propynoyl, butynoyl, pentynoyl and hexynoyl and corresponding compounds containing two or more double or triple bonds.

It should be understood that a group which is easily decomposed under

the conditions prevailing in the human or animal body includes any group that can be transformed into the . -lupeol derivative under physiological conditions.

According to a particularly preferred embodiment of the invention, R is hydrogen.

In addition, it has been found that the presence of ammonium ions provides an antiviral effect against a number of laboratory viruses, such as VSV ( = Vesicular Stomatitis Virus) and Semliki virus, as well as against for instance Rhino virus. The most probable mechanism of the antiviral effect mediated through ammonium ions is considered to be related to the fact that ammonium ions interfere with the binding of ammonium-sensitive viruses to virus receptors on the target cell and therefore improve the capacity of the host or the environment of eliminating the virus via non-specific cell processes, or via neutralization by means of suitable antibodies. Such viruses include for instance HIV-virus, hepatitis virus, usual cold viruses (such as Rhino virus, influenza virus etc.) or other infectious ammoniumion-sensitive viruses.

Based on preliminary experiments it appears that ammonium ions have an effect exclusively on the receptor level through membrane-like interactions, as said ammonium ions must be constantly present at the time when the virus is introduced in the cell cultures in order to provide an optimum antiviral effect.

Therefore another aspect of the invention is to provide a pharmaceutical composition comprising a Mupeol derivative of the above formula I as well as an ammonium ion releasing compound.

The ammonium ions are preferably derived from a salt of a pharma¬ ceutically acceptable inorganic or organic acid. Any pharmaceutically

acceptable acid can be used, and examples thereof are hydrochloric acid, sulphuric acid, phosphoric acid, carbonic acid, acetic acid, and tartaric acid. Ammonium chloride, ammonium sulphate, ammonium hydrogen carbonate or monoammonium dihydrogen phosphate are preferably used.

The ammonium ions may furthermore be derived from a compound of the general formula II

*3

where X^X^ which may be identical or different, are selected from hydrogen; C-,_ ₆ alkyl, which may be straight-chained or branched, saturated or unsaturated and may optionally contain one or more substituents selected from halogen, hydroxy, C.,_ ₄ -alkoxy or amino; aryl, which is optionally substituted with C ₁ _ ₄ alkyl, halogen, hydroxy, C.,_ ₄ -alkoxy or amino, and

Y is a physiologically acceptable salt-forming anion, preferably selected from F ^" , Cl ^" , Br ^" and I ^" .

It has been found that the combination of ? -lupeol and ammonium ions provides a synergistic antiviral effect against a number of viruses, such as

VSV, Rhino virus and probably also influenza virus.

A third aspect of the invention relates to a pharmaceutical composition as defined above and further comprising one or more mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof, including compounds with heparin or heparan structure, which do not possess essential anti-coagulant properties.

Viral infections are known to produce inflammations which are probably mediated via neutrophilic granulocytes accumulated in the affected area and causing further inflammation through the release of various substances, such as cytokines and other mediators. Furthermore, it is thought that cationic protein complexes adjacent to or situated in the neutrophilic granulocytes play an important role as they promote the inflammatory reactions causing the known cold symptoms (sore throat, pain in the joints, fever, etc.). Preliminary experiments indicate that the mere presence of a highly anionic substance related to the heparin structure, but without the anti-coagulant effect of heparin, such as the sodium salt of sucrose octasulphate (SOS), or another SOS-like component, can counteract this process because the latter may optionally "neutralize" the charge of the cationic proteins present in the accumulated neutrophilic granulocytes. The latter granulocytes are bound to the virus-infected cells through ICAM-1 -markers with the result that the usual inflammatory reactions are considerably reduced or completely suppressed.

It is known to use sulphated sugars including the aluminum complex of sucrose octasulphate, sucralphate, in the treatment of inflammations in the gastrointestinal region or for topical application on the skin for prophylaxis or treatment of inflammation, cf. for instance DK printed accepted application No. 1 65,357 and DK-PS No. 1 69,01 8. Furthermore, EP Patent Application No. 0 230 023 A2 discloses pharmaceutical compositions comprising sulphated oligosaccharides including sucrose octasulphate, for promoting ulcer healing. Thus it is assumed that SOS together with local growth factors form a biologically active complex which initiates and stabilizes, respectively, the growth factors resulting in accelerated ulcer healing processes.

- The presence of sulphated saccharides in or around the upper respiratory passages is thought to be advantageous in that these substances can accelerate the ulcer healing/curing in the throat or the oral cavity during

minor microbial infections, especially during virus infections causing inflammations, e.g. by the presence of cationic substances. The sulphated saccharides will be retained in the inflammatory areas and thereby reduce the inflammatory processes in the affected area.

A particular aspect of the present invention is therefore sulphated saccharides for use as an anti-inflammatory substance in the oral cavity and the lymphatic ring, respectively, around the lower respiratory passages (below the nasopharynx), as well as a method of treating inflammations in this area.

According to an embodiment of the present invention, the saccharide is a mono or polysulphated mono, di, tri or tetrasaccharide. According to a particular embodiment, the saccharide is a monosaccharide selected from xylose, fructose and glucose or a disaccharide selected from sucrose, lactose, maltose and cellobiose.

In a preferred embodiment the saccharide forms a complex or a salt with ammonium ions or with a metal selected from Al, Na, K, Ca, Mg, Ba, 2n, Cu, Zr, Ti, Bi, Mn, and Os, or with an amino acid.

According to a preferred embodiment of the present invention the sulphated disaccharide is sucrose octasulphate or a complex or a salt of sucrose octasulphate with ammonium ions or with a metal selected from Al, Na, K, Ca, Mg, Ba, Zn, Cu, Zr, Ti, Bi, Mn and Os, or a salt of sucrose octasulphate with an amino acid.

Among these sucrose octasulphate or the sodium, potassium or NH ₄ ⁺ salt thereof or the aluminum complex of sucrose octasulphate, sucralphate, are preferred.

Interferons usually present under ordinary virus infections, especially in

connection with colds, have been shown to intensify the antiviral effect of β- lupeol and ammonium ions. Thus it has been found that interferons in relatively low concentrations of 0.1 -2 units/ml intensify the antiviral effect.

Furthermore, it can be advantageous as a further ingredient of the pharmaceutical composition to use human or non-human immunoglo- bulines directed towards the substances contributing to intensify colds, such as microorganisms (virus) etc.

According to a particular embodiment of the invention the pharmaceutical compositions comprise therefore as a further ingredient human or non-human immunoglobulines.

The pharmaceutical composition is preferably in the form of chewing gums, lozenges, chewing tablets, resoriblets, drops, troches, gels, mouth ointments, solutions or in form of mucoadhesive formulations, preferably in the form of depot preparations. By depot preparations is in this connection to be understood preparations and formulations with a controlled, sustained release of active ingredients.

The pharmaceutical composition is preferably in the form of a chewing gum, which per piece of chewing gum having a weight of 500 to 3000 mg, preferably of approximately 1000 mg, comprises:

a) 0.01 to 2000, preferably 0.1 5-1000, particularly preferred 1 -800, such as 20-600 μg of a .-lupeol derivative/piece, calculated as β- lupeol,

b) 0 to 100, preferably 1 -50, particularly preferred 2 to 40, such as 5- 30 mg of NH ₄ ⁺ -ions/piece, calculated as ammonium chloride,

c) 0 to 1000, preferably 10-500, particularly preferred 25-250 mg of a sulphated saccharide/piece, calculated as SOS,

as well as conventional chewing gum ingredients.

The chewing gum is prepared by means of conventional chewing gum bases and conventional chewing gum additives, such as sweeteners, flavours, colorants, softeners, and texturizing substances. It may furthermore be necessary to use solubilizers or other release-controlling measures in order to release the pharmacologically active substances disclosed herein from the chewing gum. A further illustration of solubilizers can for instance be found in EP-0 486 563 B1 , in which a general mention of the preparation of chewing gum is found together with examples of applicable chewing gum ingredients.

The invention relates furthermore to the use of one or more /.-lupeol derivatives of the general formula I

in which R represents a hydrogen atom, a straight-chained or branched aliphatic C _j .g-hydrocarbyl group, which may be saturated or may contain

_ one or more unsaturated bonds selected from double and triple bonds, a

C^g-acyl group, which may be straight-chained or branched and may contain one or more unsaturated bonds selected from double and triple

bonds, or a group which is easily decomposed under the conditions prevailing in the human or animal body to release the /.-lupeol derivative for the preparation of a medicament for the prevention and/or treatment of viral infections.

Furthermore, the invention relates to the use of one or more /. -lupeol derivatives of the general formula I

in which R represents a hydrogen atom, a straight-chained or branched aliphatic C.,_ ₆ -hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C _{1 -6} -acyl group, which may be straight-chained or branched and may contain one or more unsaturated bonds selected from double and triple bonds, or a group which is easily decomposed under the conditions prevailing in the human or animal body to release the β -lupeol derivative, as well as one or more ammonium ion releasing compounds for the preparation of a medicament for the prevention and/or treatment of viral infections.

Furthermore the invention relates to the use of one or more ? -lupeol derivatives of the formula I

in which R represents a hydrogen atom, a straight-chained or branched aliphatic C^g-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C^g-acyl group, which may be straight-chained or branched and may contain one or more unsaturated bonds selected from double and triple bonds, or a group which is easily decomposed under the conditions prevailing in the human or animal body to release the /.-lupeol derivative, as well as one or more mono or polysulphated mono, oligo, or polysaccharides or analogues or derivatives thereof, for the preparation of a medicament for the prevention and/or treatment of viral infections and associated inflammations.

Finally, the invention relates to the use of one or more Mupeol derivatives of the formula I

in which R represents a hydrogen atom, a straight-chained or branched

aliphatic C^g-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C^g-acyl group, which may be straight-chained or branched and may contain one or more unsaturated bonds selected from double and triple bonds, or a group which is easily decomposed under the conditions prevailing in the human or animal body to release the Mupeol derivative, one or more ammonium ion releasing compounds, as well as one or more mono or polysulphated mono, oligo, or polysaccharides for the preparation of a medicament for the prevention and/or treatment of viral infections and associated inflammations.

Thus the invention is particularly useful in treating infections in the upper respiratory passages, especially cold viruses, such as Rhino virus, influenza virus, enterovirus, Coxsackie virus and other cold viruses.

In addition, the invention allows the use of one or more of the above mentioned active ingredients for treating HIV, hepatitis virus, cytomegalo virus, herpes virus and other viral infections as well as for treating atherosclerosis as well as for suppressing tumour cell growth.

Antivirally active substances may function in various ways:

(i) either as a substance capable of protecting the target cells provided it is present simultaneously with the virus. If the latter is a condition for producing the antiviral activity, it is very likely that the antiviral effects involve a direct binding of the antiviral substance either to the virus or the receptor thereof or a combination thereof. Many plant extracts will show this type of "non-specific", receptor-dependent antiviral activity. Most frequently, it is only possible to produce this type of antiviral activity provided the substance is present at all times, especially from the time the virus is added,

(ii) or as a substance which is capable of showing an effect without being present during the actual virus infection, such as in connection with a previous contact with the target cell, or by being present after the virus infection, but before a substantial production of viruses has taken place. It is very likely that through this type of antiviral substances more fundamental changes inside the cells are produced via the synthesis of intr acellular proteins/enzymes, which secondarily cause a relatively specific inhibition of the transcription and/or the translation of the virus in such a manner that the new intracellular proteins result in a so-called "antiviral state" of the cell. When the antiviral state has been produced in the cell, the substance need no longer be present in principle as the cells are protected for a certain period of time, although the protection must be expected to decrease gradually over time.

Ammonium ions are thought to belong to type (i) in the effect mechanism, although a certain, but weaker antiviral activity can be measured in cell cultures by the addition of NH ₄ ⁺ 2 to 4 hours after the infection.

/. -lupeol is thought to belong to type (ii) in the effect mechanism.

Mupeol is present in many plants, such as in the shell of lupin seeds, in chiccle rubber, in latex from figs and rubber plants, and in various medicinal plants, such as in extracts from bitter ginseng, .-lupeol is commercially available and may be obtained e.g. from the company Sigma.

The scope of applicability of the invention will appear from the following with reference to the drawings and the examples. It should, however, be understood that the detailed description and the specific examples are merely included to illustrate preferred embodiments, and that various alterations and modifications within the scope of protection will be obvious to persons skilled in the art on the basis of the detailed description.

Brief Description of the Drawings

The invention is explained in greater detail with reference to the drawings, in which

Fig. 1 illustrates the antiviral activity of /. -lupeol (also called B1 -g) against Rhino virus,

Fig. 2 the antiviral activity of interferon-σ (HulFN-σ) against Rhino virus,

Fig. 3 the antiviral activity of B1 -g against EMC virus,

Fig. 4A the antiviral activity of B1 -g against Rhino virus at various dilutions,

Fig. 4B the antiviral activity of B1 -g + interferon-σ against Rhino virus,

Fig. 5 the antiviral activity of NH ₄ ⁺ ions against VSV, Semliki virus and EMCIII virus,

Fig. 6 the antiviral activity of NH ₄ CI against Rhino virus,

Fig. 7A the antiviral activity of B1 -g, B1 -g + NH ₄ CI as well as B1 -g, NH ₄ CI + SOS against Rhino virus at an SOS dilution of 1 : 100 relative to a 20% SOS stock solution in water,

Fig. 7B the same at an SOS dilution of 1 :200,

Fig. 7C the same at an SOS dilution of 1 :400,

Fig. 8 the antiviral activity of B1 -g, NH ₄ CI, SOS and interferon-σ against Rhino virus, and

Fig. 9 the kinetics for the induction of an antiviral state.

Detailed Description of the Invention

The method used for determining antiviral activity is described below.

The cell cultures employed are VERO cells, WISH cells, MDBK cells and HEP cells which are common laboratory cell cultures and which are described in greater detail in Berg, K.: Purification and characterisation of murine and human interferons. A review of the literature of the 1 970s (thesis). Acta Pathol. Microbiol. Scand., Sec. C, Suppl. 279.: page 1 -1 36, 1 982. The viruses employed are VSV, EMC, Semliki virus, influenza virus and Rhino virus.

Briefly a single-layer cell culture is established in microtrays.A certain amount of the antivirally active substance in a suitable dilution is added to the cell culture together with or followed by a suitable amount of virus ("challenge virus"). A control culture receives nothing but challenge virus. The virus infected cultures are incubated until the production of virus is distinct in the virus control culture (4 to 5 days as far as Rhino virus is concerned). An MTS/PMS solution comprising 1 .0 ml MTS stock solution (1 10 /g MTS + 39.2 ml PBS, pH-value 5.6 kept at + 4°C in the dark), 2.3 ml medium and 30 μ\ PMS stock solution (1 3 mg PMS (Sigma, H5004, Lot 13, P. 9625) + 6.5 ml distilled water, kept at 4°C in the dark with a layer of paraffin oil on the top) is added, and based on OD(optical density)- readings in an OD-scanner the relative protection of the cells against viral attacks can be calculated. A high OD-reading indicates that the cells are protected against virus, and a low OD-value indicates that the cells have been killed by virus. Thus, the virus control cultures will typically have an OD-value of < 0.100, while non-infected control cell cultures will have an

OD-value > 1 .000. An antivirally active substance is thus a substance being capable in the presence of medium and challenge virus to provide

protection against the test virus in a cell culture.

As far as the MTS-methods are concerned, reference is furthermore made to Berg, K., B. H. Simonsen, M. B. Hansen, and S. Nielsen: A method for analysing a sample for the presence of a biological substance, especially a virus, use of the method for quantitative determination of biological substances and agents for use as novel substances detected by the method. PCT/DK/89/00010. 1 to 32, 1 989.

Hansen M. B., S. E. Nielsen, and K. Berg: Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods. 1 1 9: 203 to 210, 1 989.

Berg, K., M. B. Hansen and S. E. Nielsen: A sensitive bioassay for precise quantification of interferon activity as measured via the mitochondrial dehydrogenase function in cells (MTT-method). AMPIS 98: 1 56 to 1 62, 1 990.

EXAMPLES

Example 1

Antiviral activity of .-lupeol measured bv means of the MTS-svstem

500 to 1000 WISH-cells in 100 μ\ medium were seeded in wells in a microtray and incubated for 24 hours at 34°C in an atmosphere containing 5% CO ₂ . The medium was replaced by fresh medium containing dilutions of Mupeol (25 to 1 .6 g/ ml, cf. Fig. 1 ) and incubated for further 24 hours at 34°C in an atmosphere containing 5% CO ₂ . The following day challenge Rhino virus was added and after 4 to 5 days at 34°C in an atmosphere containing 5% CO ₂ MTS was added over 2 hours, whereafter the microtray was measured in an OD-scanner. A total protection against

Rhino virus was obtained at 3 /g/ml /. -lupeol ( = B1 -g). However, at high concentrations of /? -lupeol a decreasing cell number appears which must be ascribed to some toxicity of /J-lupeol at such concentrations.

Example 2

Antiviral activity of interferon-σ (rHulFN-σ-2b, "intron A") against Rhino virus

10,000 WISH cells were seeded in a microtray, and the following morning the medium was replaced by two-fold dilutions of HulFN-σ-2b ("intron A") in fresh medium containing 2% serum (cf. Fig. 2). On the following morning the medium was replaced by fresh medium containing Rhino virus. The results in Fig. 2 clearly show that Rhino virus is relatively sensitive to HulFN-σ-2b, and that a protection of approximately 90% is achieved at approximately 8 units IFN/ml. Furthermore, the toxicity of intron A appears to be negligible.

Example 3

Antiviral activity of B1 -g

10,000 WISH cells were seeded and incubated at 37°C for 24 hours as described in Example 2, and dilutions of B1 -g were added to the cultures in dilutions corresponding to the concentration range indicated in Example 1 . After 24 hours the medium was replaced by challenge virus in fresh medium while simultaneously growing challenge virus control cultures and non-infected control cultures. 24 hours later, the cultures were incubated with MTS for 2 hours at 37°C, and the tray was scanned as described above.

The results (Fig. 3) show that B1 -g has a moderate antiviral activity against EMC virus. Similar results were obtained against VSV and Semliki virus. The addition of small amounts of interferon-σ intensified the antiviral activity considerably. Thus, as little as 0.5 units of interferon resulted in almost 80% protection compared to 30% protection without interferon. It should be noted, that very often interferon is present in these amounts (0.2 to 0.6 units/ml) in patients suffering from moderate viral infections, such as ordinary cold and the like.

Example 4

Anti-Rhino virus activity of B1 -g

A corresponding experiment as described above was performed with Rhino virus. As illustrated in Fig. 4A, Rhino virus appears to be much more sensitive to B1-g at a dilution of 1 :100 - 1 :200 than for instance VSV and EMC (from a 1 mg/ml stock solution of B1 -g), as it is able to suppress the viral infection by more than 80 to 90%. Corresponding results must be expected with influenza virus. Thus it appears that B1 -g has a very strong activity towards Rhino virus compared to the effect towards VSV and EMC. This difference could not be foreseen.

The addition of 0.5 units of interferon-σ/ml intensified the antiviral activity to a significant extent, cf. Fig. 4B.

Example 5

Antiviral activity of NH^ ⁺

10,000 WISH cells were seeded in wells in a microtray for 24 hours and incubated for 24 hours at 37°C in an atmosphere containing 5% CO ₂ . Subsequently, the medium was replaced by fresh medium containing

dilutions of NH ₄ ⁺ and virus, and after incubation for 24 hours at 37°C in an atmosphere containing 5% CO ₂ , MTS was added over 2 hours at 37°C and 5% CO ₂ , whereafter the microtray was measured in a OD-scanner.

As illustrated in Fig. 5, NH ₄ ⁺ ions are capable of inhibiting VSV, and to a minor extent Semliki virus, whereas no protection appears against EMC.

Example 6

Anti-Rhino virus activity of NH^ ^"1"

As described in Example 5, the antiviral activity of NH ₄ ⁺ towards Rhino virus was tested, and after incubation for 24 hours at 37°C in an atmosphere containing 5% CO ₂ , MTS was added over 2 hours at 37°C and 5% CO ₂ , whereafter the microtray was measured in an OD-scanner.

As it appears from the results in Fig. 6, a strong antiviral effect is obtained by a dilution of a saturated NH ₄ CI-solution of 1 :900. In contrast a toxic effect appears at higher concentrations of NH ₄ ⁺ for the laboratory culture employed. The toxicity in vivo for humans is, however, as it is well- known, negligible ammonium chloride being an ingredient of inter alia liquorice.

As it appears from Figs. 5 and 6, NH ₄ ⁺ possesses a varying antiviral strength towards different viruses, and furthermore the NH ₄ ⁺ concentration varies which in each particular case provides the optimum antiviral effect.

Example 7

Antiviral activity of B1 -g. Bl -g + NH -CI as well as B1 -o + NH .CI + SOS towards Rhino virus

Tests were carried out as described in Example 5, whereby, however, the temperature was 34°C and the incubation was carried out for 4 to 5 days. The results appear from Figs. 7A, 7B and 7C.

Neither the use of SOS alone in the dilutions of 1 : 100, 1 :200 or 1 :400, NH ₄ CI alone at the dilutions of 1 : 1000 or 1 :2000 nor NH ₄ CI in combination with SOS have any significant antiviral effect.

The use of B1 -g alone reveals a good effect being intensified by the simultaneous use of NH ₄ CI, which alone at 34°C only provides a very low protection. Nevertheless, an increasing effect is obtained with an increasing NH ₄ ⁺ concentration. The additional use of SOS in the dilution of 1 : 100 provides only a moderate increase of the effect.

When comparing Figs. 7A, 7B and 7C it appears that the favourable effect of the combination of B1 -g, NH ₄ CI and SOS is most significant at an SOS- dilution of 1 :400 (Fig. 7C), where a protection of almost 95% is found corresponding to a B1 -g concentration of significantly less than 1 //g/ml. The fact that the most favourable effect is obtained at the lowest concentration of SOS tested is probably due to some toxicity of SOS towards the laboratory cells used. SOS is, however, known to be completely non-toxic to humans in all concentrations relevant in practice.

Example 8

Anti-Rhino virus activity of Bl -g, NH^CI, SOS and interferon-σ

Tests were carried out as described in Example 5, whereby all the

* substances were added simultaneously with the virus. The results appear from Fig. 8. As it appears, interferon-σ in an amount of 0.5 units/ml intensifies further the favourable effect obtained by a combination of

NH ₄ CI, B1 -g and SOS, whereby an almost total protection is obtained by the use of B1 -g, NH ₄ ⁺ ions and interferon-σ.

Accordingly, the natural presence of interferon in a human during an infection must be expected to have an intensifying effect on B1 -g and NH ₄ ⁺ . Analogous results appear with SOS in the dilutions of 1 :200 and 1 :400 (not shown). Similar results are obtained with 0.25 and 0.125 units of interferon/ml.

Example 9

Antiviral activity of Bl -g, NH _| ⁺ ions, SOS, interferons and combinations thereof

The antiviral activity was measured according to the above method. Four different viruses (EMC, VSV, Semliki Forest virus as well as Rhino virus) and three different cell lines (A-549, WISH, VERO) were used for the tests. The results appear from the Table below.

TABLE 1

VSV virus, Semliki Forest virus and influenza virus belong to the enveloped viruses; EMC and Rhino virus belong to the non-enveloped viruses. 20 ND = not determined.

As it appears from the above, Rhino virus is inhibited by ammonium ions and by B1 -g as well as by interferon-σ. Influenza virus is also assumed to be inhibited by ammonium ions. SOS appears to have some antiviral effect towards EMC, but no detectable antiviral activity towards Rhino virus. It appears clearly that Rhino virus (which exemplifies a cold virus) is inhibited by the combination of B1 -g, NH ₄ ⁺ , interferon ± SOS.

Example 10

Kinetic tests

A test was performed to examine possible differences in the antiviral effect as a function of the time for the initiation of the antiviral treatment relative to the establishment of the viral infection.

500 to 1000 WISH cells were seeded on day -1 in wells in microtrays and divided into 3 groups. To one group of cells (group -24h) was added B1 -g in various concentrations in the range of 25 to 1 .6 μg/ ml, whereafter all of the cells were incubated for 24 hours at 37°C in an atmosphere containing 5% CO ₂ . On day 0 Rhino-challenge virus was added to all of the wells, and simultaneously B1 -g was added to another group of cells (group Oh). The incubation was continued for 24 hours at 34°C and 5% CO ₂ . Subsequently, the third group of cells received B1 -g (group + 24h), and all of the cells were further incubated for 4 to 5 days at 34°C and 5% CO ₂ followed by an MTS treatment and measuring in an OD-scanner as described above.The results appear from Fig. 9.

As it appears, the antiviral effect is almost the same regardless whether the B1 -g addition is carried out 24 hours before the viral infection or simultaneously with said viral infection.

Furthermore it is seen that even if the B1 -g treatment is not initiated until

24 hours after the viral infection, i.e. at the time where the viral infection has manifested itself, a distinct antiviral effect is obtained.

While the invention has been described with reference to specific embodiments thereof, it is obvious that it can be varied in many ways. Such variations are not to be considered a deviation from the scope of the invention, and all such modifications which are obvious to persons skilled in the art are also to be considered comprised by the scope of the accompanying claims.