CN1158566A - Pharmaceutical composition for preventing and/or treating viral infection and various inflammations and its treatment method - Google Patents

Abstract

A pharmaceutical composition for the prevention and/or treatment of viral infections and various inflammatory conditions, comprising one or more derivatives of β -lupeol, optionally in combination with one or more compounds capable of releasing ammonium ions, and/or one or more mono-or polysulphated esters of one or more mono-, oligo-or polysaccharides or analogues and/or derivatives thereof. The pharmaceutical compositions may be in the form of chewing gums, lozenges, chewing tablets, resoriblets, drops, lozenges, gels, oral ointments, solutions, adhesive preparations and depot preparations. In addition, methods for preventing and treating viral infections and various inflammatory conditions by orally administering the pharmaceutical composition are also included.

Description

Pharmaceutical composition for preventing and/or treating viral infection and various inflammations and its treatment method

field of invention

The invention relates to a pharmaceutical composition for preventing and treating viral infection and various inflammations accompanied by viral infection. More specifically, the present invention relates to pharmaceutical compositions containing β-lupeol as an antiviral active ingredient. The present invention also relates to methods for preventing and treating viral infections and various inflammations by orally administering the pharmaceutical composition of the present invention to patients in need of treatment.

Background technique

So far, it is still not possible to provide an effective composition for preventing and/or treating viral infections caused by cold viruses (such as influenza virus, rhinovirus, coronavirus, etc.) or other viruses in the upper respiratory tract. Almost everyone suffers from respiratory infections, such as colds and flu, from time to time. Symptoms of these infections include sore throat, ear pain (otitis), runny nose, itchy eyes, and muscle and joint pain. These infections are caused by a variety of different viruses that are collectively called "cold viruses". While vaccines exist for a limited number of influenza strains, there is no effective prevention or treatment for most upper respiratory infections. These viral infections (such as those caused by rhinoviruses, which are associated with approximately 50% of all upper respiratory viral infections) are widespread and are of great concern to susceptible populations, such as children, the elderly, and immunocompromised patients (such as AIDS patients, cancer patients, etc.) can cause disease or directly produce fatal effects. The methods used to treat these symptoms and the infections that cause them are very important.

British Patent Application 2198041A discloses a composition comprising essentially lupeol. The composition is said to be effective in the treatment of alcohol addiction, but this effect does not appear to be attributable to lupeol.

EP-A-0287000 discloses a process for preparing a plant extract mainly containing lupeol. The extract is said to be useful in the treatment of enlarged prostate, but it is unclear whether this effect is related to lupeol.

WO90/14764 discloses various terpene ozonides having antiviral effects. But these compounds are completely different from beta-lupeol, in that they contain three oxygen atoms forming a ring of trioxolane. The antiviral effect is related to the ring system of the trioxetane.

In China, the treatment of chronic hepatitis by oral unpurified aqueous extract of Sophora flavescens has been used for years. However, the active compounds in such treatments are not known. It is therefore not foreseeable that a specific fraction, namely β-lupeol, can be extracted from Sophora flavescens, which has unexpected useful effects as described in this specification.

Summary of the invention

其中R代表氢原子；直链或支链的脂肪族C_1-6-烃基，它可以是饱和的或含有一个或多个选自双键和三键的不饱和键；C_1-6-酰基，它可以是直链或支链的，并且可以含有一个或多个选自双键和三键的不饱和键；或易于在人或动物体内的正常条件下分解释放出β-羽扇醇衍生物的基团，以及常用的可药用辅助剂、添加剂和载体。The present invention firstly relates to a pharmaceutical composition for preventing and/or treating viral infection, said composition is characterized in that it contains one or more β-lupeol derivatives of the following formula

wherein R represents a hydrogen atom; straight-chain or branched aliphatic C _1-6 -hydrocarbyl, which may be saturated or contain one or more unsaturated bonds selected from double and triple bonds; C _1-6 -acyl , which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives groups, as well as commonly used pharmaceutically acceptable adjuvants, additives and carriers.

Aliphatic C _1-6 -hydrocarbyl including methyl, ethyl, branched and straight-chain propyl, butyl, pentyl and hexyl, vinyl, branched and straight-chain propenyl, butenyl, pentenyl and hexenyl, ethynyl, branched and straight-chain propynyl, butynyl and hexynyl; and corresponding compounds containing two or more double and triple bonds.

C _1-6 -Acyl groups include formyl, acetyl, branched and straight-chain propionyl, butyryl, valeryl and hexanoyl, vinylyl, branched and straight-chain acryloyl, crotonyl, pentenoyl and hexanoyl Alkenoyl, butynoyl, branched and straight chain propiolyl, butynoyl, pentynoyl and hexynoyl; and corresponding compounds containing two or more double and triple bonds.

It should be understood that groups that are easily decomposed under normal conditions in the human or animal body include any groups that can be converted into β-lupeol derivatives 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 can produce an antiviral effect on various laboratory viruses, such as VSV (=vesicular stomatitis virus) and Semliki viruses, as well as rhinoviruses. The most likely mechanism for ammonium-mediated antiviral effects is thought to be that ammonium ions can interfere with the binding of ammonium-sensitive viruses to viral receptors on target cells thereby enhancing the host or environment through nonspecific cellular processes, or through appropriate related to the fact that the antibodies neutralize to eliminate the virus. These viruses include, for example, HIV-viruses, hepatitis viruses, common cold viruses (eg rhinoviruses, influenza viruses, etc.) or other infectious ammonium ion-sensitive viruses.

Preliminary experiments have shown that ammonium ions have an effect at the receptor level only through membrane-like interactions, so said ammonium ions must be present continuously when the virus is introduced into the cell culture medium for optimal antiviral effect.

Therefore, another object of the present invention is to provide a pharmaceutical composition comprising the β-lupeol derivative of the above molecular formula I and a compound capable of releasing ammonium ions.

Ammonium ions are preferably derived from salts of pharmaceutically acceptable inorganic or organic acids. Any pharmaceutically acceptable acid can be used, such as hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, acetic acid and tartaric acid. Preference is given to using ammonium chloride, ammonium sulfate, ammonium bicarbonate or ammonium dihydrogenphosphate.

其中X₁-X₄可以相同或不同，选自氢；C_1-6烷基，它可以是直链或支链的、饱和或不饱和的，并可以任意含有一个或多个选自卤素、羟基、C_1-4烷氧基或氨基的取代基；芳基，它被可以C_1-4烷基、卤素、羟基、C_1-4烷氧基或氨基任意取代，并且Y是可药用的成盐阴离子，优选选自F^-、Cl^-、Br^-和I^-。Ammonium ions can also be generated from compounds of general formula II

Wherein X ₁ -X ₄ can be the same or different, selected from hydrogen; C _1-6 alkyl, which can be linear or branched, saturated or unsaturated, and can optionally contain one or more selected from halogen, Substituents of hydroxy, C _1-4 alkoxy or amino; aryl, which can be optionally substituted by C _1-4 alkyl, halogen, hydroxy, C _1-4 alkoxy or amino, and Y is pharmaceutically acceptable The salt-forming anion of is preferably selected from F ^- , Cl ^- , Br ^- and I ^- .

The combination of β-lupeol and ammonium ions has been found to produce a synergistic antiviral effect against a variety of viruses such as VSV, rhinoviruses and possibly influenza viruses.

A third aspect of the present invention relates to a pharmaceutical composition as defined above, which further contains one or more mono-, oligo- or polysaccharides or analogues of mono- or polysulfates and/or derivatives thereof substances, including compounds that have a heparin or heparan-like structure but do not have anticoagulant properties.

Viral infection is known to cause inflammation, which may be caused by the accumulation of neutrophils in the infected area and causing further inflammation by releasing various substances such as cytokines and other mediators. In addition, cationic protein complexes adjacent to or within neutrophils are believed to play an important role, as they can promote inflammatory responses, causing known cold symptoms (sore throat, joint pain, fever, etc.) . Preliminary experiments have shown that the presence of small amounts of anion-rich species related to the structure of heparin but without the anticoagulant effect of heparin [such as sucrose octasulfate (SOS), or the sodium salt of other SOS-like substances] can abolish this process, because the latter Can "neutralize" the charge of cationic proteins present in aggregated neutrophils. The neutralized granulocytes bind to the virus-infected cells through the ICAM-1-marker, so that the usual inflammatory response is greatly reduced or completely suppressed.

The use of sulfated sugars, including aluminum complexes of sucrose octasulfate (sucralfate) to treat inflammation in the gastrointestinal area or topical application on the skin to prevent or treat inflammation is known, see, for example, DK Published Applications 165357 and DK-PS169018. Additionally, European Patent Application 0230023A2 discloses pharmaceutical compositions containing sulfated oligosaccharides, including sucrose octasulfate, for promoting ulcer healing. Therefore, it is speculated that SOS forms a bioactive complex with local growth factors, which can respectively trigger and stabilize growth factors to accelerate the ulcer healing process.

The presence of sulfated sugars in or around the upper respiratory tract is considered advantageous as these substances can speed up the healing of sores in the throat or mouth during periods of mild microbial infection, especially during inflammation caused by viral infections (eg by the presence of cationic species). Sulfated sugars will remain in the inflammatory area thereby attenuating the inflammatory process in the infected area.

A particular aspect of the invention is the sulfated sugar used as an anti-inflammatory substance in the oral cavity and in the lymphatic pharyngeal ring, around the lower respiratory tract (below the nasopharynx), respectively, and a method of treating inflammation in this area.

According to one embodiment of the invention, the sugar is a mono-, di-, tri- or tetrasaccharide mono- or polysulfated. According to a particular embodiment, the sugar is a monosaccharide selected from xylose, fructose and glucose or a disaccharide selected from sucrose, lactose, maltose and cellobiose.

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

According to a preferred embodiment of the invention, the sulphated disaccharide is sucrose octasulfate or sucrose octasulfate with ammonium ions or with ammonium ions selected from the group consisting of Al, Na, K, Ca, Mg, Ba, Zn, Cu, Zr, Ti , metal complexes or salts of Bi, Mn and Os, or salts of sucrose octasulfate and amino acids.

Among these sucrose octasulfate or its sodium, potassium or NH ₄ ⁺ salts or aluminum complexes of sucrose octasulfate, sucralfate is preferred.

Interferon, which usually occurs in common viral infections, especially those associated with colds, has been shown to enhance the antiviral effects of β-lupeol and ammonium ions. Interferon was found to potentiate the antiviral effect at relatively low concentrations of 0.1-2 units/ml.

Furthermore, it is advantageous to use human or non-human immunoglobulins against substances that aggravate colds [eg microorganisms (viruses) etc.] as further constituents of the pharmaceutical composition.

According to a particular embodiment of the invention, the pharmaceutical composition contains human or non-human immunoglobulins as further ingredients.

The pharmaceutical composition is preferably in the form of chewing gums, lozenges, chewable tablets, resoriblets, drops, lozenges, gels, oral ointments, solutions or adherent formulations, preferably in the form of a depot formulation. In this context, a depot formulation is understood to mean a formulation which allows controlled and sustained release of the active ingredient.

The pharmaceutical composition is preferably in the form of chewing gum, each piece of chewing gum has a weight of 500 to 3000 mg, preferably about 1000 mg, containing: a) 0.01 to 2000, preferably 0.15-1000, particularly preferably 1-800, for example 20-600 μg of β-lupeol derivatives/tablet, calculated as β-lupeol, b) 0 to 100, preferably 1-50, particularly preferably 2-40, for example 5-30 mg of _NH4 ⁺ /tablet, calculated as ammonium chloride, c) 0 to 1000, preferably 10-500, particularly preferably 25-250 mg sulfated sugar/tablet, calculated as SOS, and commonly used chewing gum ingredients.

Chewing gum is prepared with conventional chewing gum base and conventional chewing gum additives such as sweeteners, flavoring agents, coloring agents, softeners, and texturizing substances. It may also be desirable to use solubilizers or other controlled release methods to release the pharmaceutically active ingredients disclosed herein from the chewing gum. A further description of solubilizers can be found, for example, in EP0486563B1 where an overview of the preparation of chewing gums and examples of applicable chewing gum ingredients can be found.

其中R代表氢原子；直链或支链的脂肪族C_1-6-烃基，它可以是饱和的或可含有一个或多个选自双键和三键的不饱和键；C_1-6-酰基，它可以是直链或支链的，并且可以含有一个或多个选自双键和三键的不饱和键；或易于在人或动物体内的正常条件下分解释放出β-羽扇醇衍生物的基团。The present invention further relates to the use of one or more β-lupeol derivatives of general formula I in the preparation of medicines for preventing and/or treating viral infections,

Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives group of things.

In addition, the present invention also relates to the use of one or more β-lupeol derivatives of general formula I and one or more ammonium ion-releasing compounds in the preparation of drugs for preventing or treating viral infections, Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives group of things.

其中R代表氢原子；直链或支链的脂肪族C_1-6-烃基，它可以是饱和的或可含有一个或多个选自双键和三键的不饱和键；C_1-6-酰基，它可以是直链或支链的，并且可以含有一个或多个选自双键和三键的不饱和键；或易于在人或动物体内的正常条件下分解释放出β-羽扇醇衍生物的基团。In addition, the present invention also relates to one or more β-lupeol derivatives of general formula I and one or more sulfate esters of mono-, oligo- or polysaccharides or analogs or derivatives thereof in the preparation of prophylaxis and/or or the use of drugs for the treatment of viral infections and associated inflammation,

Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives group of things.

其中R代表氢原子；直链或支链的脂肪族C_1-6-烃基，它可以是饱和的或可含有一个或多个选自双键和三键的不饱和键；C_1-6-酰基，它可以是直链或支链的，含有一个或多个选自双键和三键的不饱和键；或易于在人或动物体内的正常条件下分解释放出β-羽扇醇衍生物的基团。Finally, the present invention relates to one or more β-lupeol derivatives of the general formula I, one or more compounds capable of releasing ammonium ions, and one or more mono- or polysulfates of mono-, oligo- or polysaccharides Use in the preparation of medicines for preventing and/or treating viral infections and related inflammations,

Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, containing one or more unsaturated bonds selected from double and triple bonds; or which is liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives group.

Accordingly, the present invention is particularly useful in the treatment of upper respiratory tract infections, particularly infections by cold viruses such as rhinoviruses, influenza viruses, enteroviruses, coxsackieviruses, and other cold viruses.

In addition, the present invention relates to the use of one or more of the above active ingredients for treating HIV, hepatitis virus, cytomegalovirus, herpes virus and other viral infections, as well as for treating atherosclerosis and inhibiting tumor cell growth.

Antiviral active substances can act in several ways: (i) as substances that can protect target cells (provided that it is present with the virus). If the concomitant presence of a virus is a condition for antiviral activity, it is likely that the antiviral effect involves direct binding of the antiviral substance to the virus or its receptor, or a combination of both. Many plant extracts will display this type of "non-specific", receptor-dependent antiviral activity. Most commonly, this type of antiviral activity is only possible if the substance is present at all times, in particular from the moment of virus addition, (ii) or as a non-active agent during actual virus infection (e.g. with target cells pre-exposure of the virus, or substances that can show an effect in the presence of the virus after infection but before the virus is produced in large quantities). With this type of antiviral substance, it is likely that more fundamental changes are caused within the cell by the synthesis of intracellular proteins/enzymes that subsequently cause relatively specific changes in the transcription and/or translation of the virus Inhibition, in this way, of this new intracellular protein leads to the so-called "antiviral phase" of the cell. After the antiviral phase has been produced in the cell, in theory the substance need not continue to exist, since the cell has been protected for some time, although this protection has to be gradually eliminated.

Ammonium ions were considered to be of type (i) in terms of mechanism of action, although the addition of NH ₄ ⁺ to the cell culture medium 2 to 4 hours after infection could detect definite, but very weak, antiviral activity.

β-lupeol is considered to belong to type (ii) in terms of mechanism of action.

β-lupeol is present in many plants, such as the shells of lupine seeds, chiccle rubber, the mucilage of fig and rubber plants, and various medicinal plants, such as extracts of Sophora flavescens. β-lupeol is commercially available, eg from the company Sigma.

The scope of applicability of the present invention will become clear with reference to the following figures and examples. However, it should be understood that the detailed description and specific examples are only to illustrate preferred embodiments, and that various changes and modifications within the scope of protection will be apparent to those skilled in the art from the detailed description of the application.

Figure overview

The invention is described in more detail with reference to the accompanying drawings, wherein Fig. 1 illustrates the antiviral activity of β-lupeol (also known as Bl-g) against rhinoviruses, and Fig. 2 the antiviral activity of interferon-α (HulFN-α) against rhinoviruses. Antiviral activity, the antiviral activity of Fig. 3B1-g to EMC virus, the antiviral activity of Fig. 4ABl-g to rhinovirus at various concentrations, the antiviral activity of Fig. 4BBl-g+interferon-alpha to rhinovirus, Fig. 5NH ₄ ⁺ antiviral activity of VSV, Semliki virus and EMCIII virus, Figure 6NH ₄ Cl antiviral activity of rhinovirus, Figure 7ABl-g, Bl-g+NH ₄ Cl and Bl-g, NH ₄ Antiviral activity of Cl+SOS (1:100 SOS dilution of 20% SOS stock solution in aqueous solution) to rhinovirus, Figure 7B is the same as before, using 1:200 SOS dilution, Figure 7C is the same as before, using 1:400 Figure 8 Antiviral activity of Bl-g, NH ₄ Cl, SOS and interferon-α on rhinovirus, Figure 9 Kinetics of induced antiviral phase.

Detailed description of the invention

The method used to measure antiviral activity is described below.

The cell culture media used were VERO cells, WISH cells, MDBK cells and HEP cells. These cell culture media were common laboratory cell culture media. See Berg, K. for a more detailed description: Mouse and human interferon Purification and identification. [A review of the 1970 literature (thesis). Acta Pathol. Microbiol. Scand., Part C, 279 Supplement: pp. 1-136, 1982. ] The viruses used were VSV virus, EMC virus, Semliki virus, influenza virus and rhinovirus.

Establish a monolayer of cell culture medium in microplates. A dose of an appropriately diluted antiviral active substance is added to the cell culture medium together or sequentially with an appropriate amount of virus ("challenge virus"). In the control medium, nothing was added except the challenge virus. Virus-infected media were incubated until virus production had become evident in virus control media (4 to 5 days for rhinoviruses). Add 1.0ml of MTS stock solution (110μgMTS+39.2mlPBS, pH 5.6, stored in the dark at +4°C), 2.3ml of culture solution and 30μl of PMS stock solution [13mgPMS (Sigma, H5004, Lot13, P.9625)+6.5ml of distilled water , stored in the dark at 4°C, with a layer of paraffin oil on top] of the MTS/PMS solution, based on the OD (optical density) readings on an OD scanner, the relative protection against virus attack on cells can be calculated. A high OD reading indicates that the cells are protected and a low OD reading indicates that the cells have been killed by the virus. Therefore, the OD value of the virus control medium is generally <0.100, while the OD value of the uninfected control medium is >1.000. Therefore, an antiviral active substance is a substance that can have a protective effect against the test virus in the cell culture medium, in the presence of the culture medium and the challenge virus.

Regarding the MTS method, further reference may be made to Berg, K., B.H. Simonsen, M.B. Hansen, and S. Nielsen: A method for the analysis of samples for the Uses and drugs used as novel substances detected by this method. PCT/DK/89/00010.1-32, 1989.

Hansen M.B., S.E. Nielsen, and K. Berg: Re-experimentation and further development of an accurate and rapid staining method for measuring cell growth/cell killing. J. Immunol. Methods. 119:203-210, 1989.

Berg K., M.B. Hansen and S.E. Nielsen: A sensitive bioassay (MTT method) for the accurate quantification of interferon activity as measured by the function of intracellular mitochondrial dehydrogenases. AMPIS 98: 156 to 162, 1990.

Example Example 1 Determination of the antiviral activity of β-lupeol by MTS-system

Inoculate 100 μl of culture medium containing 500 to 1000 WISH cells into the wells on the microplate and incubate for 24 hours at 34°C in an atmosphere containing 5% CO ₂ . The culture medium was replaced with fresh culture medium containing β-lupeol dilution (25 to 1.6 μg/ml, see Fig. 1) and the incubation was continued for 24 hours at 34°C in an atmosphere containing 5% CO ₂ . The next day, the aggressive rhinovirus was added, and MTS was added after 4 to 5 days at 34°C and an atmosphere containing 5% CO ₂ for 2 hours, and then the microplate was measured on an OD scanner. 3 μg/ml β-lupeol (=Bl-g) confers complete protection against rhinoviruses. However, a decrease in cell number occurred at high β-lupeol concentrations, which must be related to some toxicity of β-lupeol at such concentrations. Example 2 Antiviral activity of interferon-α (rHul FN-α-2b, "intron A") against rhinovirus

10,000 WISH cells were seeded into microplates, and the medium was replaced with a two-fold dilution of HuulFN-α-2b (“intron A”) in fresh medium containing 2% serum the next morning (see Figure 2) . The next morning the medium was replaced with fresh medium containing rhinovirus. The results in Figure 2 clearly show that rhinoviruses are sensitive to HuIFN-[alpha]-2b, with about 90% protection obtained at about 8 units IFN/ml. Furthermore, the toxicity of intron A appears to be negligible. The antiviral activity of embodiment 3B1-g

10,000 WISH cells were inoculated as described in Example 2 and incubated at 37° C. for 24 hours, and Bl-g dilution was added to the culture medium. The concentration of the dilution was the same as that given in Example 1. After 24 hours, the culture solution was replaced with the fresh culture solution added with the challenge virus, and the challenge virus control medium and the uninfected control medium were started to be cultured at the same time. After 24 hours, the medium was incubated with MTS for 2 hours at 37°C and the microplates were scanned as described above.

The results showed (FIG. 3) that Bl-g had moderate activity against EMC virus. Similar results were obtained for VSV and Semliki virus. Adding a small amount of interferon-α can significantly enhance the antiviral activity. Thus, as little as 0.5 units of interferon can induce almost 80% protection compared to 30% protection without interferon. It should be noted that these amounts of interferon (0.2 to 0.6 units/ml) are often present in patients with moderate viral infections (eg common cold, etc.). The anti-rhinovirus activity of embodiment 4B1-g

The same experiments as described above were performed with rhinoviruses. As shown in Figure 4A, at dilutions of 1:100-1:200 (from a 1 mg/ml stock solution of Bl-g) rhinoviruses appear to be much more sensitive to Bl-g than VSV and EMC due to viral suppression 80% to more than 90% are infected. The same result could be expected for influenza viruses. Thus, Bl-g appears to be very active against rhinoviruses compared to the effects against VSV and EMC. This difference cannot be foreseen.

The addition of 0.5 units/ml of interferon-α significantly enhanced the antiviral activity, see Figure 4B. The antiviral activity of embodiment 5NH ₄ ⁺

10000 WISH cells were seeded into wells on microplates for 24 hours and incubated at 37°C in an atmosphere containing 5% _CO2 for 24 hours. Replace the culture medium with fresh culture medium containing NH ₄ ⁺ dilution and virus, and after incubation for 24 hours at 37°C and an atmosphere containing 5% CO ₂ , incubate the MTS at 37°C and 5% CO ₂ for 2 hours Added within, and then the microplate was measured on the OD scanner.

As shown in Figure 5, NH ₄ ⁺ ions can inhibit VSV, have little effect on Semliki virus, and show no protective effect on EMC. The anti-rhinovirus activity of embodiment 6NH ₄ ⁺

The anti-rhinovirus activity of NH ₄ ⁺ against rhinovirus was determined as described in Example 5. After incubation for 24 hours at 37°C and an atmosphere containing 5% CO ₂ , the MTS was incubated at 37°C and 5% CO ₂ at 2 Within hours, the microplates were then assayed on an OD scanner.

As shown by the results in Figure 6, a 1:900 dilution of saturated NH ₄ Cl solution had a strong antiviral effect. At higher NH ₄ ⁺ concentrations, however, a toxic effect on the laboratory culture medium used was exhibited. However, ammonium chloride is a constituent of licorice, as is known to have negligible in vivo toxicity to humans.

As shown in Figures 5 and 6, _NH4 ⁺ has different antiviral strengths against different viruses, and the concentration of _NH4 ⁺ that provides the best antiviral effect will vary in each specific case. Example 7 Bl-g, the anti-rhinovirus activity of Bl-g+NH ₄ Cl and Bl-g+NH ₄ Cl+SOS

The assay was performed as described in Example 5, however, here the temperature was 34°C and the incubation was carried out for 4 to 5 days. The results are shown in Figures 7A, 7B and 7C.

Neither 1:100, 1:200, or 1:400 dilutions of SOS alone; 1:1000 or 1:2000 dilutions of NH ₄ Cl alone, nor the combination of NH ₄ Cl and SOS had significant antiviral effects.

Bl-g alone showed a good effect, and the simultaneous use of NH ₄ Cl (which by itself produced only a weak protective effect at 34°C) would enhance the effect. However increasing the NH ₄ ⁺ concentration produced an enhanced effect. Simultaneous use of a 1:100 dilution of SOS caused only a moderately strong increase in effect.

Comparing Figures 7A, 7B and 7C shows that the best effect of the combination of Bl-g, _NH4Cl and SOS is most pronounced at a 1:400 dilution of SOS (Figure 7C), where the protective effect is almost 95%, corresponding to a Bl-g concentration significantly lower than 1 μg/ml. The fact that the best effect was produced at the lowest SOS concentration tested may be due to some toxicity of SOS to the laboratory cells used. However, SOS is known to be completely nontoxic to humans at various concentrations tested. Example 8Bl-g, NH ₄ Cl, SOS and Interferon-alpha antirhinovirus activity

The test was carried out as described in Example 5, where all substances were added simultaneously with the virus. The results are shown in Figure 8. As shown, interferon-α at a dose of 0.5 units/ml could further potentiate the best effect produced by the combination of NH ₄ Cl, Bl-g and SOS, here, by using Bl-g, NH ₄ ⁺ ions and Interferon-α can produce almost complete protection.

Therefore, it can be expected that interferons naturally present in the human body during viral infection will necessarily enhance the effects of Bl-g and _NH4 ⁺ . Similar results were shown using 1:200 and 1:400 dilutions of SOS (not shown). The same results were obtained with 0.25 to 0.125 units of interferon/ml. Example 9Bl-g, NH ₄ ⁺ , SOS. Interferon and its combination antiviral activity

Antiviral activity was determined according to the method described above. Experiments were performed using four different viruses (EMC, VSV, Semliki Forest virus and rhinovirus) and three different cell lines (A-549, WISH, VERO). The results are shown in the table below.

Table 1

Antiviral activity against different viruses on different cell lines target cell WISH cells A-549 cells VERO cells WISH cells Virus VSV Semliki EMC VSV Semliki EMC Influenza virus A Influenza virus B rhinovirus antiviral ingredients SOS - - + - - + - - - (NH ₄ ) ₂ SO ₄ + + - + + - + + + NH ₄ Cl + - - + - - + + + SOS+(NH ₄ ) ₂ SO ₄ + + + + + + ND ND + HuIFN-α ++ ++ +++ ++ ++ ++ ND ND ++ Bl-g + -/+ + + + + ND ND ++ Bl-g+HuIFN-α + + + + + + ND ND +++ Bl-g+NH ₄ Cl ND ND ND ND ND ND ND ND +++ Bl-g+SOS ND ND ND ND ND ND ND ND ++ Bl-g+NH ₄ Cl+SOS ND ND ND ND ND ND ND ++++ Bl-g+NH ₄ Cl+HuIFN-α ND ND ND ND ND ND ND ++++ VSV virus, Semliki Forest virus, and influenza virus are enveloped viruses; EMC and rhinoviruses are non-enveloped viruses. ND = not determined.

As shown above, rhinoviruses are inhibited by ammonium ions, Bl-g, and interferon-α. Influenza virus can also be putatively inhibited by ammonium ions. SOS showed some antiviral activity against EMC but no detectable antiviral activity against rhinoviruses. Clearly, rhinoviruses (representing a type of cold virus) were inhibited by the combination of Bl-g, _NH4 ⁺ , interferon+SOS. Embodiment 10 kinetic test

The following assay was performed to determine possible differences in antiviral activity as a function of the timing of antiviral onset (relative to the time at which viral infection was established).

500 to 1000 WISH cells were seeded in the wells on the microplate on the negative day and divided into 3 groups. Add various concentrations of Bl-g ranging from 25 to 1.6 μg/ml to the cells of the first group (-24 hours group), and then incubate all cells at 37°C and an atmosphere containing 5% CO ₂ for 24 hours . Rhino-challenge virus was added to all wells on day zero, while Bl-g was added to another group of cells (0 hour group). Continue incubation for 24 hours at 34 °C and 5% CO ₂ . Bl-g was then added to the third group of cells (+24 hr group) and all cells were further incubated for 4 to 5 days at 34°C and 5% _CO , then treated with MTS as described above and scanned at OD measured on the instrument. The results are shown in Figure 9.

As shown in the figure, the antiviral effect was almost the same whether Bl-g was added 24 hours before virus infection or at the same time as said virus infection.

Furthermore, it can be seen that a significant antiviral effect can still be obtained even if the treatment with Bl-g is not started until 24 hours after the virus infection, ie when the virus infection itself is evident.

While the invention has been described in terms of specific embodiments, it will be obvious that it may be varied in many ways. These changes are not considered to be outside the scope of the invention, and all such modifications obvious to those skilled in the art should be considered to be included within the scope of the following claims.

Claims (25)

1. A pharmaceutical composition for preventing and/or treating viral infection, characterized in that it contains one or more β-lupeol derivatives of the following formula Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives The group of substances, as well as commonly used pharmaceutically acceptable adjuvants, additives and carriers. 2. A pharmaceutical composition according to claim 1, characterized in that R represents a hydrogen atom. 3. A pharmaceutical composition according to claim 1 or 2, characterized in that it further contains a compound capable of releasing ammonium ions. 4. A pharmaceutical composition according to claim 3, characterized in that the ammonium ions are generated from salts of pharmaceutically acceptable inorganic or organic acids, preferably selected from hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, acetic acid, and tartaric acid. 5. A pharmaceutical composition according to claim 3, characterized in that the ammonium ion is produced by the compound of general formula II

Wherein X ₁ -X ₄ can be the same or different, they are selected from hydrogen; C _1-6 alkyl, it can be linear or branched, saturated or unsaturated, and can optionally contain one or more selected from halogen , hydroxy, C _1-4 alkoxy or amino substituent; aryl, which may be optionally substituted by C _1-4 alkyl, halogen, hydroxy, C _1-4 alkoxy or amino, and Y is a druggable The salt-forming anion used is preferably selected from F ^- , Cl ^- , Br ^- and I ^- . 6. A pharmaceutical composition according to any one of the preceding claims, characterized in that it further contains one or more mono- or polysulfates and/or derivatives of mono-, oligo- or polysaccharides or analogues. 7. A pharmaceutical composition according to claim 6, characterized in that the saccharide is a mono-, di-, tri- or tetrasaccharide mono- or polysulfated. 8. A pharmaceutical composition according to claim 6, characterized in that the sugar is a monosaccharide selected from xylose, fructose and glucose. 9. A pharmaceutical composition according to claim 6, characterized in that the sugar is a disaccharide selected from the group consisting of sucrose, lactose, maltose and cellobiose. 10. A pharmaceutical composition according to one of the preceding claims, characterized in that the sugar is combined with ammonium ions or a compound selected from the group consisting of Al, Na, K, Ca, Mg, Ba, Zn, Cu, Zr, Ti, Bi, Mn and Os Metals or form complexes or salts with amino acids. 11. A pharmaceutical composition according to claim 9, characterized in that the sulfated disaccharide is sucrose octasulfate, sucrose octasulfate and ammonium ion or selected from Al, Na, K, Ca, Mg, Ba, Zn , Cu, Zr, Ti, Bi, Mn and Os metal complexes or salts, or salts of sucrose octasulfate and amino acids. 12. A pharmaceutical composition according to claim 11, characterized in that the sulfated disaccharide is sucrose octasulfate or its sodium, potassium or _NH4 ⁺ salt or the aluminum complex of sucrose octasulfate, sucralfate. 13. A pharmaceutical composition according to one of the preceding claims, characterized in that it further comprises one or more human or non-human immunoglobulins. 14. A pharmaceutical composition according to one of the preceding claims, characterized in that the pharmaceutical composition is a chewing gum, lozenge, chewable tablet, resoriblets, drops, lozenges, gel, oral ointment, solution, adhesive preparation and storage formulations. 15. A pharmaceutical composition according to claim 14 in the form of chewing gum, characterized in that each piece of chewing gum contains: a) 0.01 to 2000, preferably 0.15 to 1000, particularly preferably 1 to 800, for example 20 to 600 μg of beta - lupeol derivatives/tablet, calculated as β-lupeol, b) 0 to 100, preferably 1 to 50, particularly preferably 2 to 40, for example 5 to 30 mg of _NH4 ⁺ /tablet, calculated as ammonium chloride, c ) 0 to 1000, preferably 10 to 500, particularly preferably 25 to 250 mg of sulfated sugar/tablet, calculated as SOS, and commonly used chewing gum ingredients. 16. The use of one or more β-lupeol derivatives of general formula I in the preparation of medicines for preventing and/or treating viral infections, Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives group of things. 17. The use of one or more β-lupeol derivatives of general formula I and one or more ammonium ion-releasing compounds in the preparation of drugs for preventing and/or treating viral infections, Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives group of things. 18. One or more β-lupeol derivatives of general formula I and one or more sulfuric acid esters and/or derivatives thereof of one or more mono-, oligo- or polysaccharides or analogs in the preparation of prevention and/or treatment Use in medicine for viral infections and associated inflammation,

Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives group of things. 19. One or more β-lupeol derivatives of general formula I, one or more compounds that can release ammonium ions, and one or more mono-, oligo- or polysaccharides or analogs of one or more sulfates and / or its derivatives in the preparation of drugs for the prevention and / or treatment of viral infection and related inflammation, Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives group of things. 20. Use according to any one of the preceding claims 16 to 19 for the treatment of upper respiratory tract infections, in particular cold virus infections such as rhinoviruses, influenza viruses. 21. A method for the prevention and treatment of viral infections, characterized in that the method comprises oral administration of one or more β-lupeol derivatives of formula I in a pharmacological antiviral dose in a pharmaceutically acceptable carrier

Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives group of things. 22. A method for the prevention and treatment of viral infections, characterized in that the method comprises orally administering a pharmacological antiviral dose of one or more β-lupeol derivatives of formula I in a pharmaceutically acceptable carrier and a One or more compounds that release ammonium ions, Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives group of things. 23. A method for the prevention and treatment of viral infections and associated inflammation, characterized in that the method comprises oral administration of one or more β-lupeol derivatives of formula I in a pharmacological antiviral dose in a pharmaceutically acceptable carrier and one or more mono-, oligo- or polysaccharides or analogues of mono- or polysulfates and/or derivatives thereof,

Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives group of things. 24. A method for the prevention and treatment of viral infections and associated inflammation, characterized in that the method comprises oral administration of one or more β-lupeol derivatives of formula I in a pharmacological antiviral dose in a pharmaceutically acceptable carrier substances, one or more ammonium ion-releasing compounds, and one or more mono- or polysulfates of mono-, oligo- or polysaccharides,

Wherein R represents a hydrogen atom; a straight or branched aliphatic C _1-6 -hydrocarbon group, which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds; C _1-6 - Acyl, which may be linear or branched, and may contain one or more unsaturated bonds selected from double and triple bonds; or be liable to decompose under normal conditions in the human or animal body to release β-lupeol derivatives group of things. 25. A pharmaceutical composition for treating inflammation in the oral cavity and lymphatic ring, around the lower respiratory tract, characterized in that it contains one or more mono-, oligo- or polysaccharides or analogs of one or more sulfates and/or its derivative. 26. A method of treating inflammation in the oral cavity and in the lymphatic ring, around the lower respiratory tract by administering one or more mono- or polysulfates of mono-, oligo- or polysaccharides or analogues and/or derivatives thereof.

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