CA2193396A1 - A pharmaceutical composition for the prevention and/or treatment of viral infections and optionally inflammations as well as a method for the treatment thereof - Google Patents
A pharmaceutical composition for the prevention and/or treatment of viral infections and optionally inflammations as well as a method for the treatment thereofInfo
- Publication number
- CA2193396A1 CA2193396A1 CA002193396A CA2193396A CA2193396A1 CA 2193396 A1 CA2193396 A1 CA 2193396A1 CA 002193396 A CA002193396 A CA 002193396A CA 2193396 A CA2193396 A CA 2193396A CA 2193396 A1 CA2193396 A1 CA 2193396A1
- Authority
- CA
- Canada
- Prior art keywords
- beta
- chained
- straight
- contain
- lupeol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7016—Disaccharides, e.g. lactose, lactulose
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/075—Ethers or acetals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/42—Phosphorus; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
- A61K9/0058—Chewing gums
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Communicable Diseases (AREA)
- Zoology (AREA)
- Pain & Pain Management (AREA)
- Pulmonology (AREA)
- Oncology (AREA)
- Virology (AREA)
- Emergency Medicine (AREA)
- Rheumatology (AREA)
- Nutrition Science (AREA)
- Physiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
A pharmaceutical composition for the prevention and/or treatment of viral infections and optionally inflammations comprises one or more .beta.-lupeol derivatives, optionally in combination with an ammonium ion releasing compound, and/or in combination with one or more mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof. The pharmaceutical composition may be in the form of chewing gums, lozenges, chewing tablets, resoriblets, drops, troches, gels, mouth ointments, solutions, mucoadhesive formulations or depot preparations. Furthermore, a method of preventing and treating viral infections and optionally inflammations by oral administration of the pharmaceutical composition.
Description
wo 95/35103 2 1 9 3 3 9 6 Title: A ~hal 1 l làceutical co" "Jo~;lion for the r revention and/or treatment of viral infections and optionallv ill~lallllllalions as well as a method for the treatment thereof Field of the Invention 5 The present invention relates to a pharmaceutical colll,oG~;lion for the prevention and treatment of viral infections and optionally illrldllllllalions accon",d"ying viral infections. The invention relates more specifically to pha""aceutical compositions co"~ i"g ~-lupeol as the antivirally active substance. The invention relates fulll,erll,ol~ to a method of preventing 10 and treating viral infections and optionally illrlallllllaLions by oral aL~ dliul~ of the plla""dceutical composition to a person with a need thereof.
Backaround Art Until now, it has been impossible to provide an efficient ~""~.o~ilion for 15 preventing and/ortreating viral infections caused by cold virus etc, such as influenza virus, Rhino virus, Corona virus etc. or other viruses in the upper respiratory passages. Plal;lically 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 20 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 restrjcted number of influenza strains, no efficient methods are known for preventing or treating most of the infections in the upper respiratory passages. Such 25 viral infections, e.g. caused by Rhino virus, which is responsible for - a~Jpl ."d" laLc:l~r 50% of all viral infections in the upper respiratory passages, are wide-spread and can cause ili health or be directly potentially lethal for susceptible groups, such as children, elderly people, and persons suffering wo gS/35103 2 ~ 9 3 3 9 6 from a deficient immunity, such as AlDS-patients, cancer patients otc. A
method of treating these symptoms and the underlying infections would be of immense i""~o, Lance.
GB Pstent Application No. 2,198,041 A discloses compositions which i.a.
5 contain lupeol. The Cbul,uOa;LiOI-a 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 pl~,ualaLion of plant extracts, which i.a. may contain lupeol. These extracts are stated to be applicable by the treatment of prostatic hy~.~. L~ uphy, but it does not appear whether 10 the effect can be ascribed to lupeol.
WO 90/14764 discloses a number of Lt l~u~llozonideD having an antiviral effect. These compounds differ, however, e~,~.lLk~l!y from ,B-lupeol, as they contain three oxygen atoms to form a trioxycyuloptlllLalle ring. The antiviral effect is ascribed to this trioxycyclopellLalle ring system.
15 Aqueous, unpurified extracts of bitter ginseng orally adlllill;_L~ d 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. ~- lupeol, which has the unexpected 20 useful effect described in the present s,ueuiriuaLion.
Brief Des~,,iv~ion of the Invention.
The invention relates according to a first aspect to a pl,a""Dce,Jtical cOlll,uOaiLiOll for the prevention and/or treatment of viral infections, said CUIIIIUOD;L;On being ~.h~ uL~::Iiadcl by ~,o"",,ising 25 one or more ,~-lupeol derivatives of the formula wo 95/35103 2 1 ~ 3 3 ~ 6 PCTIDK95100256 _6~CH~
CH~I CH- I--CH3 I
. CH3 RO~ '><
where R ~ n~,s~lL~ a hydrogen atom, a straight-chained or branched aliphatic C1 6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1 6-acyl group, which may be straight-chained or branched and may 5 contain one or more unsaturated bonds selected from double and triple bonds, or a group, which is easily decoi",uosed under the con,liLion~
prevailing in the human or animal body to release the ,B- lupeol derivative, as well as conventional phallllaceutically acc~,uLable adjuvants, additives, and carriers.
10 The aliphatic C1 6-hydrocarbyl group includes methyl, ethyl, branched and Ul Ibl ancl1ed propyl, butyl, pentyl and hexyl, ethenyl, branched and ~",L"a"ched propenyl, butenyl, pentenyl and hexenyl, ethynyl, branched and Ul ,k, ancl-ed propynyl, butynyl and hexynyl and co~ ,uondi"g compounds ~.or,L.A;..;"g two or more double or triple bonds.
15 The C1 6-acyl group includes methanoyl, ethanoyl, branched and u,,b,c,,,-.hed propanoyl, butanoyl, pentanoyl and hexanoyl, ethenoyl, branched and u"L"a"cl-ed propenoyl, butenoyl, pentenoyl and hexenoyl, butynoyl, branched and unbranched propynoyl, butynoyl, pentynoyl and hexynoyl and con~ cl-d;"s compounds cGIlL.;.,;,,s two or more double or 20 triple bonds.
It should be understood that a group which is easily decomposed under woss/3sl03 21 9339~
the conditions prevaiiing in the human or animal body includes any group that can be Llall~rulllled into the ~-lupeol derivative under physiological con iiLions.
According to a particularly preferred ~,,ILodi.,,~,,L of the invention, R is 5 hydrogen.
In addition, it has been found that the presence of ammonium ions providcs 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 ",eul,a";..", of the antiviral 10 effect mediated through ammonium ions is con~; ie~ I:d 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 env;~u~ a~L of el;alillaLillg the virus via non-specific cell processes, or via neutralization by means of suitable 15 alllibOd; s. Such viruses include for instance HlV-virus, hepatitis virus usual cold viruses lsuch as Rhino virus, infiuenza virus etc.) or other infectious ammoniumion-sensitive viruses.
E-iased on ,u~ ~.I; a l;l lal y a~,Uc:l ;1 U C:l I L~ it appears that ammonium ions have an effectexclusivelyonthereceptorlevelthroughlllelllLlalle-likeillL~lauliolls~
20 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 phalllla~ ~utical composition comprising a ~-lupeol derivative of the above formula I as well 25 as an ammonium ion releasing compound.
The ammonium ions are preferably derived from a salt of a pharma-ceutically accel~laLI~ inorganic or organic acid. Any ,ullall"aceutically wo 9S/35103 2 1 9 3 3 9 6 I~
acce,uLable acid can be used, and examples thereof are h~dluchlolic acid, sulphuric acid, phosphoric acid, carbonic acid, acetic acid, and tartaric acid. Ammonium chloride, ammonium sulphate, ammonium hydrogen - carbonate or ,~onoallllll~ ;um dihydrogen phosphate are preferably used.
5 The ammonium ions may f~"Ll,e""o,~ be derived from a compound of the general formula 11 X
I
X4- N~- X2 ye 11 x3 where X1-X4, which may be identical or different, are selected from hydrogen; C1 6alkyl, which may be straight-chained or branched, saturated or unsaturated and may optionaily contain one or more substituents 15 selected from halogen, hydroxy, C14-alkoxy or amino; aryl, which is optionally substituted with C1 4alkyl, haiogen, hydroxy, C14-alkoxy or amino, and Y is a phy~;olo~i.,..:!y acc~l~Lable salt-forming anion, preferably selected from F-, Cl-, Br~ and 1-.
It has been found that the COIlliJ;llaLiOl~ of ,B-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 pha. . - .aceutical col l l,uG ~; Lioll as ~ 25 defined above and further cu~ ;"s one or more mono or polysulphated mono, oligo or polysac~,ha~;des or analogues and/or derivatives thereof, including compounds with heparin or heparan structure, which do not possess essential anti-coaguiant properties.
Backaround Art Until now, it has been impossible to provide an efficient ~""~.o~ilion for 15 preventing and/ortreating viral infections caused by cold virus etc, such as influenza virus, Rhino virus, Corona virus etc. or other viruses in the upper respiratory passages. Plal;lically 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 20 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 restrjcted number of influenza strains, no efficient methods are known for preventing or treating most of the infections in the upper respiratory passages. Such 25 viral infections, e.g. caused by Rhino virus, which is responsible for - a~Jpl ."d" laLc:l~r 50% of all viral infections in the upper respiratory passages, are wide-spread and can cause ili health or be directly potentially lethal for susceptible groups, such as children, elderly people, and persons suffering wo gS/35103 2 ~ 9 3 3 9 6 from a deficient immunity, such as AlDS-patients, cancer patients otc. A
method of treating these symptoms and the underlying infections would be of immense i""~o, Lance.
GB Pstent Application No. 2,198,041 A discloses compositions which i.a.
5 contain lupeol. The Cbul,uOa;LiOI-a 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 pl~,ualaLion of plant extracts, which i.a. may contain lupeol. These extracts are stated to be applicable by the treatment of prostatic hy~.~. L~ uphy, but it does not appear whether 10 the effect can be ascribed to lupeol.
WO 90/14764 discloses a number of Lt l~u~llozonideD having an antiviral effect. These compounds differ, however, e~,~.lLk~l!y from ,B-lupeol, as they contain three oxygen atoms to form a trioxycyuloptlllLalle ring. The antiviral effect is ascribed to this trioxycyclopellLalle ring system.
15 Aqueous, unpurified extracts of bitter ginseng orally adlllill;_L~ d 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. ~- lupeol, which has the unexpected 20 useful effect described in the present s,ueuiriuaLion.
Brief Des~,,iv~ion of the Invention.
The invention relates according to a first aspect to a pl,a""Dce,Jtical cOlll,uOaiLiOll for the prevention and/or treatment of viral infections, said CUIIIIUOD;L;On being ~.h~ uL~::Iiadcl by ~,o"",,ising 25 one or more ,~-lupeol derivatives of the formula wo 95/35103 2 1 ~ 3 3 ~ 6 PCTIDK95100256 _6~CH~
CH~I CH- I--CH3 I
. CH3 RO~ '><
where R ~ n~,s~lL~ a hydrogen atom, a straight-chained or branched aliphatic C1 6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1 6-acyl group, which may be straight-chained or branched and may 5 contain one or more unsaturated bonds selected from double and triple bonds, or a group, which is easily decoi",uosed under the con,liLion~
prevailing in the human or animal body to release the ,B- lupeol derivative, as well as conventional phallllaceutically acc~,uLable adjuvants, additives, and carriers.
10 The aliphatic C1 6-hydrocarbyl group includes methyl, ethyl, branched and Ul Ibl ancl1ed propyl, butyl, pentyl and hexyl, ethenyl, branched and ~",L"a"ched propenyl, butenyl, pentenyl and hexenyl, ethynyl, branched and Ul ,k, ancl-ed propynyl, butynyl and hexynyl and co~ ,uondi"g compounds ~.or,L.A;..;"g two or more double or triple bonds.
15 The C1 6-acyl group includes methanoyl, ethanoyl, branched and u,,b,c,,,-.hed propanoyl, butanoyl, pentanoyl and hexanoyl, ethenoyl, branched and u"L"a"cl-ed propenoyl, butenoyl, pentenoyl and hexenoyl, butynoyl, branched and unbranched propynoyl, butynoyl, pentynoyl and hexynoyl and con~ cl-d;"s compounds cGIlL.;.,;,,s two or more double or 20 triple bonds.
It should be understood that a group which is easily decomposed under woss/3sl03 21 9339~
the conditions prevaiiing in the human or animal body includes any group that can be Llall~rulllled into the ~-lupeol derivative under physiological con iiLions.
According to a particularly preferred ~,,ILodi.,,~,,L of the invention, R is 5 hydrogen.
In addition, it has been found that the presence of ammonium ions providcs 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 ",eul,a";..", of the antiviral 10 effect mediated through ammonium ions is con~; ie~ I:d 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 env;~u~ a~L of el;alillaLillg the virus via non-specific cell processes, or via neutralization by means of suitable 15 alllibOd; s. Such viruses include for instance HlV-virus, hepatitis virus usual cold viruses lsuch as Rhino virus, infiuenza virus etc.) or other infectious ammoniumion-sensitive viruses.
E-iased on ,u~ ~.I; a l;l lal y a~,Uc:l ;1 U C:l I L~ it appears that ammonium ions have an effectexclusivelyonthereceptorlevelthroughlllelllLlalle-likeillL~lauliolls~
20 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 phalllla~ ~utical composition comprising a ~-lupeol derivative of the above formula I as well 25 as an ammonium ion releasing compound.
The ammonium ions are preferably derived from a salt of a pharma-ceutically accel~laLI~ inorganic or organic acid. Any ,ullall"aceutically wo 9S/35103 2 1 9 3 3 9 6 I~
acce,uLable acid can be used, and examples thereof are h~dluchlolic acid, sulphuric acid, phosphoric acid, carbonic acid, acetic acid, and tartaric acid. Ammonium chloride, ammonium sulphate, ammonium hydrogen - carbonate or ,~onoallllll~ ;um dihydrogen phosphate are preferably used.
5 The ammonium ions may f~"Ll,e""o,~ be derived from a compound of the general formula 11 X
I
X4- N~- X2 ye 11 x3 where X1-X4, which may be identical or different, are selected from hydrogen; C1 6alkyl, which may be straight-chained or branched, saturated or unsaturated and may optionaily contain one or more substituents 15 selected from halogen, hydroxy, C14-alkoxy or amino; aryl, which is optionally substituted with C1 4alkyl, haiogen, hydroxy, C14-alkoxy or amino, and Y is a phy~;olo~i.,..:!y acc~l~Lable salt-forming anion, preferably selected from F-, Cl-, Br~ and 1-.
It has been found that the COIlliJ;llaLiOl~ of ,B-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 pha. . - .aceutical col l l,uG ~; Lioll as ~ 25 defined above and further cu~ ;"s one or more mono or polysulphated mono, oligo or polysac~,ha~;des or analogues and/or derivatives thereof, including compounds with heparin or heparan structure, which do not possess essential anti-coaguiant properties.
2 ! 93396 Viral infections are known to produce ;llrlal~ alions which are probably mediated via neutrophilic granulocytes accumulated in the affected area and causing further ill~lallllllalion through the release of various substances, such as cytokines and other mediators. Furthermore, it is 5 thought that cationic protein complexes adjacent to or situated in the neutrophilic granulocytes play an important role as they promote the i"rla"""dlury reactions causing the known cold symptoms Isore throat, pain in the joints, fever, etc.). P" ' llillaly experiments indicate that the mere presence of a highly anionic substance related to the heparin 10 structure, but without the anti-coagulant effect of heparin, such as the sodium salt of sucrose octasulphate ISOS), 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 15 virus-infected cells through ICAM-1-markers with the result that the usual illrlallllllaLuryreactionsarecullsidelabl~lreducedorcompletelysuppressed~
It is known to use sulphated sugars including the aluminum complex of sucroseoctasulphate,sucralphate,inthetreatmentofillrlaul,~,dLiu,,sinthe ya~LI uiuL~Liual region or for topical ,, ~i n on the skin for prophylaxis 20 or treatment of illrlaulllldLiull~ cf. for instance DK printed accepted No.165,357 and DK-PS No.169,018. Furthermore, EP Patent Application No. 0 230 023 A2 discloses l~hallllaceutical co"".o:,iLions comprising sulphated ~ ' ,, ' ~c" ides including sucrose octasulphate, for promoting ulcer healing. Thus it is assumed that SOS together with local 25 growth factors form a bioloyi~.ally active complex which initiates and stabilizes, respectively, the growth factors resulting in accelerated ulcer healing processes.
~ The presence of sulphated sd-,-,l,a,ides in or around the upper respiratory passages is thought to be advantageous in that these substances can 30 accel~dl~ the ulcer healing/curing in the throat or the oral cavity during ., . -- -- -- -- . _ _ _ _ . . .. .. . .. . .. .. ... = = ... _ . . .. _ ... _ _ . . . . ..
It is known to use sulphated sugars including the aluminum complex of sucroseoctasulphate,sucralphate,inthetreatmentofillrlaul,~,dLiu,,sinthe ya~LI uiuL~Liual region or for topical ,, ~i n on the skin for prophylaxis 20 or treatment of illrlaulllldLiull~ cf. for instance DK printed accepted No.165,357 and DK-PS No.169,018. Furthermore, EP Patent Application No. 0 230 023 A2 discloses l~hallllaceutical co"".o:,iLions comprising sulphated ~ ' ,, ' ~c" ides including sucrose octasulphate, for promoting ulcer healing. Thus it is assumed that SOS together with local 25 growth factors form a bioloyi~.ally active complex which initiates and stabilizes, respectively, the growth factors resulting in accelerated ulcer healing processes.
~ The presence of sulphated sd-,-,l,a,ides in or around the upper respiratory passages is thought to be advantageous in that these substances can 30 accel~dl~ the ulcer healing/curing in the throat or the oral cavity during ., . -- -- -- -- . _ _ _ _ . . .. .. . .. . .. .. ... = = ... _ . . .. _ ... _ _ . . . . ..
3 21 ~33 9~ r~llJ~ ~
minor microbial infections, especially during virus infections causing allllllaliunst e.g. by the presence of cationic substances. The sulphated saccha~ides will be retained in the illflallllllaluly areas and thereby reduce the illrlallallaLuly processes in the affected area.
5 A particular aspect of the present invention is therefore sulphated sac~.llalides for use as an anti-illrlallllllaLuly substance in the oral cavity and the Iymphatic ring, respectively, around the lower respiratory passages (below the nasopharynx), as well as a method of treating illrlal 1 ll llaLiOns i this area.
10 According to an embodiment of the present invention, the Sdccl,a,ide is a mono or polysulphated mono, di, tri or L~:LIa~ ,hàlid~. According to a particular e",Lo-lialal ~L, the saccharide is a monosacchal ide selected from xylose, fructose and glucose or a ~ i~e~,cl,aride selected from sucrose, lactose, maltose and cP~Iobiose 15 In a preferred ~IL~ L the sacchdlide forms a complex or a 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 e",l,c' Il~L of the present invention the sulphated d;aac~lla,ide is sucrose octasulphate or a complex or a salt of 20 sucrose octasulphate with ammonium ions or with a metai 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 NH4+ salt thereof or the aluminum complex of sucrose octâsulphate, sucralphate, are 25 preferred.
Interferons usually present under ordinary virus infections, especially in wo 9S/35103 P~l/~.~_ l ~T 93396 connection with colds, have been shown to intensify the antiviral effect of ~- lupeol and ammonium ions. Thus it has been found that i~lLelre~uns in relatively low conce"LIaLiu"s of 0.1-2 units/ml intensify the antiviral effect.
Fu,ll,e,,,~o,e:~ it can be advantageous as a further ingredient of the 5 pha""aceutical composition to use human or non-human immunoglo-bulines directed towards the substances contributing to intensify colds, such as ",ic,ooryq";D"~o (virus) etc.
According to a particular embodiment of the invention the phal ll~ac~:utica cor,,,uooiLions comprise therefore as a further ingredient human or 10 non-human immunoglobulines.
The plla,l"aceutical COIll,uOailiOll iS preferably in the form of chewing gums, lozenges, chewing tablets, leooriL~luLo, drops, troches, gels, mouth ointments, solutions or in form of mucoadl,~,~.hrc formulations, preferably in the form of depot ple:~ualdLiuns~ By depot ~ntlpalaLions is in this 15 uor",eu~io" to be ulldeloluod IJl_l~alaLiulls and formulations with a controlled, sustained release of active ingredients.
The pha""aceuticai composition is preferably in the form of a chewing yum, which per piece of chewing gum having a weight of 500 to 3000 mg, preferably of approximately 1000 mg, Cullludouo.
a) 0.01 to 2000, preferablyO.15-1000, particularlypreferred 1-800, such as 20-600 ~9 of a ~-lupeol derivative/piece, cal~ ted as ,6-lupeol, - b) O to 100, preferably 1-50, particularly preferred 2 to 40, such as 5-30 mg of NH4 +-ions/piece, calculated as ammonium chloride, WO 9S/35103 2 l ~ 3 3 ~ 6 r~
c) 0 to 1000, preferably 10-500, particularly preferred 25-250 mg of a sulphated saccha~ elpiece~ c~lcl l'atPd as SOS, as well as conventional chewing gum inyltd;cnl~
The chewing gum is prepared by means of conventional chewing gum 5 bases and conventional chewing gum additives, such as 5w~ n6.,:"
flavours, colorants, softeners, and texturizing substances. It may fu,Ll,~""o,~ be necessary to use solubilizers or other release-controlling measures in order to release the ,~I,a""~colo~: ~lly active substances disclosed herein from the chewing gum. A further illustration of sol~
10 can for instance be found in EP-0 486 563 B1, in which a general mention of the plepalalion of chewing gum is found together with examples of applicable chewing gum illylcd;~..ll~.
The invention relates fulll,~,lllor~ to the use of one or more ,~-lupeol derivatives of the general formula I
_oCH~
H3C "
RO ~CH3 15 in which R lI:pll~51~ " a hydrogen atom, a straight-chained or branched aliphatic Cl 6-hydrocarbyl group, which may be saturated or may contajn .one or more unsaturated bonds selected from double and triple bonds, a Cl 6-acyl group, which may be straight-chained or branched and may contain one or more unsaturated bonds selected from double and triple wo ss~3s103 2 ~ 9 3 3 9 ~ r~
bonds, or a group which is easily decu~.~,uosed under the uoudiLiu"s prevailing in the human or animal body to release the ~-lupeol derivative forthe,u,e,ud,t,Liollofalln;d;~ llLforthepreventionand/ortreatmentof viral infections.
5 Fu,Ll,~:""or~, the invention relates to the use of one or more,l~-lupeol derivatives of the general formula I
_~:H~
RO~CH3 in which R ,~ .lL~ a hydrogen atom, a straight-chained or branched aliphatic C1 6-hydrocarbyl group, which may be saturated or may contain 10 one or more unsaturated bonds selected from double and triple bonds, a C1 ~-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 condiLio"s prevailing in the human or animal body to release the ,~-lupeol derivative, 15 as well as one or more ammonium ion releasing compounds for the pld,udldLioll of a l"ed;~.dll,~ "L for the prevention and/or treatment of viral infections.
Furthermore the invention relates to the use of one or more ,B-lupeol derivatives of the formula l 2 1 9 ~
_6~CH
H3C "
~CH3 RO~ ><
in which R leple~e~ a hydrogen atom, a straight-chained or branched aliphatic C1 6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1 6-acyl group, which may be straight-chained or branched and may 5 contain one or more unsaturated bonds selected from double and triple bonds, or a group which is easily decor,,,uossd under the condiLiol.s prevailing in the human or animal body to release the ,~-lupeol derivative, as well as one or more mono or polysulphated mono, oligo, or polysdc.,h~l ides or analogues or derivatives thereof, for the pl e~ul c~ Lio~ ~ of 10 a ",e.l;.;a~e~lL for the prevention and/or treatment of viral infections and a:~aOciaLt:d i~rlcl,,,,,,c,Lions.
Finally, the invention relates to the use of one or more ~-lupeol derivatives of the formula I
~CH
~ ' ~ --CH3 RO ><~
in which R lel le~ellL~ a hydrogen atom, a straight-chained or branched WO 9S/35103 r~
2~ 93396 aliphatic C1 6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1 6-acyl group, which may be strai9ht-chained or branched and may contain one or more unsaturated bonds selected from double and triple 5 bonds, or a group which is easiiy decor",uosed under the conditions prevailing in the human or animal body to release the ~-lupeol derivative, one or more ammonium ion releasing compounds, as well as one or more mono or polysulphated mono, oligo, or polysscc hal ides for the preparation of a 1 ~ di~.a~ L for the prevention and/or treatment of viral infections and 10 ~.co~ id~ llllllldLilJIl:~, 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 15 mentioned active ingredients for treating HIV, hepatitis virus, cytomegalo virus, herpes virus and other viral infections as well as for treating dLl,t~ ;la,usis as well as for suppressing tumour cell growth.
Antivirally active substances may function in various ways:
(i) either as a substance capable of pluL~.,Lillg the target cells provided it 20 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 cOlllLillaLiull thereof. Many plant extracts will show this type of "non-specific', receptor-dependent antiviral activity. Most 25 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, Wo 95/35103 2 1 9 3 3 ~ S
(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.
5 It is very likely that through this type of antiviral substances more fulldalllellLcl changes inside the cells are produced via the synthesis of intracellularproteins/enzymes,whichsecondalilycausearelativelyspecific inhibition of the Llall~c,i,uLiun and/orthe Llarl~laLion of the virus in such a manner that the new intracellular proteins result in a so-called "antiviral 10 state" of the cell. When the antiviral state has been produced in the cell, the substance need no longer be present in principie as the cells are protected for a certain period of time, although the pruLeuLion must be expected to decrease gradually over time.
Ammonium ions are thought to belong to type (i) in the effect ,,,echc, ,i..""
15 although a certain, but weaker antiviral activity can be measured in cell cultures by the addition of NH4+ 2 to 4 hours after the infection.
B-lupeol is thought to belong to type (ii) in the effect l"eul,a"k"".
~-lupeol 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 vârious 20 medicinal plants, such as in extracts from bitter ginseng. ,t~-lupeol is CO u ll l lel ciclly available and may be obtained e.g. from the company Sigma.
The scope of . ,c ' ' ' :y 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 ~ 25 merely included to illustrate preferred e",L,od;."e"L~, and that various - alterations and IllodiricaLions within the scope of pluLeuLiOII will be obvious to persons skilled in the art on the basis of the detailed desu,i,uLiom WO 95/35103 2 1 9 3 3 9 6 PCTII)K95100256 Brief De~ ioll of the Drawinqs 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) against5 Rhino virus, Fig. 2 the antiviral activity of interferon-a (HulFN-a) against Rhino virus, Fis. 3 the antiviral activity of B1-a against EMC virus, Fig. 4A the antiviral activity of B1-g against Rhino virus at various dilutions, 10 Fig. 4B the antiviral activity of B1-g + interferon-a against Rhino virus, Fig. 5 the antiviral activity oF NH4+ ions against VSV, Semliki virus and EMCIII virus, Fig. 6 the antiviral activity of NH4CI against Rhino virus, Fig. 7A the antiviral activity of B1-g, B1-g + NH4CI as well as B1-g, 15 NH4CI + 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, NH4CI, SOS and interferon-a against 20 Rhino virus, and WO 95/35103 2 1 9 3 3 9 6 1 "~
Fig. 9 the kinetics for the induction of an antiviral state.
Detailed Des-.,iuLion of the Invention The method used for d~ l lg antiviral activity is described below.
The cell cultures employed are VERO cells, WISH cells, MDBK cells and 5 HEP cells which are common laboratory cell cultures and which are described in greater detail in Berg, K.: Purification and ulla~aul~ aLion of murine and human i,lL~r~,uns. A review of the literature of the 1970s (thesis). Acta Pathol. Microbiol. Scand., Sec. C., Suppl.279.: page 1-136, 1982. The viruses employed are VSV, EMC, Semliki virus, influenza virus 10 and Rhino virus.
Briefly a ,i~yle laycr cell cuiture is r~LaL~ l,ed 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.
15 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 (110~9 MTS + 39.2 ml PBS, pH-value 5.6 kept at +4~C in the dark), 2.3 ml medium and 30 ~11 PMS stock solution (13 mg PMS (Sigma, H5004, Lot 20 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 25 been killed by virus. Thus, the virus control cultures will typicall.y 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 WO 95/35103 2 1 9 3 3 9 ~ ~
ul u~ on 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 5 a virus, use of the method for quantitative d~L~nll;llalion of biological substances and agents for use as novel substances detected by the method. PCT/DKI89/00010. 1 to 32, 1989.
Hansen M. B., S. E. Nielsen, and K. Berg: Fe-ex~""i,ldLiù" and further development of a precise and rapid dye method for measuring cell 10 growth/cell kill. J. Immunol. Methods. 119: 203 to 210, 1989.
Berg, K., M. B. Hansen and S. E. Nielsen: A sensitive bioassay for precise quc"~Lir;ualiùl- of interferon activity as measured via the Illiluullolldlial deh~/-JIuy~nase function in cells ~MTT-method). AMPIS 98: 156 to 162, 1 990.
EXAMPLES
Examole 1 Antiviral activitv of ~-luoeol measured bv means of the MTS-svstem 500 to 1000 WlSH-cells in 100 ~I medium were seeded in wells in a microtray and incubated for 24 hours at 34 C in an dLIllo~,ul,e:,~ containing 20 5~~c COz. The medium was replaced by fresh medium CollLai"i"g dilutions of ,~-lupeol (25 to 1.6,ug/ml, cf. Fig.1) and incubated for further 24 hours at34ocinanallllo~ull~l~collLaillilly5%co2~Thefollowingdaychallenge Rhino virus was added and after 4 to 5 days at 34 C in an aLIl~o.,uhe~:
containing 5% C02 MTS was added over 2 hours, wl,e,t:c,rl,:r the ~ 25 microtray was measured in an OD-scanner. A total protection against WO 95/35103 2 1 9 3 3 9 6 PCT/I)K95/00256 Rhino virus was obtained at 3 ~g/ml ~-lupeol ( = B1 -g). However, at high conce"L,~Iiol,s of ~-lupeol a de,~ ;.,9 cell number appears which must be ascribed to some toxicity of ,6-lupeol at such conce"L,llLiol1s.
ExamDle 2 5 Antiviral activitv of interferon-a irHulFN-a-2b, "intron A") aqainst 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-a-2b ("intron A"~
in fresh medium co"l_:.l;"g 2% serum (cf. Fig. 2). On the following 10 morning the medium was replaced by fresh medium cor,l_;.,;"g Rhino virus. The results in Fig. 2 clearly show that Rhino virus is relatively sensitive to HulFN-a-2b, and that a protection of cl,u~uluXilll~L~ / 90% is achieved at ~I,u~ulu~dlll~hly 8 units IFN/ml. Fulll,~"",o~, the toxicity of intron A appears to be negligible.
15 ExamDle 3 Antiviral activitv of B1-q 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 co" ~,uul IJ;.,g to the cc."c~"L~ ~Lion range indicated in Example 20 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 .
WO 9~35103 1 ~
The results (Fig. 3) show that B1-g has a moderate antiviral sctivity against EMC virus. Similar results were obtained against VSV and Semliki virus. The addition of small amounts of interferon-ail,L~"a;rie,i the antiviral activity considerably. Thus, as iittle as 0.5 units of interferon resulted in 5 almost 80% protection compared to 30% prul~uLiu" 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.
Examole 4 10 Anti-Rhino virus activitv of B1-q A co"~ -l;"g ~,uelilue:lll 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 15 viral infection by more than 80 to 90%. Con~.uond;.,g results must be expected with influenza virus. Thus it appears that B1-g has a very stron~3 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-a/ml i~ ":,;t;ed the antiviral activity 20 to a s;~ ,itic~..,L extent, cf. Fig. 4B.
Examole 5 Antiviral activitv of NH1 ~ 10,000 WISH cells were seeded in wells in a microtray for 24 hours and incubated for 24 hours at 37 C in an ~l~. o~,ol)e,~ containing 5% C02.
25 5uhse~ ently, the medium was replaced by fresh medium containing WO 95/35103 2 1 ~ 3 3 9 ~ PCT/DK9S/0025~
dilutions of NH4+ and virus, and after incubation for 24 hours at 37~C in an dllllo~ le co"L~ Ig 5% CO2, MTS was added over 2 hours at 37~C
and 5% CO2, whereafter the microtray was measured in a OD-scanner As iilustrated in Fig. 5, NH4+ ions are capable of inhibiting VSV, and to a 5 minor extent Semliki virus, whereas no protection appears against EMC.
Examr le 6 Anti-Rhino virus activitv of NH1 +
As described in Example 5, the antiviral activity of NH4+ towards Rhino virus was tested, and after incubation for 24 hours at 37~C in an 10 atmosphere cOIlLailli~lg 5~h CO2, MTS was added over 2 hours at 37~C
and 5% CO2, wl,t:,~arl~l 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 NH4CI-solution of 1:900. In contrast a toxic effect appears at higher conce~ ILI ~lions of NH4 + for the laboratory culture 15 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, NH4+ posses.as a varying antiviral strength towards different viruses, and flJIlllelfnol~ the NH4+
20 con1~,,L,c,lion varies which in each particular case provides the optimum antiviral effect.
~ Examole 7 Antiviral activitv of B1-a. B1-a + NH~CI as well as B1-q + 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, 5 NH4CI alone at the dilutions of 1:100() or 1:2000 nor NH4CI in combination with SOS have any Siy~iriCclllL antiviral effect.
The use of B1-g alone reveals a good effect being illLell~iFied by the simultaneous use of NH4CI, which alone at 34~C only provides a very low protection. Nevertheiess, an increasing effect is obtained with an 10 increasing NH4+ concc,,LlaLiom The additional use of SOS in the dilution of 1:100 provides only & moderate increaso of the effect.
When comparing Figs. 7A, 7B and 7C it appears that the favourable effect of the cu~ aLion of B1-g, NH4CI and SOS is most si~"irica"l at an SOS-dilution of 1:400 (Fig. 7C), where a protection of almost 95% is found 15 collea~olldillg to a B1-g co"ce"L,dLion of siy,lirica"Lly less than 1 ,ug/ml. The fact that the most favourable effect is obtained at the lowest cu"ce"i~dLiol- of SOS tested is probably due to some toxicity of SOS
towards the laboratory cells used. SOS is, however, known to be cOlll~leLely non-toxic to humans in all collce:llLlaLions relevant in practice.
20 ExamPle 8 Anti-Rhino virus activitv of B1-a, NH1CI, SOS and interferon-a 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-a in an amount of 0.5 unitslml 25 intensifies further the favourable effect obtained by a cor"i i"aLion of _ _ . . _ . .. _ _ . ... .. ~ ... , ~ _ _ .
wo 95/35103 ~ ~ q ~ r~
NH4CI, B1-g and SOS, whereby an almost total l~uL~.Iion is obtained by the use of B1-g, NH4+ ions and interferon-a.
Accordingly, the natural presence of interferon in a human during an infection must be expected to have an intensifying effect on B1-g and 5 NH4+. 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 activitv of B1-a, NH~+ ions. SOS, i"L~rr~,uns and combinations 1 0 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, VER0) were used for the tests. The results appear from the Table below.
Antiviral ctivity against dif erent virusea on di-ferent cell lines.
Target cell WISH cell A-549 cell VERO cell WISH
cell Virus VSV Semliki EMC VSV Semliki EMC Influenza A Influenza B Rhino Antiviral component SOS-- -- + ---- +
(NH4)2SO4 + + - + + _ + + +
NH4Cl + _ _ + _ +
10 SOS+(NH4)2sO4 + ND ND +
HuIFN-a ++ ++ +++ ++ ++ ++ ND ND ++
B1-g + -/+ + + + + ND ND ++
B1-g+HuIFN-a + + + + + + ND ND +++
B1-g+NH4Cl ND ND ND ND ND ND ND ND +++
15 B1-g+SOS ND ND ND ND ND ND ND ND ++
B1-g+NH4Cl+SOS ND ND ND ND ND ND ND ++++
B1-g+NH4Cl+HuIFN-a ND ND ND ND ND ND ND ++++
VSV virus, Semliki Forest virus and influenza viru8 belong to the enveloped viruses; EMC and Rhino virus belong to the non-enveloped virusec.
20 ND = not determined.
wo 95/35l03 2 1 ~ 3 3 ~ ~ PCTmK
As it appears from the above, Rhino virus is inhibited by ammonium ions and by B1-g as well as by interferon-a. Influenza virus is also assumed to be inhibited by ammonium ions. SOS appears to have some antiviral effect towards EMC, but no dc ~ antiviral activity towards Rhino virus. It 5 appears clearly that Rhino virus (which e,.d",,uliries a cold virus) is inhibited by the co"~L,i"aLion of B1-g, NH4+, interferon ~ SOS.
ExamPle 10 Kinetic tests A test was p~,~u~ued to examine possible dir~,d"ces in the antiviral 10 effect as a function of the time for the initiation of the antiviral treatment relative to the e~LaLl;..l""~"L 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 cunct:llLldLions in the range of 25 to 1.6 ,ug/ml, ~r.hele:a~L~l all of 15 the cells were incubated for 24 hours at 37 C in an dL",Gs,ul,e,t: containing 5% CO2. 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~~6 CO2.
Sl Ihserlu~rltlyt the third group of cells received B1 -g (group + 24h), and all20 of the cells were further incubated for 4 to 5 days at 34~C and 5% CO2 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 Idyaldldss whether the B1-g addition is carried out 24 hours before the viral infection or 25 simultaneously with said viral infection.
Ful~ ,,ll,ol~ it is seen that even if the B1-g treatment is not initiated until WO 9~/35103 r~ u~
~1 933~ ~
24 hours after the viral infection, i.e. at the time where the viral infection has ~ lir~:~Lt:d itself, a distinct antivirai effect is obtained.
While the invention has been described with reference to specific ernho~' "e"b thereof, it is obvious that it can be varied in many ways.
5 Such variations are not to be ~,onside, ed a deviation from the scope of the invention, and all such ",odiric~,Lions which are obvious to persons skilled in the art are also to be con aid(:l ed comprised by the scope of the acco",~-.,"ying claims.
minor microbial infections, especially during virus infections causing allllllaliunst e.g. by the presence of cationic substances. The sulphated saccha~ides will be retained in the illflallllllaluly areas and thereby reduce the illrlallallaLuly processes in the affected area.
5 A particular aspect of the present invention is therefore sulphated sac~.llalides for use as an anti-illrlallllllaLuly substance in the oral cavity and the Iymphatic ring, respectively, around the lower respiratory passages (below the nasopharynx), as well as a method of treating illrlal 1 ll llaLiOns i this area.
10 According to an embodiment of the present invention, the Sdccl,a,ide is a mono or polysulphated mono, di, tri or L~:LIa~ ,hàlid~. According to a particular e",Lo-lialal ~L, the saccharide is a monosacchal ide selected from xylose, fructose and glucose or a ~ i~e~,cl,aride selected from sucrose, lactose, maltose and cP~Iobiose 15 In a preferred ~IL~ L the sacchdlide forms a complex or a 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 e",l,c' Il~L of the present invention the sulphated d;aac~lla,ide is sucrose octasulphate or a complex or a salt of 20 sucrose octasulphate with ammonium ions or with a metai 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 NH4+ salt thereof or the aluminum complex of sucrose octâsulphate, sucralphate, are 25 preferred.
Interferons usually present under ordinary virus infections, especially in wo 9S/35103 P~l/~.~_ l ~T 93396 connection with colds, have been shown to intensify the antiviral effect of ~- lupeol and ammonium ions. Thus it has been found that i~lLelre~uns in relatively low conce"LIaLiu"s of 0.1-2 units/ml intensify the antiviral effect.
Fu,ll,e,,,~o,e:~ it can be advantageous as a further ingredient of the 5 pha""aceutical composition to use human or non-human immunoglo-bulines directed towards the substances contributing to intensify colds, such as ",ic,ooryq";D"~o (virus) etc.
According to a particular embodiment of the invention the phal ll~ac~:utica cor,,,uooiLions comprise therefore as a further ingredient human or 10 non-human immunoglobulines.
The plla,l"aceutical COIll,uOailiOll iS preferably in the form of chewing gums, lozenges, chewing tablets, leooriL~luLo, drops, troches, gels, mouth ointments, solutions or in form of mucoadl,~,~.hrc formulations, preferably in the form of depot ple:~ualdLiuns~ By depot ~ntlpalaLions is in this 15 uor",eu~io" to be ulldeloluod IJl_l~alaLiulls and formulations with a controlled, sustained release of active ingredients.
The pha""aceuticai composition is preferably in the form of a chewing yum, which per piece of chewing gum having a weight of 500 to 3000 mg, preferably of approximately 1000 mg, Cullludouo.
a) 0.01 to 2000, preferablyO.15-1000, particularlypreferred 1-800, such as 20-600 ~9 of a ~-lupeol derivative/piece, cal~ ted as ,6-lupeol, - b) O to 100, preferably 1-50, particularly preferred 2 to 40, such as 5-30 mg of NH4 +-ions/piece, calculated as ammonium chloride, WO 9S/35103 2 l ~ 3 3 ~ 6 r~
c) 0 to 1000, preferably 10-500, particularly preferred 25-250 mg of a sulphated saccha~ elpiece~ c~lcl l'atPd as SOS, as well as conventional chewing gum inyltd;cnl~
The chewing gum is prepared by means of conventional chewing gum 5 bases and conventional chewing gum additives, such as 5w~ n6.,:"
flavours, colorants, softeners, and texturizing substances. It may fu,Ll,~""o,~ be necessary to use solubilizers or other release-controlling measures in order to release the ,~I,a""~colo~: ~lly active substances disclosed herein from the chewing gum. A further illustration of sol~
10 can for instance be found in EP-0 486 563 B1, in which a general mention of the plepalalion of chewing gum is found together with examples of applicable chewing gum illylcd;~..ll~.
The invention relates fulll,~,lllor~ to the use of one or more ,~-lupeol derivatives of the general formula I
_oCH~
H3C "
RO ~CH3 15 in which R lI:pll~51~ " a hydrogen atom, a straight-chained or branched aliphatic Cl 6-hydrocarbyl group, which may be saturated or may contajn .one or more unsaturated bonds selected from double and triple bonds, a Cl 6-acyl group, which may be straight-chained or branched and may contain one or more unsaturated bonds selected from double and triple wo ss~3s103 2 ~ 9 3 3 9 ~ r~
bonds, or a group which is easily decu~.~,uosed under the uoudiLiu"s prevailing in the human or animal body to release the ~-lupeol derivative forthe,u,e,ud,t,Liollofalln;d;~ llLforthepreventionand/ortreatmentof viral infections.
5 Fu,Ll,~:""or~, the invention relates to the use of one or more,l~-lupeol derivatives of the general formula I
_~:H~
RO~CH3 in which R ,~ .lL~ a hydrogen atom, a straight-chained or branched aliphatic C1 6-hydrocarbyl group, which may be saturated or may contain 10 one or more unsaturated bonds selected from double and triple bonds, a C1 ~-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 condiLio"s prevailing in the human or animal body to release the ,~-lupeol derivative, 15 as well as one or more ammonium ion releasing compounds for the pld,udldLioll of a l"ed;~.dll,~ "L for the prevention and/or treatment of viral infections.
Furthermore the invention relates to the use of one or more ,B-lupeol derivatives of the formula l 2 1 9 ~
_6~CH
H3C "
~CH3 RO~ ><
in which R leple~e~ a hydrogen atom, a straight-chained or branched aliphatic C1 6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1 6-acyl group, which may be straight-chained or branched and may 5 contain one or more unsaturated bonds selected from double and triple bonds, or a group which is easily decor,,,uossd under the condiLiol.s prevailing in the human or animal body to release the ,~-lupeol derivative, as well as one or more mono or polysulphated mono, oligo, or polysdc.,h~l ides or analogues or derivatives thereof, for the pl e~ul c~ Lio~ ~ of 10 a ",e.l;.;a~e~lL for the prevention and/or treatment of viral infections and a:~aOciaLt:d i~rlcl,,,,,,c,Lions.
Finally, the invention relates to the use of one or more ~-lupeol derivatives of the formula I
~CH
~ ' ~ --CH3 RO ><~
in which R lel le~ellL~ a hydrogen atom, a straight-chained or branched WO 9S/35103 r~
2~ 93396 aliphatic C1 6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1 6-acyl group, which may be strai9ht-chained or branched and may contain one or more unsaturated bonds selected from double and triple 5 bonds, or a group which is easiiy decor",uosed under the conditions prevailing in the human or animal body to release the ~-lupeol derivative, one or more ammonium ion releasing compounds, as well as one or more mono or polysulphated mono, oligo, or polysscc hal ides for the preparation of a 1 ~ di~.a~ L for the prevention and/or treatment of viral infections and 10 ~.co~ id~ llllllldLilJIl:~, 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 15 mentioned active ingredients for treating HIV, hepatitis virus, cytomegalo virus, herpes virus and other viral infections as well as for treating dLl,t~ ;la,usis as well as for suppressing tumour cell growth.
Antivirally active substances may function in various ways:
(i) either as a substance capable of pluL~.,Lillg the target cells provided it 20 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 cOlllLillaLiull thereof. Many plant extracts will show this type of "non-specific', receptor-dependent antiviral activity. Most 25 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, Wo 95/35103 2 1 9 3 3 ~ S
(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.
5 It is very likely that through this type of antiviral substances more fulldalllellLcl changes inside the cells are produced via the synthesis of intracellularproteins/enzymes,whichsecondalilycausearelativelyspecific inhibition of the Llall~c,i,uLiun and/orthe Llarl~laLion of the virus in such a manner that the new intracellular proteins result in a so-called "antiviral 10 state" of the cell. When the antiviral state has been produced in the cell, the substance need no longer be present in principie as the cells are protected for a certain period of time, although the pruLeuLion must be expected to decrease gradually over time.
Ammonium ions are thought to belong to type (i) in the effect ,,,echc, ,i..""
15 although a certain, but weaker antiviral activity can be measured in cell cultures by the addition of NH4+ 2 to 4 hours after the infection.
B-lupeol is thought to belong to type (ii) in the effect l"eul,a"k"".
~-lupeol 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 vârious 20 medicinal plants, such as in extracts from bitter ginseng. ,t~-lupeol is CO u ll l lel ciclly available and may be obtained e.g. from the company Sigma.
The scope of . ,c ' ' ' :y 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 ~ 25 merely included to illustrate preferred e",L,od;."e"L~, and that various - alterations and IllodiricaLions within the scope of pluLeuLiOII will be obvious to persons skilled in the art on the basis of the detailed desu,i,uLiom WO 95/35103 2 1 9 3 3 9 6 PCTII)K95100256 Brief De~ ioll of the Drawinqs 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) against5 Rhino virus, Fig. 2 the antiviral activity of interferon-a (HulFN-a) against Rhino virus, Fis. 3 the antiviral activity of B1-a against EMC virus, Fig. 4A the antiviral activity of B1-g against Rhino virus at various dilutions, 10 Fig. 4B the antiviral activity of B1-g + interferon-a against Rhino virus, Fig. 5 the antiviral activity oF NH4+ ions against VSV, Semliki virus and EMCIII virus, Fig. 6 the antiviral activity of NH4CI against Rhino virus, Fig. 7A the antiviral activity of B1-g, B1-g + NH4CI as well as B1-g, 15 NH4CI + 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, NH4CI, SOS and interferon-a against 20 Rhino virus, and WO 95/35103 2 1 9 3 3 9 6 1 "~
Fig. 9 the kinetics for the induction of an antiviral state.
Detailed Des-.,iuLion of the Invention The method used for d~ l lg antiviral activity is described below.
The cell cultures employed are VERO cells, WISH cells, MDBK cells and 5 HEP cells which are common laboratory cell cultures and which are described in greater detail in Berg, K.: Purification and ulla~aul~ aLion of murine and human i,lL~r~,uns. A review of the literature of the 1970s (thesis). Acta Pathol. Microbiol. Scand., Sec. C., Suppl.279.: page 1-136, 1982. The viruses employed are VSV, EMC, Semliki virus, influenza virus 10 and Rhino virus.
Briefly a ,i~yle laycr cell cuiture is r~LaL~ l,ed 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.
15 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 (110~9 MTS + 39.2 ml PBS, pH-value 5.6 kept at +4~C in the dark), 2.3 ml medium and 30 ~11 PMS stock solution (13 mg PMS (Sigma, H5004, Lot 20 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 25 been killed by virus. Thus, the virus control cultures will typicall.y 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 WO 95/35103 2 1 9 3 3 9 ~ ~
ul u~ on 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 5 a virus, use of the method for quantitative d~L~nll;llalion of biological substances and agents for use as novel substances detected by the method. PCT/DKI89/00010. 1 to 32, 1989.
Hansen M. B., S. E. Nielsen, and K. Berg: Fe-ex~""i,ldLiù" and further development of a precise and rapid dye method for measuring cell 10 growth/cell kill. J. Immunol. Methods. 119: 203 to 210, 1989.
Berg, K., M. B. Hansen and S. E. Nielsen: A sensitive bioassay for precise quc"~Lir;ualiùl- of interferon activity as measured via the Illiluullolldlial deh~/-JIuy~nase function in cells ~MTT-method). AMPIS 98: 156 to 162, 1 990.
EXAMPLES
Examole 1 Antiviral activitv of ~-luoeol measured bv means of the MTS-svstem 500 to 1000 WlSH-cells in 100 ~I medium were seeded in wells in a microtray and incubated for 24 hours at 34 C in an dLIllo~,ul,e:,~ containing 20 5~~c COz. The medium was replaced by fresh medium CollLai"i"g dilutions of ,~-lupeol (25 to 1.6,ug/ml, cf. Fig.1) and incubated for further 24 hours at34ocinanallllo~ull~l~collLaillilly5%co2~Thefollowingdaychallenge Rhino virus was added and after 4 to 5 days at 34 C in an aLIl~o.,uhe~:
containing 5% C02 MTS was added over 2 hours, wl,e,t:c,rl,:r the ~ 25 microtray was measured in an OD-scanner. A total protection against WO 95/35103 2 1 9 3 3 9 6 PCT/I)K95/00256 Rhino virus was obtained at 3 ~g/ml ~-lupeol ( = B1 -g). However, at high conce"L,~Iiol,s of ~-lupeol a de,~ ;.,9 cell number appears which must be ascribed to some toxicity of ,6-lupeol at such conce"L,llLiol1s.
ExamDle 2 5 Antiviral activitv of interferon-a irHulFN-a-2b, "intron A") aqainst 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-a-2b ("intron A"~
in fresh medium co"l_:.l;"g 2% serum (cf. Fig. 2). On the following 10 morning the medium was replaced by fresh medium cor,l_;.,;"g Rhino virus. The results in Fig. 2 clearly show that Rhino virus is relatively sensitive to HulFN-a-2b, and that a protection of cl,u~uluXilll~L~ / 90% is achieved at ~I,u~ulu~dlll~hly 8 units IFN/ml. Fulll,~"",o~, the toxicity of intron A appears to be negligible.
15 ExamDle 3 Antiviral activitv of B1-q 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 co" ~,uul IJ;.,g to the cc."c~"L~ ~Lion range indicated in Example 20 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 .
WO 9~35103 1 ~
The results (Fig. 3) show that B1-g has a moderate antiviral sctivity against EMC virus. Similar results were obtained against VSV and Semliki virus. The addition of small amounts of interferon-ail,L~"a;rie,i the antiviral activity considerably. Thus, as iittle as 0.5 units of interferon resulted in 5 almost 80% protection compared to 30% prul~uLiu" 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.
Examole 4 10 Anti-Rhino virus activitv of B1-q A co"~ -l;"g ~,uelilue:lll 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 15 viral infection by more than 80 to 90%. Con~.uond;.,g results must be expected with influenza virus. Thus it appears that B1-g has a very stron~3 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-a/ml i~ ":,;t;ed the antiviral activity 20 to a s;~ ,itic~..,L extent, cf. Fig. 4B.
Examole 5 Antiviral activitv of NH1 ~ 10,000 WISH cells were seeded in wells in a microtray for 24 hours and incubated for 24 hours at 37 C in an ~l~. o~,ol)e,~ containing 5% C02.
25 5uhse~ ently, the medium was replaced by fresh medium containing WO 95/35103 2 1 ~ 3 3 9 ~ PCT/DK9S/0025~
dilutions of NH4+ and virus, and after incubation for 24 hours at 37~C in an dllllo~ le co"L~ Ig 5% CO2, MTS was added over 2 hours at 37~C
and 5% CO2, whereafter the microtray was measured in a OD-scanner As iilustrated in Fig. 5, NH4+ ions are capable of inhibiting VSV, and to a 5 minor extent Semliki virus, whereas no protection appears against EMC.
Examr le 6 Anti-Rhino virus activitv of NH1 +
As described in Example 5, the antiviral activity of NH4+ towards Rhino virus was tested, and after incubation for 24 hours at 37~C in an 10 atmosphere cOIlLailli~lg 5~h CO2, MTS was added over 2 hours at 37~C
and 5% CO2, wl,t:,~arl~l 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 NH4CI-solution of 1:900. In contrast a toxic effect appears at higher conce~ ILI ~lions of NH4 + for the laboratory culture 15 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, NH4+ posses.as a varying antiviral strength towards different viruses, and flJIlllelfnol~ the NH4+
20 con1~,,L,c,lion varies which in each particular case provides the optimum antiviral effect.
~ Examole 7 Antiviral activitv of B1-a. B1-a + NH~CI as well as B1-q + 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, 5 NH4CI alone at the dilutions of 1:100() or 1:2000 nor NH4CI in combination with SOS have any Siy~iriCclllL antiviral effect.
The use of B1-g alone reveals a good effect being illLell~iFied by the simultaneous use of NH4CI, which alone at 34~C only provides a very low protection. Nevertheiess, an increasing effect is obtained with an 10 increasing NH4+ concc,,LlaLiom The additional use of SOS in the dilution of 1:100 provides only & moderate increaso of the effect.
When comparing Figs. 7A, 7B and 7C it appears that the favourable effect of the cu~ aLion of B1-g, NH4CI and SOS is most si~"irica"l at an SOS-dilution of 1:400 (Fig. 7C), where a protection of almost 95% is found 15 collea~olldillg to a B1-g co"ce"L,dLion of siy,lirica"Lly less than 1 ,ug/ml. The fact that the most favourable effect is obtained at the lowest cu"ce"i~dLiol- of SOS tested is probably due to some toxicity of SOS
towards the laboratory cells used. SOS is, however, known to be cOlll~leLely non-toxic to humans in all collce:llLlaLions relevant in practice.
20 ExamPle 8 Anti-Rhino virus activitv of B1-a, NH1CI, SOS and interferon-a 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-a in an amount of 0.5 unitslml 25 intensifies further the favourable effect obtained by a cor"i i"aLion of _ _ . . _ . .. _ _ . ... .. ~ ... , ~ _ _ .
wo 95/35103 ~ ~ q ~ r~
NH4CI, B1-g and SOS, whereby an almost total l~uL~.Iion is obtained by the use of B1-g, NH4+ ions and interferon-a.
Accordingly, the natural presence of interferon in a human during an infection must be expected to have an intensifying effect on B1-g and 5 NH4+. 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 activitv of B1-a, NH~+ ions. SOS, i"L~rr~,uns and combinations 1 0 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, VER0) were used for the tests. The results appear from the Table below.
Antiviral ctivity against dif erent virusea on di-ferent cell lines.
Target cell WISH cell A-549 cell VERO cell WISH
cell Virus VSV Semliki EMC VSV Semliki EMC Influenza A Influenza B Rhino Antiviral component SOS-- -- + ---- +
(NH4)2SO4 + + - + + _ + + +
NH4Cl + _ _ + _ +
10 SOS+(NH4)2sO4 + ND ND +
HuIFN-a ++ ++ +++ ++ ++ ++ ND ND ++
B1-g + -/+ + + + + ND ND ++
B1-g+HuIFN-a + + + + + + ND ND +++
B1-g+NH4Cl ND ND ND ND ND ND ND ND +++
15 B1-g+SOS ND ND ND ND ND ND ND ND ++
B1-g+NH4Cl+SOS ND ND ND ND ND ND ND ++++
B1-g+NH4Cl+HuIFN-a ND ND ND ND ND ND ND ++++
VSV virus, Semliki Forest virus and influenza viru8 belong to the enveloped viruses; EMC and Rhino virus belong to the non-enveloped virusec.
20 ND = not determined.
wo 95/35l03 2 1 ~ 3 3 ~ ~ PCTmK
As it appears from the above, Rhino virus is inhibited by ammonium ions and by B1-g as well as by interferon-a. Influenza virus is also assumed to be inhibited by ammonium ions. SOS appears to have some antiviral effect towards EMC, but no dc ~ antiviral activity towards Rhino virus. It 5 appears clearly that Rhino virus (which e,.d",,uliries a cold virus) is inhibited by the co"~L,i"aLion of B1-g, NH4+, interferon ~ SOS.
ExamPle 10 Kinetic tests A test was p~,~u~ued to examine possible dir~,d"ces in the antiviral 10 effect as a function of the time for the initiation of the antiviral treatment relative to the e~LaLl;..l""~"L 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 cunct:llLldLions in the range of 25 to 1.6 ,ug/ml, ~r.hele:a~L~l all of 15 the cells were incubated for 24 hours at 37 C in an dL",Gs,ul,e,t: containing 5% CO2. 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~~6 CO2.
Sl Ihserlu~rltlyt the third group of cells received B1 -g (group + 24h), and all20 of the cells were further incubated for 4 to 5 days at 34~C and 5% CO2 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 Idyaldldss whether the B1-g addition is carried out 24 hours before the viral infection or 25 simultaneously with said viral infection.
Ful~ ,,ll,ol~ it is seen that even if the B1-g treatment is not initiated until WO 9~/35103 r~ u~
~1 933~ ~
24 hours after the viral infection, i.e. at the time where the viral infection has ~ lir~:~Lt:d itself, a distinct antivirai effect is obtained.
While the invention has been described with reference to specific ernho~' "e"b thereof, it is obvious that it can be varied in many ways.
5 Such variations are not to be ~,onside, ed a deviation from the scope of the invention, and all such ",odiric~,Lions which are obvious to persons skilled in the art are also to be con aid(:l ed comprised by the scope of the acco",~-.,"ying claims.
Claims (26)
1. A pharmaceutical composition for the prevention and/or treatment of viral infections, c h a r a c t e r i s e d in that it comprises one or more .beta.-lupeol derivatives of the formula in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, one or more ingredients selected among ammonium ion releasing compounds, mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof, and human and non-human immunoglobulines as well as conventional pharmaceutically acceptable adjuvants, additives, and carriers.
2. A pharmaceutical composition as claimed in claim 1, c h a r a c t e r i s e d by R representing a hydrogen atom.
3. A pharmaceutical composition as claimed in claim 1 or 2, c h a r a c t e r i s e d in that it comprises one or more .beta.-lupeol derivatives of the formula in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-8-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-acyl group, wich 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 .beta.-lupeol derivative, as well as an ammonium ion releasing compound.
4. A pharmaceutical composition as claimed in claim 3, c h a r a c t e r i s e d in that the ammonium ions are derived from a salt of a pharmaceutically acceptable inorganic or organic acid preferably selected from hydrochloric acid, sulphuric acid, phosphoric acid, carbonic acid, acetic acid, and tartaric acid.
5. A pharmaceutical composition as claimed in claim 3, c h a r a c t e r i s e d in that the ammonium ions are derived from a compound of the general formula II
where X1-X4, which may be identical or different, are selected from hydrogen; C1-6alkyl, which may be straight-chained or branched, saturated or unsaturated and optionally contain one or more substituents selected from halogen, hydroxy, C1-4-alkoxy or amino; aryl, which is optionally substituted with C1-4-alkyl, halogen, hydroxy, C1-4-alkoxy or amino, and Y is a physiologically acceptable salt-forming anion, preferably selected from F-, Cl-, Br-and I-.
where X1-X4, which may be identical or different, are selected from hydrogen; C1-6alkyl, which may be straight-chained or branched, saturated or unsaturated and optionally contain one or more substituents selected from halogen, hydroxy, C1-4-alkoxy or amino; aryl, which is optionally substituted with C1-4-alkyl, halogen, hydroxy, C1-4-alkoxy or amino, and Y is a physiologically acceptable salt-forming anion, preferably selected from F-, Cl-, Br-and I-.
6. A pharmaceutical composition as claimed in claim 1 or 2, c h a r a c t e r i s e d in that it comprises one or more .beta.-lupeol derivatives of the formula in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, as well as one or more mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof.
7. A pharmaceutical composition as claimed in claim 3, c h a r a c t e r i s e d in that it furthermore comprises one or more mono or polysulphated mono, oligo or polysaccharides, or analogues and/or derivatives thereof.
8. A pharmaceutical composition as claimed in claim 6 or 7, c h a r a c t e r i s e d in that the saccharide is a mono or polysulphated mono, di, tri or tetrasaccharide.
9. A pharmaceutical composition as claimed in claim 6 or 7, c h a r a c t e r i s e d in that the saccharide is a monosaccharide selected from xylose, fructose and glucose.
10. A pharmaceutical composition as claimed in claim 6 or 7, c h a r a c t e r i s e d in that the saccharide is a disaccharide selected from sucrose, lactose, maltose and cellobiose.
11. A pharmaceutical composition as claimed in any of the preceding claims, c h a r a c t e r i s e d in that the saccharide forms a complex or a 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.
12. A pharmaceutical composition as claimed in claim 10, c h a r a c t e r i s e d in that the sulphated disaccharide is sucrose octasulphate, 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.
13. A pharmaceutical composition as claimed in claim 12, c h a r a c t e r i s e d in that the sulphated disaccharide is sucrose octasulphate or a sodium, potassium or NH4+ salt thereof or the aluminum complex of sucrose octasulphate, sucralphate.
14. A pharmaceutical composition according to claim 1, c h a r a c t e r i s e din that it comprises one or more .beta.-lupeol derivatives of the formula in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, as well as one or more human or non-human immunoglobulines.
15. A pharmaceutical composition as claimed in any of the preceding claims, c h a r a c t e r i s e d in that it is in the form of chewing gums, lozenges, chewing tables, resoriblets, drops, troches, gels, mouth ointments, solutions, mucoadhesive formulations and depot preparations.
16. A pharmaceutical composition as claimed in claim 15 in the form of a chewing gum, c h a r a c t e r i s e d in that per piece of chewing gum it comprises:
a) 0.01 to 2000, preferably 0.15 to 1000, particularly preferred 1 to 800, such as 20 to 600,µg of .beta.-lupeol derivative/piece, calculated as .beta.-lupeol, b) 0 to 100, preferably 1 to 50, particularly preferred 2 to 40, such as 5 to 30 mg of NH4+ ions/piece, calculated as ammonium chloride, c) 0 to 1000, preferably 10 to 500, particularly preferred 25 to 250 mg of a sulphated saccharide/piece, calculated as sucrose octasulphate, as well as conventional chewing gum ingredients.
a) 0.01 to 2000, preferably 0.15 to 1000, particularly preferred 1 to 800, such as 20 to 600,µg of .beta.-lupeol derivative/piece, calculated as .beta.-lupeol, b) 0 to 100, preferably 1 to 50, particularly preferred 2 to 40, such as 5 to 30 mg of NH4+ ions/piece, calculated as ammonium chloride, c) 0 to 1000, preferably 10 to 500, particularly preferred 25 to 250 mg of a sulphated saccharide/piece, calculated as sucrose octasulphate, as well as conventional chewing gum ingredients.
17. The use of one or more .beta.-lupeol derivatives of the general formula I
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, and one or more ingredients selected among ammonium ion releasing compounds, mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof, and human and non-human immunoglobulines for the preparation of a medicament for the prevention and/or treatment of viral infections.
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, and one or more ingredients selected among ammonium ion releasing compounds, mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof, and human and non-human immunoglobulines for the preparation of a medicament for the prevention and/or treatment of viral infections.
18. The use as claimed in claim 17 of one or more .beta.-lupeol derivatives of the general formula I
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-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.
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-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.
19. The use as claimed in claim 17 of one or more .beta.-lupeol derivatives of the general formula I
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, as well as one or more mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof, for the preparation of a medicament for the prevention and/or treatment of viral infections and associated inflammations.
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, as well as one or more mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof, for the preparation of a medicament for the prevention and/or treatment of viral infections and associated inflammations.
20. The use as claimed in claim 17 of one or more .beta.-lupeol derivatives of the general formula I
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, one or more ammonium ion releasing compounds, as well as one or more mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof, for the preparation of a medicament for the prevention and/or treatment of viral infections and associated inflammations.
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, one or more ammonium ion releasing compounds, as well as one or more mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof, for the preparation of a medicament for the prevention and/or treatment of viral infections and associated inflammations.
21. The use as claimed in any of the preceding claims 17 to 20 for the prevention and/or treatment of infections in the upper respiratory passages, especially cold virus, such as Rhino virus and influenza virus.
22. The use of one or more .beta.-lupeol derivatives of the general formula I
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, for the preparation of a medicament for the prevention and/or treatment of infections in the upper respiratory passages, especially cold virus, such as Rhino virus and influenza virus.
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, for the preparation of a medicament for the prevention and/or treatment of infections in the upper respiratory passages, especially cold virus, such as Rhino virus and influenza virus.
23. A method for the prevention and/or treatment of viral infections, c h a r a c t e r i s e d in that it includes oral administration of a pharmacologically antiviral amount of one or more .beta.-lupeol derivatives of the formula I
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, and one or more ingredients selected among ammonium ion releasing compounds, mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof, and human and non-human immunoglobulines in a pharmaceutically acceptable carrier.
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, and one or more ingredients selected among ammonium ion releasing compounds, mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof, and human and non-human immunoglobulines in a pharmaceutically acceptable carrier.
24. A method for the prevention and/or treatment of viral infections, c h a r a c t e r i s e d in that it includes oral administration of a pharmacologically antiviral amount of one or more .beta.-lupeol derivatives of the formula I
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, as well as one or more ammonium ion releasing compounds, in a pharmaceutically acceptable carrier.
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, as well as one or more ammonium ion releasing compounds, in a pharmaceutically acceptable carrier.
25. A method for the prevention and/or treatment of viral infections and associated inflammations, c h a r a c t e r i s e d in that it includes oral administration of a pharmacologically antiviral amount of one or more .beta.-lupeol derivatives of the formula I
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, as well as one or more mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof, in a pharmaceutically acceptable carrier.
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, as well as one or more mono or polysulphated mono, oligo or polysaccharides or analogues and/or derivatives thereof, in a pharmaceutically acceptable carrier.
26. A method for the prevention and/or treatment of viral infections and associated inflammations, c h a r a c t e r i s e d in that it includes oral administration of a pharmacologically antiviral amount of one or more .beta.-lupeol derivatives of the formula I
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, one or more ammonium ion releasing compounds, as well as one or more mono or polysulphated mono, oligo or polysaccharides in a pharmaceutically acceptable carrier.
in which R represents a hydrogen atom, a straight-chained or branched aliphatic C1-6-hydrocarbyl group, which may be saturated or may contain one or more unsaturated bonds selected from double and triple bonds, a C1-6-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 .beta.-lupeol derivative, one or more ammonium ion releasing compounds, as well as one or more mono or polysulphated mono, oligo or polysaccharides in a pharmaceutically acceptable carrier.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK72294 | 1994-06-20 | ||
| DK92694 | 1994-08-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2193396A1 true CA2193396A1 (en) | 1995-12-28 |
Family
ID=26064526
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002193396A Abandoned CA2193396A1 (en) | 1994-06-20 | 1995-06-20 | A pharmaceutical composition for the prevention and/or treatment of viral infections and optionally inflammations as well as a method for the treatment thereof |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0762876A1 (en) |
| JP (1) | JPH10504279A (en) |
| KR (1) | KR970703759A (en) |
| CN (1) | CN1158566A (en) |
| AU (1) | AU689603B2 (en) |
| CA (1) | CA2193396A1 (en) |
| EE (1) | EE9600190A (en) |
| FI (1) | FI965114A0 (en) |
| NO (1) | NO965468L (en) |
| WO (1) | WO1995035103A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU4995597A (en) * | 1996-10-23 | 1998-05-15 | Vertex Pharmaceuticals Incorporated | Methods of using sucrose octasulfate to treat or prevent enveloped virus infection |
| US6251371B1 (en) * | 1997-01-09 | 2001-06-26 | Bifodan A/S | Treatment of skin or mucosa inflammation by topical treatment with preparation containing dichlorobenzyl alcohol |
| WO1998032443A1 (en) * | 1997-01-24 | 1998-07-30 | Marigen S.A. | Ultramicro-emulsions of spontaneously dispersible concentrates containing antitumorally, antivirally and antiparasitically active esters of pentacyclic triterpenes |
| US6482857B1 (en) | 1998-07-17 | 2002-11-19 | The University Of Texas Southwestern Medical Center | Compositions which contain triterpenes for regulating hair growth |
| US6124362A (en) * | 1998-07-17 | 2000-09-26 | The Procter & Gamble Company | Method for regulating hair growth |
| ES2272689T3 (en) * | 2001-01-12 | 2007-05-01 | Bsp Pharma A/S | DIHYDRO-TRITERPENS IN THE TREATMENT OF VIRAL INFECTIONS, CARDIOVASCULAR DISEASE, INFLAMMATION, HYPERSENSITIVITY OR PAIN. |
| FR2822821B1 (en) * | 2001-04-03 | 2004-05-07 | Pharmascience Lab | LUPINE SEED HULL EXTRACT CONTAINING LUPEOL, ESPECIALLY EXTRACT RICH IN LUPEOL AND PROCESS FOR PREPARING THE SAME |
| CN104761460B (en) * | 2015-03-26 | 2017-06-20 | 苏州沪云肿瘤研究中心股份有限公司 | Glaucocalyxin A derivative and preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4983637A (en) * | 1988-06-24 | 1991-01-08 | Stephen Herman | Method for treating viral infection of HIV |
-
1995
- 1995-06-20 WO PCT/DK1995/000256 patent/WO1995035103A1/en not_active Application Discontinuation
- 1995-06-20 KR KR1019960707236A patent/KR970703759A/en not_active Application Discontinuation
- 1995-06-20 JP JP8501510A patent/JPH10504279A/en active Pending
- 1995-06-20 CA CA002193396A patent/CA2193396A1/en not_active Abandoned
- 1995-06-20 EE EE9600190A patent/EE9600190A/en unknown
- 1995-06-20 AU AU27340/95A patent/AU689603B2/en not_active Ceased
- 1995-06-20 EP EP95922445A patent/EP0762876A1/en not_active Withdrawn
- 1995-06-20 CN CN95194431A patent/CN1158566A/en active Pending
-
1996
- 1996-12-19 FI FI965114A patent/FI965114A0/en unknown
- 1996-12-19 NO NO965468A patent/NO965468L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| CN1158566A (en) | 1997-09-03 |
| AU689603B2 (en) | 1998-04-02 |
| WO1995035103A1 (en) | 1995-12-28 |
| NO965468D0 (en) | 1996-12-19 |
| EP0762876A1 (en) | 1997-03-19 |
| FI965114A (en) | 1996-12-19 |
| KR970703759A (en) | 1997-08-09 |
| AU2734095A (en) | 1996-01-15 |
| EE9600190A (en) | 1997-06-16 |
| NO965468L (en) | 1997-02-19 |
| FI965114A0 (en) | 1996-12-19 |
| JPH10504279A (en) | 1998-04-28 |
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