OA10207A - Treatment of diseases caused by viruses - Google Patents

Description

1 010207

TREATMENT OF DISEASES CAUSED BY VIRUSES

BACKGROUND OF THE INVENTION 1_._Field of the invention

The présent invention relates to treatment, inclusiveof prophylaxis and therapy, of diseases caused by viruses.More particularly, this invention provides pharmaceutical 5 composition ( including pnarmaceutical composition for veterinary use) containino as an active ingrédient a combination of a cell-fusionand/or vrius-adsorption inhibitor with an antiviral agentlacking inhibitory activity against cell-to-cell 10 infection through cell fusion from virus infected cells to uninfected cells and/or adsorption of viruses to target.cells, which are usaful in the treatment of diseases causedby viruses; and a method of treating diseases caused byviruses which comprises administering s ad ‘15 combination. The combination as described above can produce antiviral synergism and conseguently permit,enhancement of antiviral effect, while reducing individuaidosages and alleviating adverse side-effects. 2 010207

Very few chemotherapeutic agents fer viral diseaseshâve been available which can exert satisfactor'1therapeutic effects, in contrast to those for bacterialdiseases. The compounds, which are effective againstviruses in vitro, often produce severe adverse side-effectswhen adm.inistered to living body, and even antiviral agents,that are considered as tolerable for practical use find, in.many instances, severely restricted application because oftheir adverse side-effects. This may be reflected, forexample, by such antiviral agents as arabinosyladenine (AraA) or Acyclovir being in recent years lourd to be effectiveagainst herpesviruses and azidothymidine (AZT) beingrecently reported to possess effects against AIDS (acquiredimmunodeficiency syndrome) related viruses (that is to sayhuman immunodeficiency virus ordinarily referred t.o brieflyas "HIV"); these antiviral agents after being given to manalso bring about side-effects such as nausea, emesis.diarrhea, éruption, anémia and phlebit.is developed locallyat the site of administration, which sometimes leads to forced discontinuation of administration. Under thesecircumstance, development of antiviral agents with reducedside effects is strongly demanded, while réduction by wayof sonie raeans of side-effects inhérent- to the conventional antiviral agents currently constitutes one of the most. - 3 - 010207 important research subjects since the latter offers bettcr chance of success.

Viral multiplication is known to proceed through 5 adsorption to and entry into host cells, uncoatino, transcription, réplication, assembly of particles ar.d budding or release by lysis of cells, and antiviral agents are considered to inhibit either of the above described steps. The présent inventors found that sulfuric esters of

polysaccharides, or mucopolysaccharides or their sulfatedÎO dérivatives (hereinafter referred to, in some instances,collect.ively as "sulfated polysaccharides") , when appliedalone to retroviruses, inhibit reverse transcriptaseactivity derived from retroviruses and also suppressinfection te cells with and multiplication in cells of 15 retroviruses ( us Patent Application Serial No. 019180).

One of passways for multiplication of AID5Ï virus, oneof retroviruses, has been demonstrated to involve fusion ofan AIDS virus infected cell with an uninfected cell, 2q followed by direct pénétration of AIDS virus into the uninfected cell. The présent inventors hâve, in the course ofextensive research on the mechanism of action provided bythe above-described sulfated polysaccharides, discoveredthat the said sulfated polysaccharides inhibit adsorption of HIV's to target cells and also block direct cell-t.o-cel 1 25 infection by HIV through fusion of HIV-infected cells with 4 010207 uninfected oells, thereby displaying capability to suppressmultiplication of AIDS virus. Noting that the sulfatedpolysaccharides manifest. guite novel suppressing mechanismfor AIDS-virus multiplication through inhibition of virusadsorprion and cell-tc-cell fusion, in sharp contrast withinhibitory action against reverse transcriptase activityprcvided by the conventionai antiviral agents, the inventorshâve found furthermore that such two different classes of compounds, when used in combination, can prodicesignificant synergism 20 to 50 times greater than could beexpected from simple addition of the individual drugeffects. In addition, it lias been found that. any of theconventionai. antiviral agents Lacking inhibitory activityagainst cel 1-to-cell infection through cell fusion fromvirus-infected cells to uninfected calls and/or adsorptionof viruses to target cells, when simply used in combinationwith any substances possessing cell-fusion and virus-adsorpotion inhibiting activity, can universally achievesuch a level of antiviral synergism, in other words, thatany such combination constitutes a raechanisn of producingantiviral synergism.

The présent invention has been completed based on thefirdings as described above. 2. Description of related art A comprehensive review on antiviral agents is given inKirk Othmer's Encyclopedia of Chemical Technology, vol. 5, 010207 - 5 - pp. 542-552 (John & Wiley & Sons, New York, USA).

Among sulfuric esters of polysaccherrides, low-molecular~weight dextran sulfates hâve benn commercialized as anantilipemic, anti-arteriosclerotic or anticoagulant ager.t,while dextran sulfates with relatively high molecuJarweights are known to exhibit suppressory activity agairstherpesviruses (European Patent Publication No. 0066379). Since herpesviruses belong to a DNA virus,nevertheless, they are fundamentally different in mode ofmultiplication from retroviruses depending entirelv onreverse transcriptase for synthesis of DNA. Accordingly, toexert effect against herpesviruses does not. necessarilymean to be effective against retroviruses. Tri addition,low-molecular-weight dextran sulfates (having molecularweights of not more than 10,000) are known to ne almostineffective against herpesviruses.

Among muchopolysaccharides or the i. r sulfates,chondroitin sulfate is commercialized as a drug forsenorineural hearing impairment, neuralgia, lumbago orchronic nephritis and also in the forn of cornea-protectiveophthalmic solution, while it lias been known that keratansulfata is obtained from cartilages; teichuronic acid fromcell walls of Bacillus subtilis; hyaluronic acid from shsrkskin, whale cartilage or human sérum; heparan sulfate fromthe bovine liver or lung; and chitin from arthropcd shells,fungi or yeasts, respectively. A process for producj ng 01020 7 sulfated dérivatives of chondroitin sulfate is described in

Japanese Patent Publication (jp, B2) No. 9570/1971.

Heparin is known to inhibât in vitro various enzymes, 5 such as DNA polymerase of phytohemaglutinin stimulatedhuman lymphocytes and reverse transcriptase of simiansarcoma virus (Cancer Research, 38, 2401 to 2407), whereasnothing has been found about whether or not heparin caninhibât infection with retroviruses at the cell level. 10

SDMMARY OF THE INVENTION

In the first aspect, the présent invention provide; amethod of treating a disease caused by virus which comprisesadministering to a subject in need cf such t.reatment (A) an antiviral agent lacking inhibitory activity (1) ^5 against cel1-to-cell infection through cell fusion from a virus infected cell to an uninfected celland/or (2) against absorption of a vicm to at.arget cell, and (B) a compound possessing cell fusion inhibitory 2Cl activity and/or virus-adsorption inhibitory activity, said antiviral agent and compound being administered ii.respective amounts so t.hat the combinée dosage t.hereof is effective to treat sard disease. 25 ...

In the second aspect, the; présent invention provides the use of a cell-fusion and/or virus-adsorption inhibitor for the manufacture of a médicament effective for treating diseases caused by viruses, which médicament is specificaliy intended to be administered in combi nat ion with an antiviral. 010207 agent lacking inhibitory activity against ce1l-to-ceï1infection through cell fusion from virus-infected cells touninfected cells and/or adsorption of viruses to target cells·». 5

In the third aspect, the présent invention provides aph a r ma c eu t i c a 1 composition coroprising s synergistically effective amount each of an antivilal agentlacking inhibitory activity against celi-to-celî infecdior: 10 through cell fusion from virus-infecte.d cells to uninfectedcells and/or adsorption of viruses to target cells and a cell-fusion and/or virus-adsorption inhibitor.

In the fourth aspect, the présent invention provides a method of lowering side effects of an antiviral agent lacking 15 inhibitory activity against cell-to-cell infecticr throughcell fusion from virus-infected cells to uninfected colisand/or adsorption of viruses to target cells, whu.ch methodcomprises administering said antiviral agent in anount lowerthan usual dose in conjunction with. a cell-fusion and/or 20 virvs-adsorption inhibitor.

In the fifth aspect, the présent, invention provides theuse of a cell-fusion and/or virus adsorption inhibitor forthe manufacture of an antiviral synergistic composition 25 comçrising an antiviral agent lacking inhibitory activity against cell-to-cell infection through cell fusion fromvirus-infected cells to uninfected cells and/or adsorption ofviruses to target cells and a cell fusion inhibitor in orderto cichieve réduction in side effects pertaining to the formerantiviral agent. 010207

BRIEF DESCRIPTION OF DRAWING

Figs. 1 and 2 each shows a relationship between a.number of viable MT-4 cells and a concentration of dextran sulfate sodium sait (hereinafter re·ferred to briefly as"DS") having a molecular weight of 7,000 to 8,000 and a 5 sulfur content of 17 to 20 %, ras was observe! when the cells were cultured with HTLV-III viruses in the presenceof AZT and/or DS at different, concentrations as is to bedescribed in Example 11,

Figs. 3 and 4 each shows a relationship between a 10 percentage of HTLV-l'II-specif ic antigen positive (IF-positive) cells (%) and a concentration of DS as wasobserved in Example 3.1 to be described below,

Figs. 5 and 6 each shows microscopie views of cocultured, Molt-4 cells and Molt-4/HTLV-III cells in the 15 presence of 0 and 10 mrcrogram/ml of DS, respect ively, aswas obtained in Example 15,

Figs. 7 and 8 each shows a number of viable cellsMolt-4/HTLV-III and HTLV-III-specific antigen positive (IF-positive) cells (%) as was observed when the cells were 20 cultured in the presence of AZT and DS, alone and inmixture, respectively, as was observed in Example 17, 9 010 2 0 7 ίο 15 20

Fig. 9 shows a microscopie view of normal Vero ceJs,Fig. 10 shows a microscopie view of Vero colis infeeted with Herpes virus type II,

Fig. 11 and 12 each shows microscopie views cf Vero cells cultured with 1 pg/ml or 10 ug/nl,. respective] y, ofdextran sulfate added simultaneously to adsorption of tierpevirus type II, and

Fig. 13 and 14 each shows microscopie views of Verocelis cultured with 1 pg/ml or 10 pç/nl, respectively, ofdextran sulfate added after adsorption Herpes virus type II

DESCRIPTION OF THE PREFERRED EKBODIMENTS

The term "treatment" as used herein is intended tocomprehend ail the managements of diseases includingprévention, sustension (i.e., prévention of aggravation),alleviation (i.e., amelioration of conditions) and therapy.

The virus diseases to be treated accord in g "o theprésent invention include diseases caused by verrousviruses. Such viruses include Poxviricae (e.g.smallpoxviruse, rabbitpoxvirus, cowpoxvirus, swinepoxvirus,equinepoxvi rus, i'owlpoxvirus), Herpesvir idae (e. g. herpessymplex virus 1 or 2, herpes zoster virus, cytomégalovirus),Adenoviridae, Orthomyxoviridae (e.g. influença virus),Paramyxoviridae (e.g. parainfluenza virus, numps virus,respiratory syncytial virus, measles virus) . Rhabdoviridae,Picornaviridae (e.g. poliovirus, coxsackie virus, echovirus,hepatitis virus, rhinovirus) , Coror: avrr.i da; ·. s rvovir idée , 25 10 (11()207

AeOVÏlîdae, To^dVÎ I .lCcK? ( Θ . g . JdpdllG S6 θ ne G pli a 1 i t i S Vl'US ,rubella virus) , and Retroviridae (e.g. HIVs (e.c. HTLV--IIT, .-AV, ARV etc.) , ovine visna virus, eçuine irfectious anemi?.virus, avian leukemia virus, avian sarcoma virus;, avian 5

reticuioeudothetiosis virus, murine breast carcinome virus,murine leukemia virus, murine sarcoma virus;, egiline type Cvirus, hamster type C virus, rat leukemia virus, felineleukemia virus, feline sarcoma virus, feline type C virus,ovine leukemia virus, bovine leukemia virus, swii-e typ‘ C 10 virus, simian leukemia virus,

Mason-Pfizer Virus, simian sarcoma virus, simianT-lymphotropic virus, baboon type C virus, adult T-cellleukemia virus (ATLV) or human T-lymphotropic virus types I and II (HTLV-I, HTLV-II), human Kawasaki disease virus), 15 etc. The most important diseases are AIDS, PGL (Persistentçeneralized lymphadenopathy), ARC (AIDS-related ; r-.plex) , LAS (Lymphadenope.t.hy syndrome) and human T-cell· leukemia.

As the antiviral agent jacking inhibitory activity against cell-to-cell infection through cell-fusion 20 from virus-infected cell to uninfected cell and/or adsorption of viruses to a target c-11 which isuseful in this invention, there can be employed any antiviralagents insofar as they lack inhibitory activity against theabove-described cell—to—cell infection, and such antiviral, 25 agents include nucleic acid type antiviral 11 010207 agents {for example, 3'-azido-3’-deoxytnymidine(azidothymidine or AZT), 2 ’ ,3 *-dideoxynucleosides(2',3'-dideoxyadenosine, -guanosine, -inosine, -cytidine, or•-thymidine), ribavirin, isoprinosin, acyclovir, ara A, ara ej T, ara C, iododeoxyuridine (IDU) , bromovinyl deoxyurid ine , fluoroiodo aracytosin, 21-amino-2'-deoxyribofuranosyladenine, trifluridine, acyclovirdérivatives (deoxy-, glycyl- or iodo-acyclovii. .dihvdroxypropoxymethy1 guanine, «10 dihydroxybnty 1 guanine), chloroethyldecxyur.i d.i ne , •icyocamycin, puromycin, etc.), pepcide type antiviral agent(e.g., suramin, distamycin A, actinomycin D, etc.),ar.samycin type antiviral agent (e.g., ansamycin, rifamycin,rifampicin, dimethylbenzyl rifampicin, streptovaricin S, «jfj etc,), polyanionic type antiviral agents (e.g., HPA-23 (NaSbgW ^Οθθ) , etc.), thiosemicarbatic.e type . i. ivir-·!agents (imuthiol, isatin-beta-thiosemicarbazone (IBT) ,,marboran, etc.), phosphoric acid type antiviral agent (e.g.,foscarnet), amantadine type antiviral figent (e.g., ;0 amantadine, rimantadine, N'-methyladamantane-spiro-3’-pyrolidine hydrochioride, etc.,), erdogenous antiviral agent (e.g., inter ferons, interleukins, Neurotropin), glycoside type ,'.ηί i · ii ai agent (e.g., glycyrrhizin, etc.) and lipid type antiviral agent 30 (e.g. AL") 21 etc. ) . - 12 - 010207

Prefsrred antiviral agents are Chose havirci reversetranscriptase inhibitory activity and effective against HIViAZT, 2 ' , 3 ' -dideoxyr.ucleosides , suraniin, ansarcycin, IIP/ .-23,foscarnet) , and other agents not having such activât'/ but. $ * effective against HIVs ( ribavirirt, <*-, B-interferons, Al//21, ampligen).

The compound possessing ceil-iusion inhibitory ac.ivit.yand/or virus-adsorption inhibitory activity to be used in the présent invention includes naturel orθ synthetic oligosaccharides or polysaccharides having at least one S-oxoacid group attached to the saccharic carbonatom through a linking group with a low molecular weight, or pharinaceutically acceptable salts thereof, whichever such oligosaccharides or polysaccharides may bej natural or synthetic. The term "natural" neans such oligo-or polysaccharides being obtainable by way of such means asextraction from natural resources, such as plants, microbesor animais. The term "synthetic" désignâtes such oligo- orpolysaccharides beir.g obtainable by synthetic procedure, 20 for example, by introducing an S-oxoacid group into naturalor non-natural (synthetic) oligo- or polysaccharides . vh chhâve or bave; not S-oxoacid group.

The term "oligosaccharide" means carbohydrates which con*-ain of 2. to about 9 monosaccharides being each iinked 25 with the other.Fôr exarple, where oligosasccharides contain - 13 - LU 0207 3 monoss.ccharides, for exemple/ one, ^wo or three of suchmonoE'.accharides may hâve at least one S-oxoacid group.

The term "polysaccharide" means carbohydrates whichcontain of tôt less than about ten monosaccharide:s beingeach linked with the other, where at least one, a minorportion, a major portion or ail of the constituentmonosaccharides may hâve at least one but normally not more than four S-oxoacid groups. 10

The above-described oligosaccharides orpolysaccharides may, in place of hydrogen atoms in part of thei:hydroxyl groups ,have short-chain groups such as lower alkyls (e.g. methyl). and rower acyls (e.g, acetyl) ormay hâve sulfate groups linked through such short-chain groups. 15 . .

The S-oxoacid group mcludes sulfo group (-SO3O) andhydroxysulfinyl group (-SO*OHj, with sulfo grcop beingpreferred.

The term "saccharic carbon atom" refers te carbonatoms which constitute a tetrahydrof ura n ring cr 0 tetrahydropyran ring contained in oligosaccharides crpolysaccharides.

The term "low-molecular-weight linking group" isintended to eomprehend oxy (-O-), imino (-NH-), thio(-S-) , methylene (-CH2-) , ethylidene (-CHfCHj)--) and thelike. The term "low wolecular weight" means moléculerweights of any linking groups ranging from about 14 to 14 0 î 0207 about 32. The preferred linking group includes oxy and imino.

The oligosaccharides or polysaccharides, which areusable in this invention, include natural polysaccharideshaving at least one hydrogensulfate croup (-O-SO3E) which are obtained from plants or microbes and syntheticpolysaccharides having at least one hydrogen sulfate group 010207 25 -13 - (-O-SO II) which are produced by esterLfying a poly -saccharide as obtained from plants or microbes,

Preferred among them are sulfated polysaccharides havi.ng r.on-aminomonosaccharide (containing acids) us a 5 repeating unit, and those containing a trace amount ofnitrogen are also included. The Monoaminoaonosaccharidesserving- as a repeating unit includes, for exemple, xylose,arabincse, rhamnose, fucose, glucose, galactose, glucurcnic acid, galacturonic acid, raannuronic acid, etc. The- naturally 10 · . . occurrmg polysaccharides include carrageenan (galactansulfate obtainable from Gigartina stellata, Chondrns crispus,etc. ) and fucoidin (polyfucose suif acte obtained from brownmarine algae of the genus Laminaria). Said caraageenan includes carrageenans having different suî cate group 15 contents, such as /f-carrageenan, X-carrageenan andcarrageenan. Examples of the synthetic polysaccharides arethose which are obtained by sulfating polysaccharides, suchas starch and its partial hydrolysates, dextran produced bythe genus Leuconostoc and its partial hydrolysates (having a 20 molecular weight of usually 500 to 2,000,000, normally 2,000to 300,000, preferably 2,000 to 10,000, most suitably 3,000to 3,000), peirtosan/llycogen, pectin, cellulose, visccus liquidsubstances of plant origin (gum arable, tragacanth, etc.),viscid substances of plant origin (those produced from okra,aloe, Brasenia schreberi, etc.), viscous liquid substances - 1b 010207 from marine and fresh-wat.er algae (alginic acid, laminarin,etc.), polysaccharides derived from microorganisms (lentinan,pullulant, mannan, xylan, etc.) or synthetic polysaccharides.Among such polysaccharides are included known ones (dextransulfates; refer to Européen Patent Publication ’;h. 0065379) and novel onas. Such novel polysaccharides car. beproduced by rhe same procedure as employed for known ones,witn an example of such production procedures being describedin the following. 10

Chlorosulfonic acid is added êropwise to pyridine involume 8 to 10 times that of chlorosul f onic acid, whilecooling, and a small amount each of formantide and dextran (inamounts about, one-fourth of that of thlorosu.lfonic acid) areadded to the mixture, followed by heating at 55 to 65°C under

S stirring). After stirring is cortinued for several hours,the solvent is distilled off, ard the residue is purified forexanple by reprecipitation, dialysis, etc. The- term"polysulfate" is intended to refer to compounds obtainedthrough additional sul.furic-acid estérification of the 20 polysaccharides having sulfate groups.

The oligosacharides or polysaccharides, which are usefulin this invention, include natural polysaccharides having atleast sulfo group (-SO3H) as may be obtained from animais orsyntheoic polysaccharides havinc' at least one sulfo group 25 (-SO3H) as may be produced by sulfuric acid estérification ofpolysaccharides obtained from animais. 17- 01020/

Preferred among them are: mucopolysacchar i des havingaminomonosaccharides (inclusive of N-acylated and snlfated (-O-SO3H or -NHSO3H) as a repeating unit which may tae furthermore able to contain monosaccharides or acids derived 5 therefrom as another rapeating unit. The arnino acids or t.heir N-ac:ylated dérivatives (preferably N-acetylated ones) seivingas a repeating unit include glucosamine, galactosamine andtheir N-acetylated dérivatives and sulfates or partialhydrolysates of the said compounds. is the mono·· 10 saccharides or acids (preferably hexulonic acid/, there may be mentioned, for example, glucose, galactose, gluculonicacid, iduronic acid and the like. The mucopolysaccharidescontaining such repeating units include, fer example,heparin, keratan sulfates, chondroitin-4-sulfate, 15 chcndroitin-6~sulfate, dermatari sulfate, te ichuronic acid,. hyaluronic acid, heparitin sulfate, c'hitiri, chitosan ;r<their partial hydrolysates and modified dérivatives (e.g.,partially acyLated dérivatives) and synthetic polysaccharideshaving such repeating units. 20 The mucopolysaccharide polysulfates are defined as the compounds which are obtained by additional sulfation of theabove mucopolysaccharides. The sulfation can be carrieci outfor example by the procedure as described in Jape.nese PatentNo. 9570/1971, and is generally conducted by reacting 25 polysaccharides with conc. sulfuric acid or iis reactive dérivatives such as chlorosulfonic acid. 10 15 20 25 -ιέ- 01020?

This reaction is carried ont normal ly in the absence orpresenci' of solvent at Iowered températures, The réactionproduct is csolated by the conventional procedures, such asneutralization, concentration, précipitation, reprecipitationand chromatography.

The mue ο1ip ids a n d mu c oprote i n s a repolysaccharides consisting of mucopolysaccharide a-.d 1 ipid orprobein, and their sulfates can be also synthesized ly aprocedure similar to the above-described one.

The terni "pharmacsutically acceptable sait" Ls intendedto designate salts which retain biologi.cal activ.ity of theparent, compcunds but do not exhibit aiy adverse toxicity atnormal dose levels. Such salts include salts with inorganicbases, such as sodium, potassium and aluminum salts, saltswith organic bases, such as diethanolamine and amino acidsalts. These. salts are produced from their corresnonding freeacids. Such mucopolysaccharides or their sulfater; and saltsthereof are usable alone and also eus a mixture with salts with such metals as zinc and aluminum.

The anti-virus antibody is obtained, as such byadministering a viral antigen (e.g., surface antigen) or afraction containing antigenic déterminant to an vertebrate(praferably mammals) to induce an immunoreactîon, followed bycollection from body fluid of the animal, or in the form ofmonoclonal antibody by utidizing animal cells. 19- «10207

The above antiviral agent lackinu ce 11--fui', ion and/or virus-adsorption inhibitory activity and the cell-f i. s ionand/or virus-adsorption inhibitor ma-j be administeredconcurrently or sequentially. However, both of them should in principle be administered on the Samo day,optionally at different points of tire in the sans day but asclose to each other as possible. Naturally, thesa two k indsof active ingrédients may be administered simultaneously inthe form of a combination of their individual pharmaceutical i préparations or they may be contained in the same, singlepharmaceut ica1 préparation.

In the présent invention, combination of the saitantiviral· agent and cell-fusion and/or virus-adsorptioninhibitor is not limited specifically, but in selecting suchcombination, adéquate considération may be given to types ofthe above antiviral agent and cell-fusion and/or virus-adsorption inhibitor depending upon the conditions ofpatients, kind of diseases, etc. For example, sélection ismade of a combination of 3'-azido-3'-deoxythymidine and 20

Acylovir as antiviral agent and sodium dextran sulfate ascell-fusion and/or virus-adsorption inhibitor,

The dosage of the antiviral agent may be such an amountas may be sufficient to realize the concentration permittingdevelopment of antiviral activity in body, even when it isadministered alone. Since the présent invention can producesynergism, nevertheless, the antiviral agent nay in some 01020? instances be. given in doses less than the amount. producingthe above described concentration, fo:~ example, 1/2 to 1/20C,1/10 to 1/100 or 1/20 to 1/50 of che said amount. Thespécifie dosage can vary largely depending upen the type andnature of the individual pharmaceutical préparations, and maybe changed with the conditions of patients, kind of diseases,and type and amount of the cell-fusion and/or vicus-adsorption inhibitor to be combined. For exemple, Araantidinmay be administered at a daily dose of 15 to 150 mg, A’ithMethisazone being given at a daily dose of 20 to 400 mg/kg.

The cell-fusion and/or virus-adsorption inhibitorcomprehends any cell-fusion and/or virus-adsorptioninhibitors, whether or not they hâve antiviraL activity. Thedosage of the cell-fusion and/or virus-adsorption inhibitorhaving antiviral activity (most of the inhibitors exemplifiedabove fall into the same scope) may be such an amount as maybe sufficient to realize the concentration permittingdevelopment of antiviral activity in body, everi when it. isadministered solely. Since the présent invention can produceantiviral synergism, however, the cell-fusion and/or virus-adsorption inhibitor may in some instances be given in dosesless than the amount producing the above-described concentra-tion, for example, 1/2 to 1/500, 1/5 to 1/200 or 1/20 to L/50of the said amount. The spécifie dosage of the cell-fusionand/or virus-adsorption inhibitor may vary depending upon thenature of individual agents, conditions of patients, kind of - 21 01020? diseases and type and amount of the antiviral agent te becombined, and generally ranges from 0.02 to 200 mg/kg,preferably 0.1 to 100 mg/kg, with the agent beingadministered to human being at a daily dose of abcut 1 mg to10 g, preferably about 5 mg to 5 g.

Each of the above pharmaceutical préparations may beadministered once a day or as divided into b.'/o to six t i.mesor as a sustained release dosage form.

The administration can be made through any optionalroutes,, such as by oral and topical application andinjection.

For administration, the active ingrédients may beadmixed with a pharmaceutical carrier, such as crganic andinorganic solid or liquid excipient, being suitable for suchadministration route as oral administration, injection, etc.and processed in the form of the conventional pharmaceuticalpréparations. Such pharmaceutical préparations include solidones, such as tablets, granules, powders and capsules, andliquid ones, such as solutions, émulsions and suspensions.The above carriers include, for example, starch,lactose, glucose, sucrose, dextrins, celluloses, parafions,fatty acid glycerides, water, alcohols and gum arable. Ifnecessary, there may be added adjuvants, stabilizers,emulsifiers, lubricants, binders, pH-regulating agents,isotonie agents and other conventional additives.

Acute toxicities of the antiviral agents to be used in 2 2 010207 this invention are generally known, while those of the ce l-fusion and/or virus adsorption inhibitors are mostly known,and this may be exemplified by t.he facts that sodium dextransulfate (having a molecular weiglit of '7,000 to 8,000 anda sulfur content of 17 to 20 %) exhiba.ts acute toxicities(LDS0) of 21,000 mg/kg and 4,500 mg/kg, when given orally andintravenously to mice, respectively, while sodium chondroitinsulfate shows acute toxicities (LD50) of 7,500 mg/kg and4,000 mg/kg, when given orally and intraperitoneally to m.ice,respect .ively, with no poisoned nor dead case being observad.Keparin sodium and heparin calcium each shows an acutetoxicity (LD50) of 1,500 to 2,000 mg/kg when injectedintravenously to mice.

The following exemples will illustrate the preser-t.invention in further detail.

The exemples are given belcw to i 1 lustrale this in-vention in more detail, with the test examples being alsodescribed to clarify the effects cf this invention, whereinunless ocherwise specified, the fcllcwing cell-culturingconditions were employed in the fcllcwing respective exem-ples ; (a) i’or culture ci RTLV-III/Molt-4 cs..ls (lumenlymphocytes);

Cell culture was ccnducted ii RPMI 1640 medium svpple- inented with lt t oi cale bovine sérum under 5 % CO? atmosphère at 37 °C, as was the case with 1 xamp Les 11 15, 1b, 17, 18, 19, 7.1, 2 2 and 2 3 2 3 010207 (b) For culture of HSV/Vero cells (monkey kidi.cy) ;

Cell culture was carried eut in Eagle's MEM mediumsv.pplemented with 5 % of calf bovine serur. under 5 ?. CCU atmosphère at. 37°C, as was the case v;..uli Exemple 2 0 . 5 (c) For culture of Influenza/MDCK cells (doc kidutv) ; 'S _t ' '

Cell culture was conducted in Eagle’s MEM ïnidum supple-mented with iû % of calf bovine serurr. ur.der i * ccuatmosphère at 37°C, as was the case with Exair.ple ; 13and 14. 10

Préparation 1

Préparation of chondroitin polysulfate frenï chondroitin sulfate 24 010207

Chondroitin sulfate (5g) was auded to 55 % uni furieacid (10 ml) cooled to below -25°C with stirring. Aitiraddition, the reaction mixture wôs stirred at the sainetempérature for 90 minutes. After the end of the period,the réaction solution was gracually poured onto ice (120 g)with stirring. To the resultr, ng sc-Lution was graduallyctdded calcium carbonate with well stirring. The 10 15 20 25 précipitâtes were iiltered off, which, then, wete washedwell with water. To the combinée! filtrâtes (24C ml) wasadded éthanol (60 ml) , and the sol u nion was kept to standovernight at 5 °C te precipitate esLeium sulfate, Theprécipitâtes were? filtered off, and the filtrate wasadjusted to pH 10 with sodium carbonate. After addition ofacetic acid to inake the solution weakly acidic, the solutionwas concentrated to about 20 ml, then d.iluted with ethanoL(100 ml), and kept to stand overnight at 5 "C. Theprécipitâtes in the solution were isolated with centrifugation, washed with éthanol , and with ether, ar.d dried under vaccum to give the whit.e poweers of: the tille compound(S-content : 13%).

Préparation 2

Préparation of keratan polysul. fate from keratan sulfate.

Préparation 1. was repeated except that keratan sulfate(100 mg) is used as a startir.g matcrial and 1 ml in place of10 mi of 95% sulfura c acid, to give the tit.ie compound(S -content : 10%) . 25 010207

Example 1 (Λ) Sodium dextran sulfate (molecular weight: 7,000-8,000, S-cen terri : .12-2()%) 30 0 iig 45 ' 3 00 " 5 " , près.s ed the 2 5 me 120 " 3 0 0 ' 5 " i. n haro procedure. 10 15

Corn starch

Lactose

Magnésium stéarate

The above ingrédients are mixed, granulatedinto tablets and enterically coated according toconventional procedure. (B) 3 ' -Azido-3 ' -deoxythymidine;

Corn starch

Lactose

Magnésium stéarate

The above ingrédients are mixed and filledgélatine capsules according to the conventional(A) and (B) are combined into a dose ferra.

Example 2

Sodium dextran sulfate (molecular weight: 7,000-8,000, S-content : 17-20%) 12 0 mg 3 '-Azido-3 ' -deoxythymidine 100 '' 0.9 % saline q.s. to 10 ml

The above ingrédients are mixed and dissolved according to the conventional procedure to form an injectable solutionSimilarly, an injectable solution can be préparée. using sodium dextran sulfate having molecular weight < f 5,QGL and 20 26 010207 S-content of 13-14% in place of the above sodium dextra; sulrate.

Example 3 20 25

Sodium heparin 2 00 uni ts 3 ' -?izi.do-3 ' -deoxythymidine 5 0 mg 0.9 % Saline g. s .. to 10 ml Example 4 Calcium heparin b 0 0 η n i r s Ara C J. 0 mg Procain hydrochloride 10 mg Water q . s - t o 10 ml Example 5 (A) Sodium keratan polysul raie 100 mg lactose 19 5 mg Magnésium stéarate 5 pac; The above ingrédients are mixed and fi lied in h ai d gélatine capsules according to th<; couventional· proceh iur (B) Amantadine 10 0 mg Corn starch 195 mg Lactose 300 mg The above ingrédients are mixed, granulated, and pressed into tablets according to the conventional procedure. Example 6 3 ' -'Azido-3 ' -deoxythymidine 100 mg Anti-HTLV-1Iï virus antibody (as IgG) LO Î| 0.9 % Saline q.s. to 1 0 ml 27 10 15 010207

The above ingrédients are mixed ami ri.’.!;ro}ved aciordirto the conventional procedure to l.'orm an rnjeci.bl<j solution.

Example 7

Sodium dextran sulfate (molecuiar weight: 7,000--8,000, S-cortent: 1 ’-OOi)3 0 0 i -,g 3 '-Azicîo-3 ’-deoxythymidine 30

Corn starch 4 5 "

Lactose 270 "

Kagnesium stéarate 5 "

The above ingrédients are mixed, granulated, pressea into tablets and enterically coated accordrng :o theconventional procedure.

Example 8

Sodium dextran sulfate (molecuiar weight: 7,000-8,000, S-content: 17-203)2 50 ;r.g 21,3'-Dideoxycytidine 5 "

Corn starch f0 "

Lactose 100 "

Magnésium stéarate 5 "

The above ingrédients are mixed and Il Lieu in hard gélatine capsules according to the convent i.ona.1. procedure.Example 9

Sodium dextran sulfate (molecuiar weight: 7,000-8,000, S-content: 17-2C1) 30 0 r-o 20 010207 2 1 , 3 1 -Dideoxyadenir.e 5 0 < i Corn starch 50 >1 Lactose 1 0 0 ït The above ingrédients are mixed, granulated, pre s s e d into tablets and enterically coated ac :cording to the conventional procedure. Example 10 Sodium dextran sulfate (molecular weight: 7 , 000 -8,OC )0 , S-content: I 7 - 2 0 i ) 2 50 ,rg 3'-Azido-31-deoxythymidine 2 0 II Acyclovir 3 0 II Lactose 5 0 11 Magnésium stéarate 10 • 1 The: above ingrédients are mixed, granulated and f i 1 led in hard gélatine capsules accoràing to the eonventiona.1.procedure.

Example 11 (Inhibition of viral infection, HTLV-III) AIDS virus HTLV-III was challenged Lo established hunanT-lymphocyte culture eells MT-4 and the ce 11 suspension andvirus particles were incubât. e<î at 37 °C fer 1 hour. Thece] ls vere then washed with phosphate buff'ered salineonce and cultured with or without various doses of thetest substances in RPMI-164C· medium ar 3 7°C under 5 h CO..,in an incubator. MT-4 cells infected with HTLV-ΙΓΙ were destructed asthe virus proliferated, resulting in decroase of viablecells. ?rior to the cytolysis by viral cytopathio etfcct,the cells infected with HTLV-III which were détectable by - 29 010207 indiret immuno fluorescence method appeared. On day ?,nutnber of the living cells was counted to knew rhe entent ; finfection, and rata of the infect ed cells was detemnned by indirect immuno-fluorescence method using an antibody having 5 specificity to the virus spécifie: antigen. As the testsubstances, 3 ' -azido-3 ' -deoxythynidine !AhT) and dexrrar:sulfate(M.W. 7000 - 8000, S-content 17 - 20%) ÎDS) we.e usée.The résulte are shown in Figs. 1 to 4.

It. is évident from these data that, by fine combined e:s- 10 of the above two substances, increase in the number ofviable cells or decrease in the number of infected cells,i.e., inhibition of infection with and réplication of AIDSvirus, could be realized even at the concentrations it whichno effect could be obtained by the sole use o i; any of these substances. 15 - 30 - o i ü;;07 Example 12 (Inhibition of viral infection eu th Herpes Simplex 'xrr.p) Λ suspension of Vero ce Ils was distribvted in 1 m iportions i.nto culture test tubes, and t.he tubes were piucedon a slant test tube stand, follov;ed by incubation overniqht.at 3 7 °C. One arbitrary test tube .vas taken eut to court numbers of cells contained thereiu. Front such number of cells, the concentration (PFU/mï) of a virus s; tsper. si’>n to be inooulated was calculated by the followi.ng i ?qua t ion : Multiplication of infection (moi) -- V/C ( 0.2) where; V is a number of viruses C is a number of cells A stock solution of HSV Type II (SAV) viruses vas diluted tea specifically cetermined virus concentrât ion using theculture solution containing 450 ml of MEM (MEM préparée! bydissolving 9.4 g of Eagle MEM Medium Ko. 1 in ICO ml ofreclistilled water) , 5 ml of a 2.6G % glutamine solution, 15ml of calf bovine sérum and 7.5 mi of a 7.0 % KaHCO^solution, and the thus diluted stock solution was inooulatedin 0.1 ml portions onto the culture test tubes. Afterinfection was allowed at 37°C for 1 hour, 0.9 ml of tue culture solution was added to the test, tubes, and .incubation was carried out at 37°C for 20 honrs, foflowed by évaluation of cytopathic effect (CPE) caused. With varions antiviral agents and drugs ( ξ u 1 fa tel polysaccharides) being picked up is test, subst. onces, nhe abeve test procedures were conducted xn the fo llowing t.hrce modifiée, manners: namely, (i) the fitsf ou itu.ro. solution fo 3.1 010207 dilution of the SAV stock solution and the second culture solution for incubation both were exployed without additionof any test substances to run contre! tests, (2) both the first and second culture solutions were incorporated with 5 test substance(s) to carry out tests on virus-adsorption inhibitory activity (Method 1), and (3) the first. culture solution vas used without addition of test substances(s I , with the second one being admixed with test substance (si , te conduct tests on cell-fusion inhibitory activity (Method 1).

W

The cytopathic effect (CPE) as evaluated vas expressedby the following rating symbols: ++ : Nearly the same as in the case of co.ut.rol (100 %infection) t : 10 to 40 % of cytopathic effect observed. 15 : Above 40 % of cytopathic effect observed.

Plague assay

After évaluation of cytopathic effect, the test tubes,vure subjected to three réfrigération (dry ice/acetone)~thawing cycles, and respective test specimens were diluted 20 with Hank's solution in step-wise te· 10 to 100,000 times andeach distributed in equal portions into two Pétri, dishes (35mu diameter) containing 2.5 ml of Vero cell suspension,followed by incubation. Upon unilayer formation ci cellgrowth, the culture broths were almost removed by suction,and the diluted SAV stock solution vas inoculated in 0.1ml/dish to the Pétri dishes. After infection threughadsorption at 37°C for 1 hours, the dishes were overlaidwith about. 2.5 ml of the above culture solution, admixed with 32 010207 2 % inethylcel lulose, subjected to ’.ncubation at 37°c ior 3days and incorporated with 1,5 ml of 0.015 % Neutral Fedsolution, followed by culture overnight at 37°C to covnt thenunbers of plaques formed. Based on the numbers of plaques,the virus yeilds (PFlJ/ml) were determined, with the relativeviral réplication supressinç activities of treated specimensagainst Controls being tabulated in the following tables.

Calculation ; 10 /PFU'\

Inhibition Value' _ '

Yield of virus (Control) 15 • PFU/ Yield oï virus (Treated)

As the test substances, the following compounds v;er used : - alginic acid sulfate chondroitin-4-sulfate polysulfate(A)chondroi tin-4-sulfate polysul fa te (B)chondroitin-6-sulfate polysulfate(A)chondroitin-6-sulfate polysulfate(B) S = 1 41 S = 6 % 3=16 % 3 = 8% S = 15% 33 .. 010207 chitin sulfate chitosan sulfate 9% 13¾

Dextran sulfate, molecular weight. 5,000(DS-S) S ~ 14%Dextran sulfate, molecular weight 500,000(DS-L·) £ - 14%(Note) St S-content (%)

Inhibition values for each combination of test sgerts are shownin the Table below. Méthod 1 * 10 15 ---------------------Antiviral Agent(yg/ml) Ara C i/.l} Compound (y^Tînï) ü 0.5 1 . Û 0 (1) S 6 5 DS-S C.5 1 13 9 4 DS-S l.C 1 3 8 30 3 DS-L 0.5 1 18 8 5 ύ Method 1 ---------._Antiviral Age n t(y g/m3 1 Ama ntadir L Compound" Û^TmH 0 10 5 0 1G 0 (1) 1 6 DS-S 1 2 4 16 ?

Method 1 20

Antiviral Agent(yg/ml) Ara A

Compound (ug/mlV 0

DS-S

Method 1 (1) 1 8 0 450 25 -______Antiviral Agent (yg/ml) ______Cœy»und Ug/jniy-____q___ 0 ( 1 ) DS-S 1 2. A cyc '1 ovi r

L 130 9 0 0 3 0 0 0 34

Methoà 1

Antiviral Agent (iJ g/rl )

Compound______( P g / ;r 1 ) _ 0_ _ 0 ( 1 )

DS-S

DS-L 10 15 20

Method 2 _____Antiviral Agent (Ug/ml )

Compound ( μ g / jj

Dextran sulfate 0

T

Jl 10

Method 2 _____Ajrtiviral Agent(Ug/ni) _______Compound________(Uq7mT) 0 DS-S 10

Method 1 ____Antiviral Agent(Ug/ml)

Compound ( μ <£/ m ï~) 0 (1) 0 ( 1 i ·? 011) 2 0 7

ihU _1_....... 1 0.1

Ara T 110 2 75 440

Ü A 4 17 17 j.jj l'J 0 4 40 1 10 0 ! . 9 14 5 5 7 9 ij ; 3 5' ô chondroitin-4-su.l fate polysulfate (A)chondroitin-4- sulfate polysulfate (B)

Acyc l.o vi r 0 ( i :· 8 5' 35 010 2 0 10

Melrhod J. _________Antiviral Agent (P g/ml)

Cornpound {μ g/ml ) 0 ehondroitm 6-sulfate 1 pol.ysulfate (A) chondroitin-6-sulfate 1 polysulfate (B)

Meldiod 1 ___________Antiviral Agent (Pg/ml)

Compound(Pg/ml)

Acyclovir _Jj _ ___' ( 1 ) 5 -1

1 1.6 G 1 mi chitosan sulfate

Acyclovir _C______________________i ( 1 ) 5 1 1 18 1

Method 1

Virus yield (untreated/treated) ——--------Antiviral Agent (pg/ml) Ata ( ^5 Compound (Pg/ml) 0 0, 0 1 li 50 0 alginic acid sulfate 1 1 2 6 6 7 chondroitin-4-sulfate 1 7> 20000 polysulfate (A) chondroitin-4-suifate 1 X 3 εοοο polysulfate (B) cbondi oitin-6-sulfate Ί 5 250 0 polysulfate (A) chondroitin-6-sulfate 1 ί 0 000 1.0 13 3 3 4 4 44

2 000 (J 1 3 3 3 3 5 ο o i, ο ο ο Ί polysulfate (B) 36 OIO2O7 cnit in sulfate 100 .3 5(OjO 4000 chitosan sulfate 1 1 1150 4 0 0 0

It ;.s évident from the above resu. 1 ts :..>t the effect cou.Ld be significantly enhanced by combining amantadine, acyclovir, ara A, ara C, ara T and 1DU, whichare anti-virus agents having r.o ce.Ll fusion inhibitoryactivity, with alginic acid sulfate, ebond roi t.in sulfate 1ü 15 20 25 polysulf ates, chitin sulfate, chitosan sulfate, or âe>.tran sulfate.

Example 13 (Inhibition of viral infection, influença virus) MDCK cells derived from dog kidney were allowed t.oorcliferate sufficiently in a culture test tube ( to fora amono layer), and washed once with phosphate bufferedsaline(PBS(-)) . Type-A influença virus vas diluted with aculture medium to 1.0 X 10 PFU/ml. and 0.2 ml of theobtained suspension was inoculated onto tbe cells to allovinfection. In an experiment. us.ing dextran sulfate with amolecular weight of 500C (S-content 14%) (DS--S) or 500000 (S-content 14%) (DS-L) togetiier with s-ruantadiré , these substances at various concentrations shown in uhe table below were added to the virus suspension immmediately before the use for infection. The mixture was allowed te star.d atroora teirperatrure for 30 minutes to aiiew infectiort ofvirus, followed by addition of a culture rcediuiri (1 ad)containing the said substances at the concentra fions she-wnbelow. After incubation at 37 °C for 2 4 hours, judgements were made in such ntanner that when the rate or the forinedCPE (Cytopathic effect) was 0 - 11 %, the resint was h·, 37 010207 when it was 10 - 40 %, the resuit was + , and vdu n it was greater than 40 %, the resuit was (There was found more than 80 % CPE formed in the culture (est tube without the test substances.) The results are shown in thefollowinç table. ——— Antiviral Agent(pg/ml) Amantadine

Compound(ug/ml) 0 0.1 1 10 100 0 - — -P -n- ψ DS-S 1 - -i' v II 10 - + +4- 1« 100 - H* 4--r ++ + 4- 11 1000 - + 1- + ++ + 4- —--------Antiviral Agent(pg/ml) Ama n t a gi ι Compound(pg/ml) 0 0.01 1 10 100 û -, + DS-L 0.1 - t - II 1 — 0 + + 4

It is évident from the above results that the antiviraleffect was synergistically improved by the comtined use of amantadine and dextran sulfate.

Example 14 (Inhibition of viral infection) MDCX cells derived from dog kidney were sufficiently proliferated (to form a mono layer) in a 96-well micrcplate,and washed once with phosphate buffered saline (PBS(-)).Type-A influenza virus was dilutec with a culture mediura to1.0 X 10J PFU/ml, and 0.025 ml of the obtained suspens ionwas inoculated onto the cells to allow infection. Thesubstances were added in the saine amount as shown in thetable below to the virus suspension at the speoified 38 10 0 I (J 2 0 7 concentrations immédiate!y before ihe use for .infection.

Ihe mixture was allowed to stand al toom ter.-.pei.aturt 3 minutes to allow infection of virus, fo'ilowed by addition ;3 culture medium (100 ul) containing the test substances a!

the concent.rations shown belcw. After incubation st 37’C under 5 3. C0o for 44 hours, judgemonts were mafi e in the sainianner as Kxample 9, The results are sbown in the xlbwin· tab]e,

Antiviral Agent (ug/ml) Aria etnaine

Compound (ug/ml) ___________ 0 _ C . 1 t __ i0__ _-0_2 û - - t t alginic acid sulfate 1 - - - + î (Sulfur content= 14%) " K) - 7- t + 7- + 100 ++ +4 + r +·(· l··-

Antiviral Agent(ug/ml) Arnantadin

Compound ( ug/ml ) ____' __0 _0_. 1 I_ 10 100 0 - - .......t ehondroitin-4-sulfate 1 - - - - + (Sulfur content=16%) " 10 - - · ~ t " 100 --- + (- " 1000 - 7 3 25

It is évident from uhe above results that -c.be untivir effect was synergistica11y improved by the combinée! use ofamantadiee and alginic acid sulfate or ehoncroitin su’fab? polysulf.ites -

Example 15 (Inhibition ol: ce) ! fusion) 39' 01020/

By means of a cell to cell infection System usineestablished huraan T-cell Molt-4 (OKT 4+) and its AIDS virusproducing cell line Molt-4/HTLV-III, activity cl dextransulfate having a molecular weight of 7,000 - <3,000 and asulfur content of 17 -20 % (DS) açjainst the cell to cell infection was evaluated. The cultivation v;as eonducted as follows: -

Molt-4 cells (uninfected) and Molt-4/liTLV- I ! i ccj ls(virus productive) were mixed in a cell ratio of 9 : ; air; incubated in an RPMI medium with or without dextran sulfate.

In this system, the Molt-4/HTLV-III cells successively fusewith Molt-4 cells to form giant cells and theri perish. h'I.enthe Molt-4 cells and the Molt-4/HTLV-III cells were ccncurrently cultured without dextran sulfate, giant cellsinfected through cell fusion appeared (abeut cells in ’h·.visual field) as shown in Fig.5. When the concurrentculture was carried out by adding 10 yg/ml of DS,, no giantcell was observed as shown in Fig. 6. This was indicative of the fact that the dextran sulfate inhibited the cell fusion between the cells infected with virus and the cells not infected with virus. In other words, the dextran

sulfate inhibited the direct cell-to-cell infection of AIDS virus.

Example 16 (Suppression of Virus release ammount)HTLV-III producing Molt-4/HTLV-III cells (ali beug positive to antigen having specificity to virus) and theuninfected Molt-4 cells were mixed at a cell ratio of 1:9,and the mixture was incubated in a culture medium containir/; - 40 - 9 I 9 2 0 10, 50, and .100 P g/ml of dextrap. sulfate (no 1 x-cv 1 a r weieht.7,000 - 8,000 , sulfur content. 17 - 13 %) (DS! . The culluremedium were renewed every three days. On day 9, nurnber ofthe livir.g cells vas counted, and tbe virus release amovntvas determined by reverse transcriptase activity (theviruses re.Leased intc, the medium were separated by centrifugation and dilut.ed t.o rerr.ove possible 5 nbibitor··effect. of DS which nay be présent in the rem a in in y medium) .Also, the rates of cells positive te virus aiTiger: (infectred cells) were determined by an indirect immunof luo rorescence method . The results are showr. in -,he following table. ____DS (Pg/ml)_______

Virus release amount (RT activity(xlO" cpm/ml)

Rate of infected cells (%) ’rocecure addec to Î'T-4 cells. p ............10 5 0 )J. )0 4 0 5 161 h 5 ; G 100 100 1 1 esuit.s th.: ·, x. 4·· ; t- t .be vu'.'. 4 sed and the r i '.· n - i il· f e t DS conta : i n .1 : io mec in n' f 1 fa t e i ii h abat :.od t bi o : e .1 i 1 a 1 s . ; d s o r p l i on ! ed to a : :u lt n re me du Όί 1 s, ch ich w a s i. mdi' e <11 . i- t im 4 a i h Z c loi' h J a nd the S 11 ’ Ί .. r- rj ;; t r. ' removed. Viruses which were r.ot adsorbeû to tlu? cells werf 41 010207 removed by washing twice with physiological phosphatebuffer. Fresh culture medium vas added to the washericells and the culture was incubated at 37°C under 5% (Ό., fo3 days. On day 3, the effects were compared by deternining 5 the rate of infected cells (%) by indirect fluorescent antibody technique.

Results

Compound M.W. S-content’’· Rate of infected cells* (%) 10 Non (un infectedcontrol) - - 0 Non (infectedcontrol) 100 15 λ-carrageenan - sl6 <5 alginic acidsulfate 50,000 ^300,000 sl4 < 5 chitosan sulfate 18 <5 dextran sulfate 500,000 16 <: 5 20 dextran sulfate 7,000 οδ,οοο 17^20 <5 chcncroitinpolysulfate 5,000 o8,0C0 13 <5 * S-content is originated from sulfate and suifonate groupattached to the saccharic chain. ** Rate of infected cells=fluorescent positivecells*10Q/total cells

From the above results, it can be clearly seen t.bat fhtest coinpounds, dextran sulfate and other sir.ii.Lar sulfatedpolysaccharide strongly inhibited the process of acsorption 42 Ο 10 2 0? cf HIV virion to the receptor of tnrgst cell, ? ..c. th<·fcrsi step of the establishment of viral iniect

Conséquently, ït can be inferi'ed th3t ihe col 1 £·;·;ίοηinhibiting activity of dextran sulfate arc the?: i;ii?il.,rsuifated polysaccharide was resulted from tin ïnl·.ibitin<jactivity against the bonding of HIV tuitigen prerumably cienerated on the surface of the .in::ect.ed coll t<» the receptor of the uninfected cell.

Example 18 (Inhibition of vira.'· iniectv r?

Procedure ,'n the same procedure as in Expample Ί, the MT-4 cü!infected with HTLV-J3I were cultured in medium erd thechanges in the number of viable cel.'s and thc: rare of twinfected cells were recorded in tirno course. Four kinds of culture médiums were used, i.e., wiLbout. addition, with 1 >nK.AZT, with 5 ug/ml dextran sulfate, and co-exi-tence h:nM.AZT and 5 ug/ml dextran sul fate (?Ί .W. t000 - 2303,. S-eoncent 17 - 20%). Résulté 20 25

As sbown in Fig. 7 (number of viable cells) and Fig., δ(rate of infected cells) , in the case or the soie use of iünid, AZT or 5 ug/ml dextran sulf ate, complète infectionslightly retarded in comparison with the case of non-addition, but 100 % infection was autauied as .·,η fi cC3se of non-addition on day 9. However, in the case oicombinée use of 10 nM.AZT and 5 ug/ml aextir-n sulfate, nochange was seen in the number of viabl.n- coi '.s compaieowith the case of non-infection up to day 12. uiw rate tinfected ceII was also defenately low. 43 - 01020?

Example 19 (Inhibition of viral infection)

Procedure

In the same procedure as in Example 7, the MT-4 ce il ainfected with HTLV-III were cultured in medium containingdextran sulfate(M.W. 7000 - 8000, S-content 17 -- 20%) (DS) and antiviral agents (21,31-dideoxythymidine and 21,3'-dideoxyadenosine) in various concentration. On day12, rate of infected cells (%) wss determined by an indirec- immunoflucrescence method in a mannar similar to tkat i;i

Example 7. Résulta

In the following Tables, values indicate the rate of infected cells.

1) Combination of 23’-deoxythymidine and DS _Antiviral Agent(nM) 23'-deoxythymidine

Compound ~~~--- q y g c,g DS Opg/ml 100 100 5pg/ml 100 50

2) Combination of 21,31-deoxyadenosine and DS

Antiviral Agent(nM) 21,3'-deoxyadenosine

ComjDound 50 DS Opg/ml 100 100 5pg/ml 100 90

3) Combination of suramin and DS _Antiviral Agent suramin. ~~~~Opg/ml ..ompouna 200 100 4 5 100 0 ] 00

0.1P 2/ m 1 /_nnL

CS Opg/mL5pg/mL 100 100 100 100 10 il 44 010207

4) Combination oi HPA-23 and DS

Antiviral Agent Ε1ΡΛ ·· 2 3 c"· or.nd ~ - · Opg/rr.i ... 1θϋ3_/ηιΙ. 4 U y g / m 1 y DS 0μg/ml 1 00 100 1 G 0 5pg/nl 100 8 0 2 0

Fron the above results, it can )ze clear/Ly i.een tint i.beffect of DS as a synergist is exerted not on l.y in -il·.combination with 3 ' -azidc-3 ' -deoxy thym i,<ii ae but aise wi-.J· other anti-Z^IDS antiviral agents.

Examp le 2 0 (Inhibition of cel. fusion)

Fusion of Vero cells derived frora African qreen uonkeykidney cari be induced when monolayer of said cells are.infected with îierpes virus type 11 (Fig. 9, 10) . Activitéof dextran sulfate (M.W. 5,000) (D£-S) against. ce I L fueiér· 15 45 (11 0207 ίο 20 25 inducible. by viral infection with Herpes virus was determined using the above infection system.

In a culture system in which DS-S vas added.simultanecusly with viral adsorption on Vero ceils,cytopathic effect due to viral infection as weil asincidence of cell fusion were observée, as sho«:i in Fig. lï,in the presence cf 1 yg/ml DS-S.

With 10 yg/ml dextran sulfate, however, no oo’iinfection was observed as shown ir Fig. 12.

In another System in which dextran si'fcr· was aedeiafter one hour from the viral adsorption on Vero cell:., aisecytopathic effect due to viral injection as weli as incidence of cell fusion were observed, as shown in Fig. 13,in the presence of 1 yg/ml dextran sulfate,. W’itn 10 ng/mldextran sulfate, however, incidence of cell fusion was ne',observed while cytopathic effect due to viral rnfectionappeared (Fig. 14).

These results indicate that the activity of cext.mnsulfate inhibiting viral infection of Herpes virus includesa step inhibiting cell fusion alone between stops of noinhibition and of complété inhibition depending on conditioiof application and/or concentration of dextran sulfate.

Thus, dextran sulfate has an activity inhibiting cell fusicrin the course of infection of celLs with Herpes virus.

Example 21 (Inhibition of viral infection)

Procedure MT-4 cells, infected with HTLV-III accordinç t o Examp 1<· 7, were cultured in the medium with or without varicus . 010 2 0 of cn.ll fusion inhibitors (cûridroii·.in-4-su t : ate polycn fa (S-ccntent 16%) and chitosan sul f a e ( t-cori l.< i ,0V.) -unidr.tiviral agents ( 3 ’-a ido-3 1 -deox>thymidine ai b2',3'-didecxycytidine). On day 12, rate o? inb-eted cnil(¾) was determined by the fluorescent mitibnay metbod asbxarrip le 7. be su 11 s 1) Use of 1'-azido-3'-deoxythymidiie aïid coud roitin-4-suifate polysulfate

1C '-· 3'-azido-3'- ·'. .. deoxy thymidine· condroitl n- 4- sulfate polysulfate

Ont·'. J lit

1 0 nM 0 μο/ιηΐ 5 μçî/ml 2) Use of 2. ‘,3' i;clysul fate 10 0 100 10 (i e and cordroitin-l-sulfati 2. ' , 3 ' - d i d e o x y cy t i d i n e

On K 10 ni·’.

d;r.E condroitin-4- ' .sulfate polysulfate 0 μα/ι.'ιΐ 5 pçf/ml· 100 100

. 0 G fi !>

Ose of 3 '-azido-3 '-deoxytbyir.idinechitosan sulfate arij 20 01020/ 10 20 31-azido-3'-deoxythymidine chitosan sulfate 0 μg/ml5 pg/ml

OnM 100 100

10 r.M 100 5 0 50 ni· 4) Use of 23'-dideoxycytidine and chitosan sulfate ,3'- dideoxycytidine chiuosan sulfate

ÛnM

1 nM

1.0 r.M 100 10 0 ycj/ml 100 100 5 pg/ml 100 55

Example 22 (Inhibition of viral infection)

Procedure MT-4 cells, infected with ETLV-IIÏ according to Example7, were cultured in the medium with or without varions dosesof dextran sulfate (MW: 7000-8000, £.==17-20%) and entiviralagents (acyclovir, glycyrrhizin and neurotropin!. On day12, rate of infected cells (%) wss determinea by thefluorescent antibody method as in Sxample 7.

Results ——__acyclovir dextran sodium sulfate ΟμΜ 8 ΟμΜ 0 μ g/ml5 yg/ml 100 100 100 10 —lycyrrhizin dextcan sodium sulfate Ûyg/aïl ΙΟΟμο/ηΡ G 0 0: 1 ; i i- 0 uc/ml 5 pc/nil 100 100 ' - . peurctrop ir iextran sodium sulfate 0 μ g/ml5 μ g/ml

Ou g/' ml

1001 OG i 0 0 μ g/n 1 G Op g/ml 1G0

Example 2 3 (Inhibition of viral Lnfectvionj

Procedure MT-4 cells, infected with HTLV-ΙΠ accordirg co Exemple7, were cultured in the medium with or without varions dosesof oeil fusion inhibitors and antiviral agents as skown inthe following Tables. On day 12, rate of infected cells 'i’was determined by the fluorescent antibody roethod as inEx amp le '' . 15 49 010207 s a o oo

O O O en m -t o o oe t" ·τ o o o © co o

Antiviral Agent 3'—azido—3'— 2',3'- acyclovir a o Ό· ΰ

•H Ό

-H μ

O >1

X

O

C '0

•H g;

G C 2G G•H OΌ in

S

-H

.G μ >1

X

O 0) Ό a

G o

rH O O o cri r- r— © o o oo r-· © ο ο o © JO oo o © © o © © rH r—(

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Claims (58)

1. 50 01020? ν,ΉΛΤ IS CLAIMED 18:

   1. A pharinaceutical corcpcsition fo;. treatiinj a oiseusecaused by virus comprising (A) ar. antiviral agent lacking inhibitory activât’/ (1)against ce 11-t.o-cell infection tbrough cell fusion from a virus infected cell to an ur.infected ceii and/or (2) against adsorption of a viras to atarget cell, and (B) a compound possessing cell fusion iniiiuitoryactivity and/or virus-adsorption inhibitoryactivity, said antiviral agent and compound being acininistered inrespective amounts so that the combined dosage thereof is effective to treat said disease.
2. 2. The composition according to claim 1, wherein the saidantiviral agent is an antiviral agent lacking inhibitoryactivity against. celi-to-cell infection tbrough cell fusion from a virus-infected cell to an rninfected oeil.
3. 3. The composition according to claim 2, wherein the saidanti.viral agent is combined with a cell-fusion inhibitor.
4. 4. The composition according to claim 2, wherein the sai.dantiviral· agent is combined with a compound serving the dualpurposes as a cell-fusion inhibitor and a virus-absorption inhibitor.
5. 5. The composition according to claim 1, wherein the saidantiviral agent rs an antiviral agent lacking inhibitoryactivity against adsorption of a virus to a. target cei.l> 51 010207
6. 6. The composition according to claim 5, whereio Lhe iidantiviral agent is combined with a virus-adsorption inhibitor.
7. 7. The composition according to claim 5, wherein the said 5 antiviral· agent is combined with compound serving the dual· purposes as a cell-fusion inhibitoi’ and a virus-adsorption inhibitor·.
8. 8. Ihe composition according to claim 1, wherein the saidantiviral agent is an antiviral agent lacking inhibitory 10 activities both against cell-to-cell infection through celi fusion froin a virus-infected cell to an uninfected celi and against adsorption of a virus to a target cell.
9. 9. The composition according to claim 8, wherein the saidantiviral agent is combined with a cell-fusion inhibitor.
10. 10. The composition according to claim 8, wherein the saidantiviral agent is combined with compound serving the dualpurposes as a cell-fusion inhibitor and a virus-adsorption inhibitor.
11. 11. The composition according to claim 1, wherein saieantiviral· agent and said compound are présent in respectiveamounts which are individually effective to treat saie disease.
12. 12. The composition according to claim 1, wherein theantiviral agent and said compound are présent in respectiveamounts, one of which is less than the dosage thereof whichwould be effective to treat said disease. 25 52 «1020? 10 15 20
13. 13. The composition according to daim 1, wiierdn sa iciantiviral agent and sait! compound <.·. re présent il ;.espe< oie.a;.iouiits which provide a synergistic eifect j.n tk. t.real menJof s disease caused by a virus.
14. 14. The composition according to c. a ira i, wi.ereui the : a.· ..iantiviral agent is présent in an amount lower than i fs-,effective dose by jtself agaiist virus.
15. 15. The composition according to ci air.· 1, vd.ere:.d the ; aiucell-fusion inhibitor and/or virus-· aosorptit>n irhibitoilack(s) antiviral activity.
16. 16. The composition according to daim 13, whfeiein the sa ifcell-fusion inhibitor and/or a virus-adsorption inhibiiorand are (is) présent: in an amount Lower than their (its)lowest effective dose by itself against virus.
17. 17. The composition according to daim i, wherdn the sa.idantiviral agent is an Amantadin type antivirai agent.
18. 18. The composition according to daim 1, wkerein the :.aidantiviral agent is a nucleic-acid type antivira.l; agent.
19. 19. The composition according to daim 1, where.in the sa idantrviral agent is a peptide type antiviral, agent.
20. 20. The composition according to daim i, wk-rein the ;.iidantiviral agent is « polyanicn type antiv.irai agent . 25
21. 21. The composition according to daim 1, wherdn the .-aid a n t i v i r a 1 agent is > a phosphoric-acid type uid.iv Irai agent. 22. The composition according to daim 1, v/herem iht -aid antiviral agent is . a eridogenous antiviral ngent . t 3 . .L? b g c omposition according to clam wherdn the said antiviral agent is a glycoside type i ra l·. e '.eiil., 53 010207
22. 24. The composition according to claim 1, where?n the saidantiviral agent is a lipid type antiviral agent.
23. 25. The composition according to daim 1, whemn the saie,antiviral agent is a Ansamycin type antiviral agesit.
24. 26. The composition according to claim. 1, wherem the saidantiviral agent is a thiosemicarbazide type antnviral agent.
25. 27. The composition according to claim 18, wherein the saidnucleic-acid type antiviral agent is .3 ' -azido-3 ' -deoxythymidine (AZT), 2 1 , 3 '-dideoxythymidine , 2 ' , 3 1-dideoxyadenosine, 2',3'-dideoxyguanosine, 2 ' , 3 '-dideoxyinosine, 2 ’ , 3 '-dideoxycytidine , Ara A, Ara C,Ara T, icdodeoxyuridine or Acyclovir.
26. 28. The composition according to claim 1, wherein the saidcompound possessing cell-fusion inhibitor activity and/orvirus-adsorption inhibitory activity is a compcund having asaccharic carbon atom, said compound being selected from thegroup consisting of: a) a natural oligosaccharide, b) s. synthetic oligosaccharide, c) a. natural polysaccharide, d) a synthetic polysaccharide, and e) a pharmaceutically acceptable sait of any of theitems, a, b, c or d. a.nd having at least one S-oxoacid group linked to eithei ofits saccharic carbon atoms through a linking group with alow molecular weight.
27. 29. The composition according to claim 28, wherein the s,nid S-oxoacid group is a sulfo group (-SO^H). 54 1)10207 JO 20
28. 30. The composition according to claïm 28, vher-sr. the uai<i .linking group is an oxy (-O-) or iniiio çrr-up (-Nï-i) .
29. 31. The composition according te· claim 28, rflmuhn "ho cale conipcund is selected from the yroup eo;:si.:.,ting of: a) a suifetted natural polysaccharide, b) a sulfated synthetic polysaccharide ano o) a pharmaceutica31y acceptable sait oî :ty of ihoitems, a ar.d b.
30. 32. The composition according to cldim 31,. whcioin the sac1corcpound is selected i'rom the group consistinç ci: a, a naturel polysaccharide having at least cr.e hydrogen sulfate group (-o-SO.^Iî) which rs obtainablefror.i a plant or microorganism, b) a synthetic polysaccharide; having at least onchydrogen sulfate group (-C’-SO^H) produced byesterifying with a sulfafmg agent a polysaccharidewhich is obtainable from a plant or r/c c coorganisa., and c) a pharmaceutically acceptable sait oi anv of theitems, a and b.
31. 33. The composition according to daim 32, vhe rein the saidccicpcur.d is a polysaccharide selected frorc. the groupcor.sisting of: dextran sulfate, alginic acid sulfate,· 25 1er. tir an i sulfate and pullulan su 1Late. 34 . The composition according to :laim 33, whe i rein t h e sai de:-: t r £ r sulfate has n mclecula r w-cight of. ri o o i to 2,003,000 3 5 . The composâti on according to . j ctjri 34. whe ne in tli ;; sa.l dextran sulfate bas a nolecular wc-ight of 19 G to 2 0 3,300. 55 01020? 10 15 20 25
32. 36. The composition according to cl.aim 35, wherein the saiddextran sulfate has a inol.ecu3.ar weight of 2,000 to 10,000.
33. 37. The composition according to claim 36, wherejn the saiddextran sulfate has a molecular weight of 3,000 to 8,000.
34. 38. The composition according to claim 33, wherein the said dextran sulfate has a sulfur content of 5 to 23 %.
35. 39. The composition according to claim 32, wherein the saidnatural polysaccharides include CÆtrrageenan or fucoicin.
36. 40. The composition according to claim 31, wherein the saidcompound is selected from the group consisting of: a) a natural polysaccharide having at least onesulfo group (-SO^H) which are obtainablefrom an animal, b) a synthetic polysaccharide having at least onesulfc group (-SO^H) which is produced byesterifying with a sulfating agent a polysaccharideobtained from an animal, and c) a pharmaceutically acceptable sait of any of theitems, a and b.
37. 41. The composition according to claim 40, wherein the saidcompound is mucopolysaccharides.
38. 42. The composition according to claim 40, wherein the saidcompound is a sulfated synthetic inucopolysaccharide piroducedby esterifying a natural mucopolysaccharide with a sulfatingagent.
39. 43. The composition according to claim 40, wherein the saidcompound is a natural polysaccharide selected from the group 56 01020? consisting of heparin, chondroitin sulfate, dermatansulfate, heparitin sulfate, keratan sulfate, hyii lurcn i racid, teichuronic acid, chitin, ch:.tosan, mucolipid andmucoprotein or a pharmaceutically acceptable sait theieoi.
40. 44. The composition according to claim 40, wherein the saidcompound is a synthetic polysaccharide selected from thegroup consisting of chondroitin pofysulfate, cermatanpolysulfate, heparitin polysulfate„ keratan pclysai fate,hyaluronic acid sulfate, teichuronic acid sulfate, chitinsulfate and chitosan sulfate or a pharmaceutica.i lyacceptable sait thereof.
41. 45. The composition according to claim 1, wherein the saidcompound possessing cell-fusion inhibitor activity and/cr avirus-adsorption inhibitor activity is an cntiviralant.ibody.
42. 46. The composition according to claim 1, wherein the sainantiviral agent is 31-azido-31-deozythymidine, and the saidcompound possessing cell-fusion inhibitory -activity and/orvirus-adsorption inhibitory activity is dextrar:· sulfate.
43. 47. The composition according to claim 1, wherein the ;aidantiviral agent is 2 ’ , 3 1 -dideoxycyfcidine , and the saidcompound possessing cell-fusion innibitory activity and/crvirus-adsorption inhibitory activity lis dextran sulfate.
44. 48. The composition according to claim 1, wherein the saidantiviral agent is 2·,3’-dideoxyadenosine, and the saidcomoound possessing cell-fusion inhibitory activity and/orvirus-adsorption inhibitory activiry is dextran sulfate. Î7 010207
45. 49. The composition according to claim 1, wherein the saiddisease caused by virus is brought about by retrovirus,herpesvirus or influenza virus.
46. 50. The composition according to claim 49, wherein the saidretrovirus is a human or animal retrovirus.
47. 51. The composition according to claim 50, wherein the saidhuman retrovirus is selected from the group consisting ofETLV-I, HTLV-II, HTLV-III, LAV, ARV and the Kawasaki-Disease '0 ·?*» causative virus.
48. 52. The composition according to claim 50, wherein the saiddisease is caused by HIV.
49. 53. The composition according to claim 50, wherein the saiddisease is caused by herpesvirus.
50. 54. The composition according to claim 50, wherein the saiddisease is caused by influenza virus.
51. 55. The composition according to claim 50, wherein the saidanimal retrovirus is avian myelobiêistosis virus or Friendmurine leukemia virus.

    55. The composition according to claim 1, wherein the saiddisease caused by viruses is lymphadenopathy (LAS),persistent generalized lymphadenopathy (PGL), AIDS, AIDSrelated complex (ARC), adult T-cell leukemia (ATL), KawasakiDisease, herpes or influenza.
52. 57. A pharmaceutical composition for enhancing the effect ofan antiviral agent lacking inhibitory activity againstcell-to-cell infection through cell fusion from a 5*? 010207 10 15 20 25 virus-infected cell to an uninfected ceil and/or inhibi tcryactivity against adsorption of a virus to a target oeilccmprising a compound possessing cell fusion inhibitoryactivity and/or virus-adsorption inhibitory activity.
53. 58. The composition according to claim 57, wherein the saidantiviral agent is 3 1 -az.ido-3 ' -deexythymidïne , and the saidcompound possessing cell-fusion inhibitory activity and/orvirus-adsorption inhibitory activity is dextran sulfate.
54. 59. The composition according to claim 57, wherein the saieantiviral agent is 2’,3’-dideoxycytidine, and the saidcompound possessing cell-fusion inhibitory activity arm/orvirus-adsorption inhibitory activity is; dextran sulfate.
55. 60. The composition according to claim 57, wherein the saidantiviral agent is 2’,31-dideoxyadenosine, and the saidcompound possessing cell-fusion inhibitory activity and/orvirus-adsorption inhibitory activity is dextran sulfate.
56. 61. The composition according to claim 57, wherein the saidantiviral agent is Ara A, Ara C or Ara T, and the saidcompound possessing cell-fusion inhibitory activity and/orvirus-adsorption inhibitory activity is dextran sulfate.
57. 62. The composition according to claim 57, wherein the saidantiviral agent is iododeoxyuridine, and the said compoundpossessing cell-fusion inhibitory activity and/or virus-adsorption inhibitory activity is dextran sulfate.
58. 63. The composition according to c„aim 57, wherein the saidantiviral agent is Acyclovir, and the said compoundpossessing cell-fusion inhibitory activity and/orvirus-adsorption inhibitory activity is dextran sulfate.