CA1277239C - Treatment of diseases caused by retroviruses - Google Patents
Treatment of diseases caused by retrovirusesInfo
- Publication number
- CA1277239C CA1277239C CA000532772A CA532772A CA1277239C CA 1277239 C CA1277239 C CA 1277239C CA 000532772 A CA000532772 A CA 000532772A CA 532772 A CA532772 A CA 532772A CA 1277239 C CA1277239 C CA 1277239C
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- Prior art keywords
- polysaccharide
- sulfate
- composition according
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- group
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H11/00—Compounds containing saccharide radicals esterified by inorganic acids; Metal salts thereof
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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
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
Abstract
Abstract The present invention is directed to a pharmaceutical composition for the treatment of diseases caused by retro-viruses comprising an antivirally effective amount of a natural or synthetic oligo- or polysaccharide having at least one S-oxoacid group attached to the saccharic carbon atom through a linking group of lower molecular weight or a pharmaceutically acceptable salt thereof as an active ingredient in association with a pharmaceutically acceptable carrier, diluent or excipient.
Description
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Treatment of diseases caused by retroviruses The present invention relates to the prevention, therapy and treatment of diseases caused by retroviruses.
~ore particularly, the present invention provides a medi-cament (including a veterinary medicament) containing as-active ingredient a natural or synthetic oligo- or polysaccharide having at least one S-oxoacid group attached to the saccharic carbon atom through a linking group of lower molecular weight or a pharmaceutically acceptable salt thereof and a method of prevention and therapy, etc. of the diseases caused by retroviruses, especially AIDS (acquired immune deficiency syndrome), ARC (AIDS-related complex), PGL (persistant generalized lymphadenopathy) and AIDS-virus carrier using such medicament.
Retroviruses refer to a family of virus which has RNA and reverse transcriptase (RNA-dependent DNA poly-merase), which is essential to the first stage of its self-replication for synthesizing complementary DNA on the base of template RNA of the virus.
Retroviruses include various oncoviruses, for exam-ple, avian leukemia virus, avian sarcoma virus, avian ~ ~ ' ' . ' ' .
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reticuloendotheliosis virus, murine mammary cancer virus, murine leukemia virus, murine sarcoma virus, guinea pig type C virus, hamster type C virus, rat leukemia virus, feline leukemia virus, ~eline sarcoma virus, feline type C virus, ovine leukemia virus, bovine leukemia virus, swine type C virus, simian leukemia virus, Mason-Pfizer virus, simian sarcoma virus, simian T-lymphotropic virus, baboon type C virus, and the like. Among those infective to humans, important ones include adult T-cell leukemia virus (ATLV), or human T-lymphotropic virus type I
~HTLV-I), and type II (HTLV-II). Adult T-cell leukemia abounds in Japan, especially in the west part, but effective treatment including prevention and therapy of the disease has not been established.
On the other hand, retroviruses also include those having no oncogenecity, for example, visna virus, ovine progre~siv~ pneumonia virus, ovine maedi virus, simian T-lymphotropic virus type III (STLV-III), eguine infec-tious anemia virus, and the like~ The viruses isolated from humans as being the causative agents for AIDS or ARC etc. (HTLV-III, LAVl, LA~2, ARV, and other so-called AIDS-viruses) belong to this subfamily. Recently, the AIDS-causative viruses have been called HIVs ~human immune deficiency viruses). Further, as the third subfamily, there is known a spumavirinae to which simian foaming virus belongs. Also, a retrovirus has been isolated recently as a causative virus for Kawasaki disease (mucocutaneous lymphonode syndrome).
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World-wide interests have been focused on AIDS due to its unfavorable prognosis. It is a clinical syndrome characterized by recurrent opportunistic infections, (e.g.
preumocysti.s carinii preunonia, cryptococcal meningitis, disseminated toxoplasmosis), lymphadenopathy, and an aggressive Kaposi's sarcoma, and induces a mortali~y rate of more than 90% by the disregulation of immune system.
It is also known that the helper-T cells are specifically destroyed by the infection of the virus.
In order to find pharmaceutical agents effective for the treatment of AIDS, ARC, PGL, and AIDS-virus carrier, the present inventorst using a cell line of MT-4 estab-lished from T-cells o~ adult T-cell leukemia patients and HTLV-III which is a causative virus for AIDS, examined the effects of various substances on the infection and replication of HTLV-III.
The above ~T-4 cell line is absolutely susceptible to the infection with HTLV-III followed by causing cell lysis (experimental HTLV-III infection). The present inventors ~ound that when certain polysaccharides having a sul~onate group (-SO3) or mucopolysaccharides having a sulonate group or their additionally sulfuric acid esterified sub-stances were added to the experimental HTLV-~II infection system, the infection of HTLV-III on MT-4 cells and viral replication were strongly inhibited without any accompany-ing toxicity to the cells.
Further, th~e present inventors demonstrated that the above polymerized sugar inhibits the reverse transcriptase . . .
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of the retrovirus in_vitro, and thereby suppresses the replication oE the virus.
Among the sul~uric acid esters of polysaccharides, dextran sulfate with a lower molecular weight has long been commercialized as an antilipemic or anti-arteriosclerosis agent. Also, de~tran sulfate with a relatively higher molecular weight is known to have an inhibitory action against the herpes virus. (European Patent Laid-Open Publication No. 0066379.) However, since the herpes virus is a DNA virus, its replication is absolutely different from that of the retrovirus which depends entirely on reverse transcriptase for synthesis of DNA. Accordinglyt effectiveness on herpes virus does not necessarily mean effectiveness on retrovirus alike.
Furthermore, dextran sulfate with a molecular weight less than 10,000 was found to be almost ineffective on herpes viruses.
Among the mucopolysaccharides or their sulfates, chondroitin sulfate is commercialized as a drug for sensorineural hearing impairment, neuralgia, lumbago and chronic nephritis, and also as a cornea-protective ophthalmic solution. Reratan sulfate is obtainable from the cartilage, teichronic acid from the cell walls of Bacillus subtilis, hyaluronic acid from shark skin, whale cartilage, or from human serum, heparan sulfate from bovine liver or lung, and chitin from arthropod or from fungus or yeast, respectively. The preparation process for the further sulfuric acid esterified compound of ~ .- . . . .
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chondroitin sul~ate is described in Japanese Patent Publication (JP, B2) No. 9570/1971.
EIeparin is known to inhibit various en~ymes in vitro, e.g., DNA polymelase of phytohemagglutinin stimulated human lymphocytes and reverse transcriptase of simian sarcoma virus (Cancer Research, 38, 2401 - 2407), but it has not been proven to inhibit the viral infection of cells.
In one aspect, the presen-t invention provides a method for the treatment of diseases caused by human retroviruses which comprises administering an effective amount of a natural or synthetic oligo- or polysaccharide h~ving at least one S-oxoacid group attached to the saccharic carbon atom through a linking group of lower molecular weight or a pharmaceutically acceptable salt thereof to a subject in need of such treatment.
In another aspect, the present invention provides a use of the above oligo- or polysaccharide or a salt thereof for the manufacture of a medicament for the treatment of diseases caused by human retroviruses.
In a further aspect~ the present invention provides a pharmaceutical composition comprising the above oligo- or polysaccharide or a salt thereof as an active ingredient in association with a pharmaceutically acceptable carrier, diluent or excipient.
In the drawings which illustrate embodiments of the invention, Figs. 1-7 show the reverse transcriptase inhibition ~, : ' ' . , :- ' :
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activities of the test substances in Example 1.
Fig. 8 shows the reverse transcriptase inhibition activity of the test substance in Example 2.
Figs. 9-15, 16-22, and 23-29 show the effects of the test substances on cell growth, viability, and infected cell rate (%) of MT-4 cells infected with HT~V-III, respectively, in Example 3.
Figs. 30-33 show the reverse transcriptase inhibition activities of the test substance in Example 5.
Figs. 34-37, 38-~1, 42-45 show the effects of the test substances or cell growth, viability, and infected cell rate (%) of MT-4 cells infected with HTLV-III 7 respectively, in Example 6.
Figs. 46-48 show the effects of heparin on cell growth, viability and infected cell rate of MT-4 cells infected with HTLV-III, respectively, in Example 7.
The term 'ttreatment" herein is intended to cover all aspects of disease treatment including prevention, sustention (i.e. prevention of aggravation), reducing (i.e. alleviation of conditions) and therapy.
The retroviruses includes all viruses having RNA and reverse transcriptase as the basic components including those exemplified above.
The diseases referred to herein cover all the diseases caused by retroviruses, including those bearing or not bearing the aforementioned virus name. Particularly im-portant diseases are the diseases caused by AIDS-viruses.
The oligo- or polysaccharide usable in the present - - ~ , .
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invention are those having at least one S-oxoacid group attached to the saccharic carbon atom through a linking group of lower molecular weight. Such an oligo- or polysaccharide may be natural or synthetic. The term "natural" is intended to mean that the oligo- or poly-saccharide is obtainable from a natural source, for example, plant, microorganism or animal by extraction and other means. The term "synth~etic" is intended to mean that the oligo- or polysaceharide is obtainable synthetieally, for example, by introdueing S-oxoaeid group into another oligo- or polysaeeharide whieh has or has not S-oxoaeid group and whieh is natural or unnatural (and synthetie~.
The term "oligosaceharide" refers to a earbohydrate eontaining from two up to about nine monosaceharides linked together. For example, when an oligosaeeharide eontains three monosaeeharides, one, two or three of the monosaeeharides may have at least one S-oxoaeid group.
~he term "polysaeeharide" refers to a earbohydrate eontaining about ten or more monosaeeharides linked together. At least one and a minor or major part or all of the monosaecharides may have at least one and normally up to four S-oxoaeid groups.
The S-oxoaeid group includes a sulfo group (-SO3H) and a hydroxysulfinyl group (-SO.OH). Preferably the S-oxoacid group is a sulfo group.
The term '7saccharic earbon atom" refers to a ear-bon atom which is a member of a tetrahydrofuran or a -. -: . . .
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~ 8 tetrahydropyran ring of a monosaccharide contained in the oligo- or polysaccharide.
The linking group of the lower molecular weight includes oxy (-O-), imino (-NH-), thio (-S-), methylene (-CH2-), ethylidene (-CH(CH3)-) groups and the like. The term "lower molecular weight" is intended to mean that the group has a molecular weight from about 14 up to about 32. Preferably the linking group is an oxy or an imino group.
One class of the oligo- or polysaccharide is a natural polysaccharide having at least one hydrogen sulfate group (-O-SO3H) and is obtained from a plant or a microorganism, or a synthetic polysaccharide having at least one hydrogen sulfate group (-O-SO3H) and is formed by esterifying a polysaccharide obtained from a plant or a microorganism.
Within this class, a preferable subc~ass is a poly-saccharide composed o non-amino monosaccharide (including sugar acid) as the repeating unit. This polysaccharide~
however, may contain a trace amount of nitrogen. Examples of the non-amino sugar repeating units include xylose, arabinose, rhamnose, frucose, glucose, galactose, glu-curonic acid, galacturonic acid, mannuronic acid, etc.
The natural polysaccharide includes carrageenan (galactan sulfate o~tainable from Gigartina tenella, etc.) and -fucoidin (polyfucose sulfate obtainable from Laminaria brown seaweed). Carrageenan includes ~-carragheenin, ~-carrageenan, I-carrageenan, etc. which have a different content of the hydrogen sulfate group. The synthetic . .
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g polysaccharide includes those to be obtained by sul~uric acid esterification of polysaccharides, e.g., starch and partial hydrolyzate thereof, dextran which is produced by ~euc_n_stoc__p. and partial hydrolyzate thereof (usually having the molecular weight oE 500 - 2,000,000, ordinar-ily 2,000 and 300,000, pre~erably 2,000 - 10,000, most suitably 3,000 - 8,000, e.g., 7,000 - 8,000), glycogen, pectin, cellulose, plant viscous liquids (gum arabic, tragacanth gum, etc.), plant mucilage products ~those obtainable from Hibiscus esculentus, Aloe, Brasenia schreberi, xylan, etc.), viscous li~uids of marine and fresh water algae ~alginic acid, laminarin, etc.~ or polysaccharide derived from microorganism (lentinan, pluran, mannan, etc.). They include known ones (dextran sulfate, cf., European Patent Laid-Open Publication No.
0066379) and novel ones. The novel ones may be produced in the same manner as in the known ones. ~n example of the preparation process is shown, as follows:-Chlorosulfonic acid is added dropwise to dry pyridin of 8-10 fold volume while cooling. To the mixture are added small amounts of formamide and dextran (about 1/4 weight of chlorosulfonic acid), and the mixture is heated to 55-65C under stirring. After stirring the mixture for several hours, the solvent is distilled off, and the residue is purified for example by recrystallization, dialysis, etc. Within the synthetic polysaccharide, those obtained by further sulfuric acid esterification are represented by the term "polysulfate".
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Another class of the oligo- or polysaccharide is a natural polysaccharide having at least one hydrogen sulfate group (-O-SO3H) and is obtained from an animal, or a synthetic polysaccharide having at least one hydrogen sulfate group (-O-SO3~1) and is formed by esterifying a polysaccharide obtained from an animal.
Within this class, a prefera~le subclass includes mucopolysaccharides, which are composed of repeating units of amino monosaccharides (including N-acyl or NH-SO3H).
These may further contain as another repeating unit a non-amino sugar or an acid derivative thereof. The repeating amino-sugar unit or its N-acylated (preferably N-acetylated) derivatives include glucosamine, galactos-amine, N-acetylated derivatives of them, and the sulfuric acid ester or partial hydrolyzate of the above compound.
Examples of monosaccharide or acid (preferably, hexu-lonic acid) include glucose, galactose, glucuronic acid, iduronic acid, etc. The mucopolysaccharides containing such a repeating unit include heparin, keratan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, teichuronic acid, hyaluronic acid, heparitin sulfate, chitin, and their partial hydrolyzates, modified derivatives (e.g., partial acylated products), and syn-thetic polysaccharides containing the repeating unit as mentioned above.
The mucopolysaccharide polysulfates are defined as the products which are synthesized by additional sulfuric acid esterification of the above mucopolysaccharides having a ~4 ' : ~ ~,.' ' ', "
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~Z7~39 sulfate group. This esterification may be carried out, for example, according to the procedure described in Japanese Patent Publication No. 9570/1971. In general, the esteri~ication is carried out by the treatment of the mucopolysaccharides with one sulfating reagent, for example, concentrated sulfuric acid or chlorosulfonic acid.
These reactions are usually carried out with or without a solvent at low temperature. The reaction product is separated by any conventional procedure, e.g., neutralization, concentration, precipitation, recrystallization, chromatography, etc.
The term "pharmaceutically acceptable salt" is intended to mean that the salt has the biological acti-vity of the parent compound and is not unusably toxic atthe administration level. Such a salt includes the salt of an inorganic base e.g., sodium salt, potassium salt, ammonium salt, etc., and the salt of an organic base e.g., diethanolamine salt, cyclohexylamine salt, amino acid salt, etc. These salts are produced from the cor-responding acids by conventional procedures. The above oligo- or polysaccharides and their salts may be used alone or as a mixture with metal salts e.g. zinc, aluminum, etc. The oligo- or polysaccharide should be ~5 administered at a dose sufficient to produce the effect for the desired treatment. For example, the dosage of the sulfates of the above polysaccharide or their salts sufficient to produce blood concentration for anti-virus .. .
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activity is generally 0.2-200 mg/kg, preferably 0.5-100 mg/kg. In the case of humans, an amount of about 10 mg - 10 g/day, preferably about 50 mg - 5 g/day, is admini-stered in 1-4 divisions a day, or as a sustained release form. The administration route is optional and can be oral, rectal, nasal, local (including sublingual), injection (including subcutaneous, intracutaneous, intra-muscular and intravenous), inunction etc. The preferred route is selected depending on various factors including the kind of active ingredient, conditions and age of the patient, severity of infection etc.
The dosage of the mucopolysaccharides or their poly-sulfates or the salts thereof sufficient to produce a concentration for anti-virus activity is generally 0.2-20~ mg/k~, preferably 0.5-100 mg/kg. In the case of humans, an amount of about 10 mg - 20 g/day, preferably about 50 mg - 10 g/day is administered in 1-4 divisions a day, or as a sustained releas~ form.
The administration route is optional and can be oral, local, injection, inunction, etc.
For administration, the effective ingredient may be mixed with a pharmaceutical carrier, for examp~e, organic or inorganic solid or liquid excipient e.g. suitable for internal administration or injection, and administered in the form of a conventional pharmaceutical preparation.
Such preparations include solids (e.g., tablet, granule, powder, capsule, etc.), liquids (e.g., liquid, emulsion, suspension, etc.), and ointments. The above carriers ~.
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: . ' ' 7~3g include starch, lactose, glucose, sucrose, dextrin, cellulose, paraffin, fatty acid glycerider water, alcohol, gum arabic, etc. If necessary, ordinary use auxiliaries, stabilizers, emulsiEiers, lubricants, binders, pH regulat-ing agents, isotonicity agents, and other additives may be used.
The toxicity o~ the above oligo- or polysaccharide is extremely low. For example, the acute toxicity (LD50) of sodium dextran sulfate tmolecular weight 7,000-8,000, S-content 17-20%) is 21,000 mg/kg when orally, and 4,500 mg/kg when intravenously administered to miceO
- The acute toxicity (LD50) of sodium chondroltin sulfa~e is 4,000 mg/kg or more when intraperitoneally, and 7,500 mg/kg or more when orally administered to mice.
The acute toxicity tLD50) of sodium heparin is 1,500-2~000 mg/kg when intravenously injected to mice.
The following examples will illustrate the present inventiun in further detail.
Preparation l Preparation of chondroitin polysulfate from chondroitin sulfate Chondroitin sulfate (5 9) was added to 95~ sulfuric acid (lO ml) cooled at below -25~C with stirring. After the addition, the reaction mixture was stirred at the same temperature for 90 minutes. After the 90 minutes had lapsed, the reaction solution was gradually poured onto ice (120 g) with stirring. Calcium carbonate was gradually addecl to the resulting solution with vigorous ; ~
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stirring. The precipitates were filtered o~f and washed well with water. Ethanol (60 ml) was added to the combined filtrates ~240 ml), and the solution was allowed to stand overnight at 5C to precipitate calcium sulfate.
The precipitates were filtered off, and the filtrate was adjusted to pH 10 with sodium carbonate. After the addition of acetic acid to make the solution weakly acidic, the solution was concentrated to about 20 ml, then diluted with ethanol (100 ml), and allowed to stand overnight at 5C~ The precipitates in the solution were isolated with centrifugation, washed with ethanol, and with ether, and dried under vacuum to give the white powder of the title compound.
Preparation 2 Preparation of keratan polysulfate from keratan sulfate.
To give the title compound preparation 1 was re~ea~ed except that keratan sulfate (100 mg) was used as a start-ing material and 1 ml in place of 10 ml of 95~ sulfuric acid was used.
Formulation 1 Sodium dextran sulfate (molecular weight: 7,000 -8,000, S-content: 17 - 20%) 150 my Corn starch 45 mg Lactose 300 mg Magnesium stearate 5 mg The above ingredients were mixed, granulated, and ;
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pressed according to conventional tablet making procedures and then enterically coated.
Formulation 2 _ ______ Sodium dextran sul~ate (molecular weight: 7,000 -8,00(), S-content: 17 - 20&) 600 mg Physiological saline q.s,. to 10 ml.
Formulation 3 Sodium dextran sulfate (molec:ular weight: 5,000, S-content: 13 - 14~) 600 mg Physiological saline q.s. to 10 ml.
Formulation 4 Sodium salt of chondroitin sulfate150 mg Corn starch 45 mg Lactose 300 mg Magnesium stearate 5 mg The above ingredients were mixed, granulated, and pressed according to conventional tablet making procedures and then en~erically coated.
Formulation 5 Sodium salt o keratan polysulfate400 mg Lactose 195 mg Magnesium stearate 5 mg The above ingredients were mixed according to the conventional procedures and filled in hard gelatine capsules.
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~277;~39 Formulation 6 Sodium salt of chondroitin polysulfate 300 mg Physiological saline q.s. to 10 ml.
Formulation 7 __ __ .
Sodium heparin 25,000 units Physiological saline q.s. to 10 ml.
Formulation 8 ____ Calcium heparin 5,000 units Procain hydrochloride 10 mg Water q.s. to 10 ml.
Example 1 (Inhibition of reverse transcriptase activity) Test substances were assayed for inhibition against the enzyme activity of reverse transcriptase (authentic sample) derived from Avian Myeloblastosis Virus (abbrev.
AMV), a kind o retrovirus.
Five microliters of ~YA)n(template RNA), 4 ~1 of (dT)l~ 18(primer DNA), and 1 ~1 of water were mixed with 5 Yl of 0.5M Tris-~Cl (pH 8.4) including 0.1% Triton*
X-100, 5 ~1 of lnM-MgC12, 5 ~1 of 20 mM-DDT, 5 ~1 of water, and [3H]-TTP (tritium labeled thymidine triphos-phate). To this mixture, test substances in solutions (final concentrations: 1, 0.1, and 0.01 ~g/ml, 5 ~1) or buffer solutions (control, 5 ~1) at various doses were added. Then, 5 ~1 (one unit) of the authentic reverse transcriptase clerived from AMV was added and the reac-tion mixture was incubated at 37C for 30 minutes. The * Trade mark :;:
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reaction was stopped by the addition of trichloroacetic acid, and after filtering the reaction mixture, the radioactivity o~ the polymerized (3H-T)n retained on the filter was measured using a liquid scintillation counter.
As the test substances, sodium dextran sulfate (molecular weight: 5,000), same (molecular weight: 8,000), same (molecular weight: 500,000), fucoidin, K-carrageenan, ~-carrageenan, and l-carrageenan were used. The results are shown in Figs. 1-7.
Figs. 1-7 show that the enzyme inhibition increases with the increasing doses of the above test substances.
ExamE~e 2 (Inhibition of reverse transcriptase activity) The assay procedure of Example 1 was repeated using disrupted HTLV-III virions as a crude reverse trans-criptase in order to evaluate the reverse transcriptase inhibitory effect of dextran sulfate (DS, molecu~ar weight 7,000 - 8,000, S-content 17 - ~0%). The result is shown in Fig. 8.
Fig. 3 shows that DS has an inhibitory effect against the reverse transcriptase derived from AIDS-virus, HTLV-III.
Example 3 (Anti-AIDS virus activity~
To MT-4 cells (30 x 10 /ml) cultured in RPMI-1640 medium containing 10% bovine serum, was inoculated HTLV-III, and the suspension was incubated at 37C for 1 hour to cause the adsorption of the virus. The cell:virus ratio was 500:1. The cells were then washed, and cultured .. ~ , .
~2~77~9 with or without various doses of the test substances (same as those of Example 1) at 37C under 5~ C02 for 3 days, after which cell growth, viability, and percentage of infected cells were recorded. The infected cells were distinguished from the uninfected cells by indirect immuno-fluorescence method. Thus, the cultured cells were fixed with cold methanol on a slide glass, reacted with the antibody to the HTLV-III-specific antigens, and further with the secondary antibody (having fluorescent label~. The results are shown in Figs. 9-29, wherein, V, and ~ show the controls without virus, ~, ~ and ~ show the infection experiments with HTLV-III. The cell growth is indicated in number of cells, the viability (%) in number of viable cells x 100/number of total cells, and the infected cell rate (%) in number of fluorescent-positive cell~ x 100/number of total cells.
Figs. 9-22 demonstrate that when no test compound was added to the medium, the cells did not grow and were killed by the viral infection~ whereas depending on the increase in the dose of the test substance, the number of cells and viability came near to the values of the control without virus. Also, it is shown from Figs. 23-29 that when the test substance is not added, almost all cells are infected (-100~), whereas depending on the increase in the dose of the test substances, the infection of cells was strongly inhibited.
Accordingly, it is evident that the test substances have excellent inhibiting activities against infection .
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of AIDS virus to host cells and viral proliferation.
Exam~le 4 (Cytotoxicity) As the anti-virus substances often show toxicity to the host cells, the following experiment was conducted to know whether or not the test substances (used in Examples 1 and 3) would induce cytotoxicity.
MT-4 cells were cultured with or ~ithout 1-100 ~g/ml of each test substance which is the same as in Examples 1 and 3 and the proliferation and viability of the cells were recorded. The results are shown in the following Table.
Substance (~g/ml) Cell number Viability (x104 cells/ml (%) Sodium dextran sulfate (molecular weight: 5,000, S-content: 13%) o 124 87 Sodium dextran sulfate (molecular weight: 7,000 - 8,000, S-content: 17 - 20%) Sodium dextran sulfate (molecular weight: 500,000 S-content: 16%) :
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Fucoidin ~-Carrageenan 80% + ~-Carragheenin 20~
~-Carrageenan 1 147 8~
o 124 ', 87 -Carrageenan .
The above results show that the test substances have little cytotoxicity.
Example 5 (Inhibition of reverse transcriptase activity) The effects of the test substances on the reverse transcriptase activity of AMV were evaluated by the method described in Example 1. The test substances used are chondroitin sulfate (S-content: 6.2-6.9%), chondroitin polysulfate (S-content: 11.6-12.1~), keratan sulfate (S-content: 7.0-8,0%), and keratan polysulfate (S-content:
9.7%). The resul-ts are shown in Figs. 30-33.
Figures 30-33 indicate that the enzyme inhibition .. . .
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increases with the increasing doses of the above test substances. The above results also demonstrate that the reverse transcriptase inhibitory activity of the test substances is closely related to the number o~ sulfate 5 groups in the molecule, as evidenced by the fact that the synthetic substances (e.g. chondroitin polysulfate and kera~an polysulfate) have stronger activity than the natural substances (e.g. chondroitin sulfate and keratan polysulfate).
Example 6 (Anti-AIDS virus activity) Test substances were assayed for the anti-AIDS virus activity in the same manner as in Example 3 using cell culture. The test substances are the same as those used in Example 5. The results are shown in Figs. 34-45, wherein v,~ and ~ show the controls without virus, ~, and ~ show the infection tests with virus.
Fiqs. 34-41 demonstrate that, without the test sub-stances, the cells did not grow and were killed by viral infection, whereas depending on the increase in the dose of the test substance, the decrease in the number of cells and loss of viability were prevented. Also, Figs.
~2-45 demonstrate that when the test substance was not present, almost all of the cells were infected (- 100%) with HTLV-III, whereas depending on the increase in the dose of the test substances, the infected cell rate (%) was significantly reduced.
The above results also indicate that the synthetic mucopolysaccharide polysulfates having higher S-content ., , ~ ' b .
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had stronger anti-AIDS virus activities than those of the natural products.
Exam~le_7 (Anti-AIDS virus activity) The anti-AIDS virus activity of heparin was evaluated in the same manner as in Example 3. The results are shown in Figs. 46-48, wherein v,~ and a show the controls without virus, and v, ~, and ~ show the infection tests with virus.
Figs. 46 and 47 show that without heparin, the cells did not grow and were killed by viral infection, whereas depending on the increase in the dose of heparin, the number of cells and viability were maintained to that of the control. It was also shown from Fig. 48 that when heparin was not present, almost all of the cells are infected, whereas depending on the increase in the dose of heparin, they become Less susceptible to the viral infection.
Example 8 (Cytotoxicity) As the anti-virus substances often show toxicity to the host cells, an experiment was conducted to know whether or not heparin would induce such cytotoxicity.
Without the virus, MT-4 cells were cultured in the same manner as in Example 4 except the test sample was heparin, and proliferation and viability of cells were recorded. The results are shown in the following Table.
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Heparin Cell number Viability (~g/ml~ (x104 cells/ml) (%) o 12~ 87 The above results demonstratë that heparin has little cytotoxicity.
ExamE~e 9 ~Anti-AIDS activity3 In order to examine the correlation between the anti-AIDS virus activity and the molecular structure (especially, molecular weight and S-content or number of sulfate groups in this case) of various test substances including those used in Examples 1-7, the anti-AIDS acti-vities were evaluated for the various naturally occurring polysaccharides, polysaccharides having sulfate groups, mucopolysaccharides, mucopolysaccharide sulfate, and muco-polysaccharide polysulfate. Further, similar experiments were carried out with various other sulfates which were synthetically obtained. The experimental procedures employed are identical to that in preceding experiments.
The cultured MT-4 cells were infected with HTLV-III and the inhibitory effects of various test substances on the infected cell rate (number of fluorescent cell x 100/total cell, ~) were determined on the bth day. The results are shown in the following Table.
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5) Other polysaccharides None of pectin, chromic acid, inulin, raffinose, and methylcellulose showed any anti-AIDS virus activities.
From the above results, it can be clearly seen that the anti-AIDS virus activity is closely related to the S-content or number of sulfate groups in this case rather than to the molecular weight. Substances without sulfate groups showed no anti-AIDS activity. Further, the anti-AIDS activity was intensiEied with increasing S-content (number of sulfate groups) of the molecule. With respect to the relation with the molecular weight, there was no effect at all in the monosaccharides. However, in the substances having molecular weights of 5,000 and higher, an increase in the molecular weight did not affect the anti-AIDS virus activity as seen in e.g. dextran sulfate.
This is quite different from the pattern of manifes-tation of heretofore known activities of polysaccharide sulfates against the herpes virus.
In view of the fact that the polysaccharides with 2G higher molecular weights and their sulfates are known to have high toxicities to human beings and animals, the experimental evidence obtained in the present invention that the dextran sulfates with lower molecular weights show sufficient anti-AIDS activity, is extremely import-ant in developing it as a medicament for the preventionand therapy ~f viral diseases.
Among ~he above test substances, those which showed particularly strong anti-AIDS virus activities are dextran 7~23~3 sulEate, ~-carrageenan, alginic acid sulfate, chitosan sulfate, chondroitin polysulfates, further sulfated chondroitin-4-sulfate and -6-sulfate, heparin, etc.
having an S-content of more than 10%.
Exam~le 10 ~Anti-Friend leukemia virus ~F-MuLV) activity) (Procedure 1) Anti-FMuLV activity of dextran sulfate (molecular weight: 7,000 - 8,000, S-content: 17 - 20~) was deter-mined by a XC-plaque assay method. BALB3T3 cells were cultured in adhesive form in a 35 mm-dish at 5x104 cells/dish (2 ml). After removing the culture medium, a fresh medium with or without indicated concentrations of the test substance (1 ml each) and 0.2 ml of the virus preparation were charged, and the cells were cultured overnight. On the following day, the culture media were replaced with those (2 ml) containing or not containin~
the above substances, the incubation was continued for three additional days to allow the infection to progress and for replication of the virus. After the removal of the medium, the further progression of viral replication was stopped by W irradiation. To this dish, the suspen sion of XC-cells (2 ml) was added and cultured for three days and the plaque formation produced by the virus par-ticle induced cell-fusion~ was observed. The number of plaques was shown in the following Table.
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~ 30 -Tabl_ Anti-Friend leukemia virus activity by Procedure l ._ ____~ _ ___ _____ DS Number of plaques Inhibition (~g/ml) _ _ per dish 5Control 168 l 14 92 ll 93 lOlO0 6 96 As observed from the above Table, DS inhibi~ted 90%
or more the formation of plaque at the concentrations of l-lO0 ~g/l, indicating that the infection and replication of the virus was strongly inhibited. The plaque formation was not detected at l,000 ug/ml of DS~
DS at l-lO0 ug/ml did not show any cytotoxicity to BALB 3T3 cells.
(Procedure 2) Procedure l was repeated except that after adsorption of the virus in the medium without DS, the non-adsorbed viruses were removed and the culture was carried out in the medium (2 ml) containing or not containing DS. The results are shown in the following table.
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Table Anti-Friend leukemia vlrus acti_ity_~y Procedure 2 DS Number of plaq-les Inhibition ~ mll_____ _ ---E~r dlSh _ ~ %) 5Control 35 (o) O . 01 33 6 0.1 19 45 The above results indicate that, also in Procedure 2, DS inhibited the infection and replication of the virus by about 60~ at the concentration of 1 ~g~ and almost 15 completely at 500 ~g/ml.
From the above results, it is evidenced that DS
inhibits the inection and replication of the oncogenic virus (Oncovirinae) including F-MuLV, as well as the cytolytic virus (Lentivirinae) including AIDS-virus.
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Treatment of diseases caused by retroviruses The present invention relates to the prevention, therapy and treatment of diseases caused by retroviruses.
~ore particularly, the present invention provides a medi-cament (including a veterinary medicament) containing as-active ingredient a natural or synthetic oligo- or polysaccharide having at least one S-oxoacid group attached to the saccharic carbon atom through a linking group of lower molecular weight or a pharmaceutically acceptable salt thereof and a method of prevention and therapy, etc. of the diseases caused by retroviruses, especially AIDS (acquired immune deficiency syndrome), ARC (AIDS-related complex), PGL (persistant generalized lymphadenopathy) and AIDS-virus carrier using such medicament.
Retroviruses refer to a family of virus which has RNA and reverse transcriptase (RNA-dependent DNA poly-merase), which is essential to the first stage of its self-replication for synthesizing complementary DNA on the base of template RNA of the virus.
Retroviruses include various oncoviruses, for exam-ple, avian leukemia virus, avian sarcoma virus, avian ~ ~ ' ' . ' ' .
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reticuloendotheliosis virus, murine mammary cancer virus, murine leukemia virus, murine sarcoma virus, guinea pig type C virus, hamster type C virus, rat leukemia virus, feline leukemia virus, ~eline sarcoma virus, feline type C virus, ovine leukemia virus, bovine leukemia virus, swine type C virus, simian leukemia virus, Mason-Pfizer virus, simian sarcoma virus, simian T-lymphotropic virus, baboon type C virus, and the like. Among those infective to humans, important ones include adult T-cell leukemia virus (ATLV), or human T-lymphotropic virus type I
~HTLV-I), and type II (HTLV-II). Adult T-cell leukemia abounds in Japan, especially in the west part, but effective treatment including prevention and therapy of the disease has not been established.
On the other hand, retroviruses also include those having no oncogenecity, for example, visna virus, ovine progre~siv~ pneumonia virus, ovine maedi virus, simian T-lymphotropic virus type III (STLV-III), eguine infec-tious anemia virus, and the like~ The viruses isolated from humans as being the causative agents for AIDS or ARC etc. (HTLV-III, LAVl, LA~2, ARV, and other so-called AIDS-viruses) belong to this subfamily. Recently, the AIDS-causative viruses have been called HIVs ~human immune deficiency viruses). Further, as the third subfamily, there is known a spumavirinae to which simian foaming virus belongs. Also, a retrovirus has been isolated recently as a causative virus for Kawasaki disease (mucocutaneous lymphonode syndrome).
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World-wide interests have been focused on AIDS due to its unfavorable prognosis. It is a clinical syndrome characterized by recurrent opportunistic infections, (e.g.
preumocysti.s carinii preunonia, cryptococcal meningitis, disseminated toxoplasmosis), lymphadenopathy, and an aggressive Kaposi's sarcoma, and induces a mortali~y rate of more than 90% by the disregulation of immune system.
It is also known that the helper-T cells are specifically destroyed by the infection of the virus.
In order to find pharmaceutical agents effective for the treatment of AIDS, ARC, PGL, and AIDS-virus carrier, the present inventorst using a cell line of MT-4 estab-lished from T-cells o~ adult T-cell leukemia patients and HTLV-III which is a causative virus for AIDS, examined the effects of various substances on the infection and replication of HTLV-III.
The above ~T-4 cell line is absolutely susceptible to the infection with HTLV-III followed by causing cell lysis (experimental HTLV-III infection). The present inventors ~ound that when certain polysaccharides having a sul~onate group (-SO3) or mucopolysaccharides having a sulonate group or their additionally sulfuric acid esterified sub-stances were added to the experimental HTLV-~II infection system, the infection of HTLV-III on MT-4 cells and viral replication were strongly inhibited without any accompany-ing toxicity to the cells.
Further, th~e present inventors demonstrated that the above polymerized sugar inhibits the reverse transcriptase . . .
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of the retrovirus in_vitro, and thereby suppresses the replication oE the virus.
Among the sul~uric acid esters of polysaccharides, dextran sulfate with a lower molecular weight has long been commercialized as an antilipemic or anti-arteriosclerosis agent. Also, de~tran sulfate with a relatively higher molecular weight is known to have an inhibitory action against the herpes virus. (European Patent Laid-Open Publication No. 0066379.) However, since the herpes virus is a DNA virus, its replication is absolutely different from that of the retrovirus which depends entirely on reverse transcriptase for synthesis of DNA. Accordinglyt effectiveness on herpes virus does not necessarily mean effectiveness on retrovirus alike.
Furthermore, dextran sulfate with a molecular weight less than 10,000 was found to be almost ineffective on herpes viruses.
Among the mucopolysaccharides or their sulfates, chondroitin sulfate is commercialized as a drug for sensorineural hearing impairment, neuralgia, lumbago and chronic nephritis, and also as a cornea-protective ophthalmic solution. Reratan sulfate is obtainable from the cartilage, teichronic acid from the cell walls of Bacillus subtilis, hyaluronic acid from shark skin, whale cartilage, or from human serum, heparan sulfate from bovine liver or lung, and chitin from arthropod or from fungus or yeast, respectively. The preparation process for the further sulfuric acid esterified compound of ~ .- . . . .
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chondroitin sul~ate is described in Japanese Patent Publication (JP, B2) No. 9570/1971.
EIeparin is known to inhibit various en~ymes in vitro, e.g., DNA polymelase of phytohemagglutinin stimulated human lymphocytes and reverse transcriptase of simian sarcoma virus (Cancer Research, 38, 2401 - 2407), but it has not been proven to inhibit the viral infection of cells.
In one aspect, the presen-t invention provides a method for the treatment of diseases caused by human retroviruses which comprises administering an effective amount of a natural or synthetic oligo- or polysaccharide h~ving at least one S-oxoacid group attached to the saccharic carbon atom through a linking group of lower molecular weight or a pharmaceutically acceptable salt thereof to a subject in need of such treatment.
In another aspect, the present invention provides a use of the above oligo- or polysaccharide or a salt thereof for the manufacture of a medicament for the treatment of diseases caused by human retroviruses.
In a further aspect~ the present invention provides a pharmaceutical composition comprising the above oligo- or polysaccharide or a salt thereof as an active ingredient in association with a pharmaceutically acceptable carrier, diluent or excipient.
In the drawings which illustrate embodiments of the invention, Figs. 1-7 show the reverse transcriptase inhibition ~, : ' ' . , :- ' :
:~27723~
activities of the test substances in Example 1.
Fig. 8 shows the reverse transcriptase inhibition activity of the test substance in Example 2.
Figs. 9-15, 16-22, and 23-29 show the effects of the test substances on cell growth, viability, and infected cell rate (%) of MT-4 cells infected with HT~V-III, respectively, in Example 3.
Figs. 30-33 show the reverse transcriptase inhibition activities of the test substance in Example 5.
Figs. 34-37, 38-~1, 42-45 show the effects of the test substances or cell growth, viability, and infected cell rate (%) of MT-4 cells infected with HTLV-III 7 respectively, in Example 6.
Figs. 46-48 show the effects of heparin on cell growth, viability and infected cell rate of MT-4 cells infected with HTLV-III, respectively, in Example 7.
The term 'ttreatment" herein is intended to cover all aspects of disease treatment including prevention, sustention (i.e. prevention of aggravation), reducing (i.e. alleviation of conditions) and therapy.
The retroviruses includes all viruses having RNA and reverse transcriptase as the basic components including those exemplified above.
The diseases referred to herein cover all the diseases caused by retroviruses, including those bearing or not bearing the aforementioned virus name. Particularly im-portant diseases are the diseases caused by AIDS-viruses.
The oligo- or polysaccharide usable in the present - - ~ , .
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invention are those having at least one S-oxoacid group attached to the saccharic carbon atom through a linking group of lower molecular weight. Such an oligo- or polysaccharide may be natural or synthetic. The term "natural" is intended to mean that the oligo- or poly-saccharide is obtainable from a natural source, for example, plant, microorganism or animal by extraction and other means. The term "synth~etic" is intended to mean that the oligo- or polysaceharide is obtainable synthetieally, for example, by introdueing S-oxoaeid group into another oligo- or polysaeeharide whieh has or has not S-oxoaeid group and whieh is natural or unnatural (and synthetie~.
The term "oligosaceharide" refers to a earbohydrate eontaining from two up to about nine monosaceharides linked together. For example, when an oligosaeeharide eontains three monosaeeharides, one, two or three of the monosaeeharides may have at least one S-oxoaeid group.
~he term "polysaeeharide" refers to a earbohydrate eontaining about ten or more monosaeeharides linked together. At least one and a minor or major part or all of the monosaecharides may have at least one and normally up to four S-oxoaeid groups.
The S-oxoaeid group includes a sulfo group (-SO3H) and a hydroxysulfinyl group (-SO.OH). Preferably the S-oxoacid group is a sulfo group.
The term '7saccharic earbon atom" refers to a ear-bon atom which is a member of a tetrahydrofuran or a -. -: . . .
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~ 8 tetrahydropyran ring of a monosaccharide contained in the oligo- or polysaccharide.
The linking group of the lower molecular weight includes oxy (-O-), imino (-NH-), thio (-S-), methylene (-CH2-), ethylidene (-CH(CH3)-) groups and the like. The term "lower molecular weight" is intended to mean that the group has a molecular weight from about 14 up to about 32. Preferably the linking group is an oxy or an imino group.
One class of the oligo- or polysaccharide is a natural polysaccharide having at least one hydrogen sulfate group (-O-SO3H) and is obtained from a plant or a microorganism, or a synthetic polysaccharide having at least one hydrogen sulfate group (-O-SO3H) and is formed by esterifying a polysaccharide obtained from a plant or a microorganism.
Within this class, a preferable subc~ass is a poly-saccharide composed o non-amino monosaccharide (including sugar acid) as the repeating unit. This polysaccharide~
however, may contain a trace amount of nitrogen. Examples of the non-amino sugar repeating units include xylose, arabinose, rhamnose, frucose, glucose, galactose, glu-curonic acid, galacturonic acid, mannuronic acid, etc.
The natural polysaccharide includes carrageenan (galactan sulfate o~tainable from Gigartina tenella, etc.) and -fucoidin (polyfucose sulfate obtainable from Laminaria brown seaweed). Carrageenan includes ~-carragheenin, ~-carrageenan, I-carrageenan, etc. which have a different content of the hydrogen sulfate group. The synthetic . .
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g polysaccharide includes those to be obtained by sul~uric acid esterification of polysaccharides, e.g., starch and partial hydrolyzate thereof, dextran which is produced by ~euc_n_stoc__p. and partial hydrolyzate thereof (usually having the molecular weight oE 500 - 2,000,000, ordinar-ily 2,000 and 300,000, pre~erably 2,000 - 10,000, most suitably 3,000 - 8,000, e.g., 7,000 - 8,000), glycogen, pectin, cellulose, plant viscous liquids (gum arabic, tragacanth gum, etc.), plant mucilage products ~those obtainable from Hibiscus esculentus, Aloe, Brasenia schreberi, xylan, etc.), viscous li~uids of marine and fresh water algae ~alginic acid, laminarin, etc.~ or polysaccharide derived from microorganism (lentinan, pluran, mannan, etc.). They include known ones (dextran sulfate, cf., European Patent Laid-Open Publication No.
0066379) and novel ones. The novel ones may be produced in the same manner as in the known ones. ~n example of the preparation process is shown, as follows:-Chlorosulfonic acid is added dropwise to dry pyridin of 8-10 fold volume while cooling. To the mixture are added small amounts of formamide and dextran (about 1/4 weight of chlorosulfonic acid), and the mixture is heated to 55-65C under stirring. After stirring the mixture for several hours, the solvent is distilled off, and the residue is purified for example by recrystallization, dialysis, etc. Within the synthetic polysaccharide, those obtained by further sulfuric acid esterification are represented by the term "polysulfate".
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Another class of the oligo- or polysaccharide is a natural polysaccharide having at least one hydrogen sulfate group (-O-SO3H) and is obtained from an animal, or a synthetic polysaccharide having at least one hydrogen sulfate group (-O-SO3~1) and is formed by esterifying a polysaccharide obtained from an animal.
Within this class, a prefera~le subclass includes mucopolysaccharides, which are composed of repeating units of amino monosaccharides (including N-acyl or NH-SO3H).
These may further contain as another repeating unit a non-amino sugar or an acid derivative thereof. The repeating amino-sugar unit or its N-acylated (preferably N-acetylated) derivatives include glucosamine, galactos-amine, N-acetylated derivatives of them, and the sulfuric acid ester or partial hydrolyzate of the above compound.
Examples of monosaccharide or acid (preferably, hexu-lonic acid) include glucose, galactose, glucuronic acid, iduronic acid, etc. The mucopolysaccharides containing such a repeating unit include heparin, keratan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, teichuronic acid, hyaluronic acid, heparitin sulfate, chitin, and their partial hydrolyzates, modified derivatives (e.g., partial acylated products), and syn-thetic polysaccharides containing the repeating unit as mentioned above.
The mucopolysaccharide polysulfates are defined as the products which are synthesized by additional sulfuric acid esterification of the above mucopolysaccharides having a ~4 ' : ~ ~,.' ' ', "
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~Z7~39 sulfate group. This esterification may be carried out, for example, according to the procedure described in Japanese Patent Publication No. 9570/1971. In general, the esteri~ication is carried out by the treatment of the mucopolysaccharides with one sulfating reagent, for example, concentrated sulfuric acid or chlorosulfonic acid.
These reactions are usually carried out with or without a solvent at low temperature. The reaction product is separated by any conventional procedure, e.g., neutralization, concentration, precipitation, recrystallization, chromatography, etc.
The term "pharmaceutically acceptable salt" is intended to mean that the salt has the biological acti-vity of the parent compound and is not unusably toxic atthe administration level. Such a salt includes the salt of an inorganic base e.g., sodium salt, potassium salt, ammonium salt, etc., and the salt of an organic base e.g., diethanolamine salt, cyclohexylamine salt, amino acid salt, etc. These salts are produced from the cor-responding acids by conventional procedures. The above oligo- or polysaccharides and their salts may be used alone or as a mixture with metal salts e.g. zinc, aluminum, etc. The oligo- or polysaccharide should be ~5 administered at a dose sufficient to produce the effect for the desired treatment. For example, the dosage of the sulfates of the above polysaccharide or their salts sufficient to produce blood concentration for anti-virus .. .
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activity is generally 0.2-200 mg/kg, preferably 0.5-100 mg/kg. In the case of humans, an amount of about 10 mg - 10 g/day, preferably about 50 mg - 5 g/day, is admini-stered in 1-4 divisions a day, or as a sustained release form. The administration route is optional and can be oral, rectal, nasal, local (including sublingual), injection (including subcutaneous, intracutaneous, intra-muscular and intravenous), inunction etc. The preferred route is selected depending on various factors including the kind of active ingredient, conditions and age of the patient, severity of infection etc.
The dosage of the mucopolysaccharides or their poly-sulfates or the salts thereof sufficient to produce a concentration for anti-virus activity is generally 0.2-20~ mg/k~, preferably 0.5-100 mg/kg. In the case of humans, an amount of about 10 mg - 20 g/day, preferably about 50 mg - 10 g/day is administered in 1-4 divisions a day, or as a sustained releas~ form.
The administration route is optional and can be oral, local, injection, inunction, etc.
For administration, the effective ingredient may be mixed with a pharmaceutical carrier, for examp~e, organic or inorganic solid or liquid excipient e.g. suitable for internal administration or injection, and administered in the form of a conventional pharmaceutical preparation.
Such preparations include solids (e.g., tablet, granule, powder, capsule, etc.), liquids (e.g., liquid, emulsion, suspension, etc.), and ointments. The above carriers ~.
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: . ' ' 7~3g include starch, lactose, glucose, sucrose, dextrin, cellulose, paraffin, fatty acid glycerider water, alcohol, gum arabic, etc. If necessary, ordinary use auxiliaries, stabilizers, emulsiEiers, lubricants, binders, pH regulat-ing agents, isotonicity agents, and other additives may be used.
The toxicity o~ the above oligo- or polysaccharide is extremely low. For example, the acute toxicity (LD50) of sodium dextran sulfate tmolecular weight 7,000-8,000, S-content 17-20%) is 21,000 mg/kg when orally, and 4,500 mg/kg when intravenously administered to miceO
- The acute toxicity (LD50) of sodium chondroltin sulfa~e is 4,000 mg/kg or more when intraperitoneally, and 7,500 mg/kg or more when orally administered to mice.
The acute toxicity tLD50) of sodium heparin is 1,500-2~000 mg/kg when intravenously injected to mice.
The following examples will illustrate the present inventiun in further detail.
Preparation l Preparation of chondroitin polysulfate from chondroitin sulfate Chondroitin sulfate (5 9) was added to 95~ sulfuric acid (lO ml) cooled at below -25~C with stirring. After the addition, the reaction mixture was stirred at the same temperature for 90 minutes. After the 90 minutes had lapsed, the reaction solution was gradually poured onto ice (120 g) with stirring. Calcium carbonate was gradually addecl to the resulting solution with vigorous ; ~
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stirring. The precipitates were filtered o~f and washed well with water. Ethanol (60 ml) was added to the combined filtrates ~240 ml), and the solution was allowed to stand overnight at 5C to precipitate calcium sulfate.
The precipitates were filtered off, and the filtrate was adjusted to pH 10 with sodium carbonate. After the addition of acetic acid to make the solution weakly acidic, the solution was concentrated to about 20 ml, then diluted with ethanol (100 ml), and allowed to stand overnight at 5C~ The precipitates in the solution were isolated with centrifugation, washed with ethanol, and with ether, and dried under vacuum to give the white powder of the title compound.
Preparation 2 Preparation of keratan polysulfate from keratan sulfate.
To give the title compound preparation 1 was re~ea~ed except that keratan sulfate (100 mg) was used as a start-ing material and 1 ml in place of 10 ml of 95~ sulfuric acid was used.
Formulation 1 Sodium dextran sulfate (molecular weight: 7,000 -8,000, S-content: 17 - 20%) 150 my Corn starch 45 mg Lactose 300 mg Magnesium stearate 5 mg The above ingredients were mixed, granulated, and ;
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pressed according to conventional tablet making procedures and then enterically coated.
Formulation 2 _ ______ Sodium dextran sul~ate (molecular weight: 7,000 -8,00(), S-content: 17 - 20&) 600 mg Physiological saline q.s,. to 10 ml.
Formulation 3 Sodium dextran sulfate (molec:ular weight: 5,000, S-content: 13 - 14~) 600 mg Physiological saline q.s. to 10 ml.
Formulation 4 Sodium salt of chondroitin sulfate150 mg Corn starch 45 mg Lactose 300 mg Magnesium stearate 5 mg The above ingredients were mixed, granulated, and pressed according to conventional tablet making procedures and then en~erically coated.
Formulation 5 Sodium salt o keratan polysulfate400 mg Lactose 195 mg Magnesium stearate 5 mg The above ingredients were mixed according to the conventional procedures and filled in hard gelatine capsules.
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~277;~39 Formulation 6 Sodium salt of chondroitin polysulfate 300 mg Physiological saline q.s. to 10 ml.
Formulation 7 __ __ .
Sodium heparin 25,000 units Physiological saline q.s. to 10 ml.
Formulation 8 ____ Calcium heparin 5,000 units Procain hydrochloride 10 mg Water q.s. to 10 ml.
Example 1 (Inhibition of reverse transcriptase activity) Test substances were assayed for inhibition against the enzyme activity of reverse transcriptase (authentic sample) derived from Avian Myeloblastosis Virus (abbrev.
AMV), a kind o retrovirus.
Five microliters of ~YA)n(template RNA), 4 ~1 of (dT)l~ 18(primer DNA), and 1 ~1 of water were mixed with 5 Yl of 0.5M Tris-~Cl (pH 8.4) including 0.1% Triton*
X-100, 5 ~1 of lnM-MgC12, 5 ~1 of 20 mM-DDT, 5 ~1 of water, and [3H]-TTP (tritium labeled thymidine triphos-phate). To this mixture, test substances in solutions (final concentrations: 1, 0.1, and 0.01 ~g/ml, 5 ~1) or buffer solutions (control, 5 ~1) at various doses were added. Then, 5 ~1 (one unit) of the authentic reverse transcriptase clerived from AMV was added and the reac-tion mixture was incubated at 37C for 30 minutes. The * Trade mark :;:
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reaction was stopped by the addition of trichloroacetic acid, and after filtering the reaction mixture, the radioactivity o~ the polymerized (3H-T)n retained on the filter was measured using a liquid scintillation counter.
As the test substances, sodium dextran sulfate (molecular weight: 5,000), same (molecular weight: 8,000), same (molecular weight: 500,000), fucoidin, K-carrageenan, ~-carrageenan, and l-carrageenan were used. The results are shown in Figs. 1-7.
Figs. 1-7 show that the enzyme inhibition increases with the increasing doses of the above test substances.
ExamE~e 2 (Inhibition of reverse transcriptase activity) The assay procedure of Example 1 was repeated using disrupted HTLV-III virions as a crude reverse trans-criptase in order to evaluate the reverse transcriptase inhibitory effect of dextran sulfate (DS, molecu~ar weight 7,000 - 8,000, S-content 17 - ~0%). The result is shown in Fig. 8.
Fig. 3 shows that DS has an inhibitory effect against the reverse transcriptase derived from AIDS-virus, HTLV-III.
Example 3 (Anti-AIDS virus activity~
To MT-4 cells (30 x 10 /ml) cultured in RPMI-1640 medium containing 10% bovine serum, was inoculated HTLV-III, and the suspension was incubated at 37C for 1 hour to cause the adsorption of the virus. The cell:virus ratio was 500:1. The cells were then washed, and cultured .. ~ , .
~2~77~9 with or without various doses of the test substances (same as those of Example 1) at 37C under 5~ C02 for 3 days, after which cell growth, viability, and percentage of infected cells were recorded. The infected cells were distinguished from the uninfected cells by indirect immuno-fluorescence method. Thus, the cultured cells were fixed with cold methanol on a slide glass, reacted with the antibody to the HTLV-III-specific antigens, and further with the secondary antibody (having fluorescent label~. The results are shown in Figs. 9-29, wherein, V, and ~ show the controls without virus, ~, ~ and ~ show the infection experiments with HTLV-III. The cell growth is indicated in number of cells, the viability (%) in number of viable cells x 100/number of total cells, and the infected cell rate (%) in number of fluorescent-positive cell~ x 100/number of total cells.
Figs. 9-22 demonstrate that when no test compound was added to the medium, the cells did not grow and were killed by the viral infection~ whereas depending on the increase in the dose of the test substance, the number of cells and viability came near to the values of the control without virus. Also, it is shown from Figs. 23-29 that when the test substance is not added, almost all cells are infected (-100~), whereas depending on the increase in the dose of the test substances, the infection of cells was strongly inhibited.
Accordingly, it is evident that the test substances have excellent inhibiting activities against infection .
:,, : ' .
7~
of AIDS virus to host cells and viral proliferation.
Exam~le 4 (Cytotoxicity) As the anti-virus substances often show toxicity to the host cells, the following experiment was conducted to know whether or not the test substances (used in Examples 1 and 3) would induce cytotoxicity.
MT-4 cells were cultured with or ~ithout 1-100 ~g/ml of each test substance which is the same as in Examples 1 and 3 and the proliferation and viability of the cells were recorded. The results are shown in the following Table.
Substance (~g/ml) Cell number Viability (x104 cells/ml (%) Sodium dextran sulfate (molecular weight: 5,000, S-content: 13%) o 124 87 Sodium dextran sulfate (molecular weight: 7,000 - 8,000, S-content: 17 - 20%) Sodium dextran sulfate (molecular weight: 500,000 S-content: 16%) :
~' :
' ' ' ' , ' ' '. ' `
--~'7723~
Fucoidin ~-Carrageenan 80% + ~-Carragheenin 20~
~-Carrageenan 1 147 8~
o 124 ', 87 -Carrageenan .
The above results show that the test substances have little cytotoxicity.
Example 5 (Inhibition of reverse transcriptase activity) The effects of the test substances on the reverse transcriptase activity of AMV were evaluated by the method described in Example 1. The test substances used are chondroitin sulfate (S-content: 6.2-6.9%), chondroitin polysulfate (S-content: 11.6-12.1~), keratan sulfate (S-content: 7.0-8,0%), and keratan polysulfate (S-content:
9.7%). The resul-ts are shown in Figs. 30-33.
Figures 30-33 indicate that the enzyme inhibition .. . .
- , ~
, .
2~
increases with the increasing doses of the above test substances. The above results also demonstrate that the reverse transcriptase inhibitory activity of the test substances is closely related to the number o~ sulfate 5 groups in the molecule, as evidenced by the fact that the synthetic substances (e.g. chondroitin polysulfate and kera~an polysulfate) have stronger activity than the natural substances (e.g. chondroitin sulfate and keratan polysulfate).
Example 6 (Anti-AIDS virus activity) Test substances were assayed for the anti-AIDS virus activity in the same manner as in Example 3 using cell culture. The test substances are the same as those used in Example 5. The results are shown in Figs. 34-45, wherein v,~ and ~ show the controls without virus, ~, and ~ show the infection tests with virus.
Fiqs. 34-41 demonstrate that, without the test sub-stances, the cells did not grow and were killed by viral infection, whereas depending on the increase in the dose of the test substance, the decrease in the number of cells and loss of viability were prevented. Also, Figs.
~2-45 demonstrate that when the test substance was not present, almost all of the cells were infected (- 100%) with HTLV-III, whereas depending on the increase in the dose of the test substances, the infected cell rate (%) was significantly reduced.
The above results also indicate that the synthetic mucopolysaccharide polysulfates having higher S-content ., , ~ ' b .
, .
~7;2~
had stronger anti-AIDS virus activities than those of the natural products.
Exam~le_7 (Anti-AIDS virus activity) The anti-AIDS virus activity of heparin was evaluated in the same manner as in Example 3. The results are shown in Figs. 46-48, wherein v,~ and a show the controls without virus, and v, ~, and ~ show the infection tests with virus.
Figs. 46 and 47 show that without heparin, the cells did not grow and were killed by viral infection, whereas depending on the increase in the dose of heparin, the number of cells and viability were maintained to that of the control. It was also shown from Fig. 48 that when heparin was not present, almost all of the cells are infected, whereas depending on the increase in the dose of heparin, they become Less susceptible to the viral infection.
Example 8 (Cytotoxicity) As the anti-virus substances often show toxicity to the host cells, an experiment was conducted to know whether or not heparin would induce such cytotoxicity.
Without the virus, MT-4 cells were cultured in the same manner as in Example 4 except the test sample was heparin, and proliferation and viability of cells were recorded. The results are shown in the following Table.
-.,~
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Heparin Cell number Viability (~g/ml~ (x104 cells/ml) (%) o 12~ 87 The above results demonstratë that heparin has little cytotoxicity.
ExamE~e 9 ~Anti-AIDS activity3 In order to examine the correlation between the anti-AIDS virus activity and the molecular structure (especially, molecular weight and S-content or number of sulfate groups in this case) of various test substances including those used in Examples 1-7, the anti-AIDS acti-vities were evaluated for the various naturally occurring polysaccharides, polysaccharides having sulfate groups, mucopolysaccharides, mucopolysaccharide sulfate, and muco-polysaccharide polysulfate. Further, similar experiments were carried out with various other sulfates which were synthetically obtained. The experimental procedures employed are identical to that in preceding experiments.
The cultured MT-4 cells were infected with HTLV-III and the inhibitory effects of various test substances on the infected cell rate (number of fluorescent cell x 100/total cell, ~) were determined on the bth day. The results are shown in the following Table.
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5) Other polysaccharides None of pectin, chromic acid, inulin, raffinose, and methylcellulose showed any anti-AIDS virus activities.
From the above results, it can be clearly seen that the anti-AIDS virus activity is closely related to the S-content or number of sulfate groups in this case rather than to the molecular weight. Substances without sulfate groups showed no anti-AIDS activity. Further, the anti-AIDS activity was intensiEied with increasing S-content (number of sulfate groups) of the molecule. With respect to the relation with the molecular weight, there was no effect at all in the monosaccharides. However, in the substances having molecular weights of 5,000 and higher, an increase in the molecular weight did not affect the anti-AIDS virus activity as seen in e.g. dextran sulfate.
This is quite different from the pattern of manifes-tation of heretofore known activities of polysaccharide sulfates against the herpes virus.
In view of the fact that the polysaccharides with 2G higher molecular weights and their sulfates are known to have high toxicities to human beings and animals, the experimental evidence obtained in the present invention that the dextran sulfates with lower molecular weights show sufficient anti-AIDS activity, is extremely import-ant in developing it as a medicament for the preventionand therapy ~f viral diseases.
Among ~he above test substances, those which showed particularly strong anti-AIDS virus activities are dextran 7~23~3 sulEate, ~-carrageenan, alginic acid sulfate, chitosan sulfate, chondroitin polysulfates, further sulfated chondroitin-4-sulfate and -6-sulfate, heparin, etc.
having an S-content of more than 10%.
Exam~le 10 ~Anti-Friend leukemia virus ~F-MuLV) activity) (Procedure 1) Anti-FMuLV activity of dextran sulfate (molecular weight: 7,000 - 8,000, S-content: 17 - 20~) was deter-mined by a XC-plaque assay method. BALB3T3 cells were cultured in adhesive form in a 35 mm-dish at 5x104 cells/dish (2 ml). After removing the culture medium, a fresh medium with or without indicated concentrations of the test substance (1 ml each) and 0.2 ml of the virus preparation were charged, and the cells were cultured overnight. On the following day, the culture media were replaced with those (2 ml) containing or not containin~
the above substances, the incubation was continued for three additional days to allow the infection to progress and for replication of the virus. After the removal of the medium, the further progression of viral replication was stopped by W irradiation. To this dish, the suspen sion of XC-cells (2 ml) was added and cultured for three days and the plaque formation produced by the virus par-ticle induced cell-fusion~ was observed. The number of plaques was shown in the following Table.
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~ 30 -Tabl_ Anti-Friend leukemia virus activity by Procedure l ._ ____~ _ ___ _____ DS Number of plaques Inhibition (~g/ml) _ _ per dish 5Control 168 l 14 92 ll 93 lOlO0 6 96 As observed from the above Table, DS inhibi~ted 90%
or more the formation of plaque at the concentrations of l-lO0 ~g/l, indicating that the infection and replication of the virus was strongly inhibited. The plaque formation was not detected at l,000 ug/ml of DS~
DS at l-lO0 ug/ml did not show any cytotoxicity to BALB 3T3 cells.
(Procedure 2) Procedure l was repeated except that after adsorption of the virus in the medium without DS, the non-adsorbed viruses were removed and the culture was carried out in the medium (2 ml) containing or not containing DS. The results are shown in the following table.
~'77;~3~
Table Anti-Friend leukemia vlrus acti_ity_~y Procedure 2 DS Number of plaq-les Inhibition ~ mll_____ _ ---E~r dlSh _ ~ %) 5Control 35 (o) O . 01 33 6 0.1 19 45 The above results indicate that, also in Procedure 2, DS inhibited the infection and replication of the virus by about 60~ at the concentration of 1 ~g~ and almost 15 completely at 500 ~g/ml.
From the above results, it is evidenced that DS
inhibits the inection and replication of the oncogenic virus (Oncovirinae) including F-MuLV, as well as the cytolytic virus (Lentivirinae) including AIDS-virus.
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Claims (25)
1. A pharmaceutical composition for the treatment of diseases caused by human retroviruses comprising an antivirally effective amount of a natural or synthetic oligo- or polysaccharide having at least one S-oxoacid group attached to the saccharic carbon atom through a linking group of lower molecular weight or a pharmaceuti-cally acceptable salt thereof as an active ingredient in association with a pharmaceutically acceptable carrier, diluent or excipient.
2. The composition according to claim 1, wherein the said S-oxoacid group is a sulfo group (-SO3H).
3. The composition according to claim 1, wherein the said linking group is an oxy group(-O-) or an imino group (-NH-).
4. The composition according to claim 1, wherein the said human retroviruses are selected from HIVs.
5. The composition according to claim 1, wherein the said human retroviruses are selected from HTLV-III, LAV
and ARV.
and ARV.
6, The composition according to claim 1, wherein the said human retroviruses are selected from HTLV-I, HTLV-II
and Kawasaki disease causative retroviruses.
and Kawasaki disease causative retroviruses.
7. The composition according to claim 1, wherein the said oligo- or polysaccharide is natural or synthe-tic polysaccharide sulfate ester or a pharmaceutically acceptable salt thereof.
8. The composition according to claim 7, wherein the said natural or synthetic oligo- or polysaccharide is a natural polysaccharide having at least one hydrogen sulfate group(-O-SO3H) obtained from a plant or a microorganism, or a synthetic polysaccharide having at least one hydrogen sulfate group(-O-SO3H) formed by esterifying a polysaccharide obtained from a plant or a microorganism with a sulfating agent, or a pharmaceutically acceptable salt thereof.
9. The composition according to claim 8 , wherein the said synthetic polysaccharide is selected from dextran sulfate alginic acid sulfate, lentinan sulfate and pururan sulfate.
10. The composition according to claim 9, wherein the said dextran sulfate has a molecular weight between 500 and 2,000,000.
11. The composition according to claim 9, wherein said dextran sulfate has a molecular weight between 2,000 and 300,000.
12. The composition according to claim 9, wherein the said dextran sulfate has a molecular weight between 2,000 and 10,000
13. The composition according to claim 9, wherein the said dextran sulfate has a molecular weight between 3,000 and 8,000.
14. The composition according to claim 9, wherein the said dextran sulfate has a sulfur content between 5-and 22%.
15. The composition according to claim 9, wherein the said dextran sulfate has a sulfur content between 10 and 20%.
16, The composition according to claim 9, wherein the said dextran sulfate has a sulfur content between 15%
and 20%.
and 20%.
17. The composition according to claim 8, wherein the said natural polysaccharide is selected from carrageenan, fucoidin.
18. The composition according to claim 7, wherein the said natural or synthetic oligo- or polysaccharide is a natural polysaccharide having at least one sulfo group (-SO3H) obtained from an animal, or a synthetic polysaccharide having at least one sulfo group(-SO3H) formed by esterifying a polysaccharide obtained from an animal with a sulfating agent, or a pharmaceutically acceptable salt thereof.
19. The composition according to claim 18, wherein the said polysaccharide is a natural polysaccharide selected from mucopolysaccharide or a synthetic poly-saccharide selected from mucopolysaccharide sulfate produced by esterifying a natural mucopolysaccharide with a sulfating agent.
20. The composition according to claim 18, wherein the said polysaccharide is heparin or a pharmaceutically acceptable salt thereof.
21. The composition according to claim 18, wherein the said polysaccharide is a natural polysaccharide selected from chondroitin sulfate, dermatan sulfate, heparitin sulfate, keratan sulfate, hyaluronic acid, teichronic acid, chitin and chitosan or synthetic polysaccharide selected from chondroitin polysulfate, dermatan polysulfate, heparitin polysulfate, keratan polysulfate, hyaluronic acid sulfate, teichronic acid sulfate, chitin sulfate and chitosan sulfate, or a pharmaceutically acceptable salt thereof.
22 A process for preparing a pharmaceutical compo-sition in ready-to-use drug form for treating PGL, ARC, AIDS, ATL or Kawasaki disease which process is character-ized by incorporating in the pharmaceutical composition as active ingredient an antivirally effective amount of a natural or synthetic oligo- or polysaccharide having at least one S-oxoacid group attached to the saccharic carbon atom through a linking group of lower molecular weight or a pharmaceutically acceptable salt thereof.
23 . A process for preparing a pharmaceutical compo-sition in ready-to-use drug form for the prevention of infection from human retroviruses which process is characterized by incorporating in the pharmaceutical composition as active ingredient an antivirally effective amount of a natural or synthetic oligo- or polysaccharide having at least one S-oxoacid group attached to the saccharic carbon atom through a linking group of lower molecular weight or a pharmaceutically acceptable salt thereof.
24. The use of a pharmaceutical composition as defined in claim 1 for the treatment of PGL, ARC, AIDS, ATL or Kawasaki disease.
25. The use of a pharmaceutical composition as defined in claim 1 for the prevention of infection from human retroviruses.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP078470/1986 | 1986-04-04 | ||
| JP7847186 | 1986-04-04 | ||
| JP7847086 | 1986-04-04 | ||
| JP078471/1986 | 1986-04-04 | ||
| JP9301986 | 1986-04-21 | ||
| JP093019/1986 | 1986-04-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1277239C true CA1277239C (en) | 1990-12-04 |
Family
ID=27302728
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000532772A Expired - Fee Related CA1277239C (en) | 1986-04-04 | 1987-03-23 | Treatment of diseases caused by retroviruses |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US4840941A (en) |
| EP (1) | EP0240098A3 (en) |
| AU (1) | AU597635B2 (en) |
| CA (1) | CA1277239C (en) |
| MY (1) | MY101207A (en) |
| OA (1) | OA08581A (en) |
| PH (1) | PH25964A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0012115A1 (en) * | 1978-12-04 | 1980-06-11 | Ciba-Geigy Ag | Pharmaceutical compositions for topical treatment of virus infections |
| EP0066379A3 (en) * | 1981-05-15 | 1983-06-08 | Riker Laboratories, Inc. | Composition for combating herpes virus |
| US4374832A (en) * | 1981-12-28 | 1983-02-22 | American Cyanamid Company | Modulators of the complement system comprising polyhexaose arylene sulfate derivatives |
| US4522814A (en) * | 1982-11-05 | 1985-06-11 | Raphael Pappo | Composition of matter from Cryptosiphonia woodii useful for the treatment of herpes simplex virus |
| US4590181A (en) * | 1982-12-20 | 1986-05-20 | Board Of Regents Of The University Of Nebraska | Synthetic immunoregulators and methods of use and preparation |
| US4710493A (en) * | 1984-08-14 | 1987-12-01 | Albert Landsberger | Therapeutic agent for the use in cancer treatment |
| FR2570081B1 (en) * | 1984-09-10 | 1987-01-09 | Pf Medicament | MEMBRANE POLYSACCHARIDES USEFUL IN PARTICULAR AS A MEDICAMENT AND PROCESS FOR THEIR PREPARATION |
| DE3601136A1 (en) * | 1986-01-16 | 1987-07-23 | Max Planck Gesellschaft | INHIBITORS OF REVERSE TRANSCRIPTASE FOR PROPHYLAXIS AND THERAPY OF RETROVIRUS INFECTIONS IN MAMMALS |
-
1987
- 1987-01-14 EP EP87300282A patent/EP0240098A3/en not_active Ceased
- 1987-03-23 CA CA000532772A patent/CA1277239C/en not_active Expired - Fee Related
- 1987-04-02 OA OA59102A patent/OA08581A/en unknown
- 1987-04-03 PH PH35103A patent/PH25964A/en unknown
- 1987-04-03 AU AU71074/87A patent/AU597635B2/en not_active Ceased
- 1987-04-03 MY MYPI87000426A patent/MY101207A/en unknown
-
1988
- 1988-01-15 US US07/144,131 patent/US4840941A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| EP0240098A3 (en) | 1989-05-10 |
| EP0240098A2 (en) | 1987-10-07 |
| AU7107487A (en) | 1987-10-08 |
| MY101207A (en) | 1991-08-17 |
| OA08581A (en) | 1988-09-30 |
| AU597635B2 (en) | 1990-06-07 |
| US4840941A (en) | 1989-06-20 |
| PH25964A (en) | 1992-01-13 |
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