SE448604B - PHARMACEUTICAL PREPARATION CONTAINING HEPARIN, DEXTRANSULPHATE OR POLYVINYL SULPHATE AND ZINCION - Google Patents

Description

448 604 and plaque test. The samples were diluted in Hanks' solution + 0.05% albumin applied to the cell layer of the chicken embryo fibroblasts. After 90 minutes of virus absorption, the cultures were washed twice, added with 4 ml of "LY-Agaroverlay" {Hanks' solution with 0.5% lactalbumin hydrolyzate and 0.01% "Yeastolate" (yeast extract)} and then incubated at 3500 until counting. of plaques (after 96 hours) was performed.

Table Ia Preparation, ng / ml: ZHSO4 Heparin sodium salt * .7 H 2 O. 33 100 0 5.10 aï 5.04 5.00 33 4.89 4.76 4.95 100 4.76 2.60 2.62 300 4.68 <1.00 <1.00 a) Virus content: The values denotes log 10 of the number of PFU / ml ("Plaque Forming Units / pro ml"). i: 160 IU / mg (from Bíofac AS, Copenhagen, Denmark) Table Ib Polysulphate salt Concentration in ZnSO4-7H20 ug / ml Concentration in ng / ml 0 11 '33 100 Inte: 1 0 6.06 a) 5.33 4.06 4.65 Potassium salt of 11 5.34 3.20 2.84 2.50 polyvinyl sulfate.

Molecular weight 33 5.27 _2.95 2.39 2.27 about 8-104 100 4.78 2.74 2.30 2.17 Sodium salt of 11 5.39 4.72 4.63 3.74 dextran sulfate.

Molecular weight 33 5.22 4.05 3.32 2.69 about 5-105 100 4.61 3.14 2.39 2.54 Sodium salt of 11 5.49 4.54 4.30 3.69 dextran sulfate.

Molecular weight 33 5.30 4.05 3.62 3.02 about 2-106 100 4.67 3.43 2.95 2.90 a) Compare table Ia. 10 448 604 2. Inhibition of plaque formation by "HVH2 / Ang." in chicken embryo fibroblasts Cell layer was infected with 100 PFU "HVH2 / Ang." under 90 min. Each time 4 ml of the preparations incorporated in "LY-Agaroverlay" (with S% feed serum, 0.5% "OXO-L-28-Agar", without diethylaminoethyldextran) were applied to the infected cell cultures and incubated at 3500 until counting of plaques (after 96 hours] was performed.

Table 11a Preparation, ng / ml: ZnSO4.7H2O Heparin sodium salt * 0 1.5 3 6 0 100 a) sz s? 76 2-3 b) 2 ~ 3 2-3 1-2 3 104 65 57 42 1-3 1 ~ 2 0,5-1 0,5 6 64 31 11 2 lZ 0,5-1 0,5 0, 5 12 43 8 Z 0.5-1 0.5 0.5 0.5 25 15 5 2 1 0.5 0.5 0.5 0.5 a) Average plaque count from S bowls for each concentration in% of the plaque number of the control test. b) Plaque size ø in mm (approx.). * 160 IU / mg ("Biofac"). 10 15 448 604 4 Table IIb Polysulfate Salt Concentra- ZnSO4.7H2O tion in K. . / ml oncentration 1 Hg lig / ml 0 3 6 12 24: mer o maa) 100 92 s? 66 Potassium salt of polyvinyl- _ 1,5 100 89 87 69 57 â: lšató4Molecular weight1 88 75 59 48 9 6 43 33 7 5 1 Sodium salt of dextran- 1,5 102 103 96 81 34 sulphate.

Molecular weight approx. 5-105 ä go ïâ 7š 61 g Sodium salt of 1.5 96 96 94 75 52 dextran sulphate.

Moiekyivikt ca 2-106 ä gå 128 1? Eye 5% a) Compare Table IIa. Average plaque count from 6 bowls for each concentration. With the number of plaques, their diameter also decreases from about 2-3 mm in the control test to <0.5 mm in the tests with the most effective combination, 3. Inhibition of replication of "HVH2 / Ang." in "VBRO" cells at the beginning of pre-infectious treatment Confluent single layers of "VERO" cells were washed with "F15-Medium" {"Minimum Essential Medium (Eag1e)", Gibco Bio. Cult Ltd. Paísley, Scotlandl and then coated with each batch 4 ml of the various concentrations of the preparations in "F15-Medium". The cultures were infected after 60 min with 1000 PFU "HVH / Ang." in 0.1 ml of medium. After incubation for 20 hours at 35 ° C, the condition of the cells was assessed microscopically and the virus content of the cell lysates (cells with 1 ml of bidistilled water twice frozen and thawed) was determined by titration on chicken embryo fibroblasts (plaque formation). 10 15 20 25 448 604 5 Table III Preparation, ng / ml Sodium ZnSO .7H O Heparin salt * 4 Z 0 6 o 4,581) 1.84 6 3.59 <1.00 12 3.32 <1.00 25 2.82 <1.00 1) Log 10 PFU / ml cell lysate * 160 IU / mg ("Biofac").

Conclusions drawn from the results of the three series of tests The above test results clearly show the synergism of the combinations according to the invention of zinc sulphate (ZnSO 4 .7H 2 O) and heparin-sodium salt according to Tables Ia, IIa and III. one according to Table Ia. In particular, virus-inactivation-tested concentrations were practically ineffective, while combining each time one third of the maximum concentration of the individual components already reduced the virus content of the chicken embryo fibroblasts by 102. Table IIa shows, among other things. a. that heparin-sodium salt has only a weak effect, but that nevertheless both heparin-sodium salt and also zinc sulphate in the lowest concentration together with each concentration of the second component shows a higher effect than the next highest With a combination of 6 ng / ml zinc sulphate and 3 pg / ml the heparin-sodium concentration of the second component alone. salt already has the same effect as with 25 pg / ml zinc sulphate alone, and with 6 ug / ml zinc sulphate and 6 ng / ml heparin sodium salt as well as with 12 pg / ml zinc sulphate and 3 ng / ml heparin sodium salt almost the same effect as with a combination of 25 ng / ml zinc sulphate and 6 ng / ml heparin sodium salt. Virus replication is only moderate according to Table III by means of zinc sulphate at the highest concentration of 25 ng / ml, but is with heparin in the only tested concentration of 6 ng / ml, corresponding to the highest in the other study series, due to the strong absorption inhibitors clearly inhibit the action of the heparin sodium salt; however, all tested combinations show an effect at least 10 times stronger than the individual components.

The results given in Tables Ib and IIb also show a synergism in the combination of zinc sulphate with the sodium salts of two dextran sulphates with different molecular weights as well as with the potassium salt of polyvinyl sulphate. Of particular note are the results in the form of virus inactivation according to Table I, in that the components alone in the highest concentration of 100 μg / ml reduced the virus content by only about 101 ° 5, ie. equal or less strongly than the combination of each one-ninth of the maximum concentration of the individual components and that on the other hand the combination of each one-third of the maximum concentration of the individual components reduced the virus content by 2'5 to 103.5. Table 11b shows that the effect of polysulphate salts, which alone are clearly stronger, ie. with about 10 t.o.m. at the highest concentration of 6 pg / ml did not show any strong effect, significantly increased by the addition of zinc sulphate in concentrations of 6 ng / ml and 12 ug / ml, which alone are ineffective respectively. almost inactive. 4. Effect on by infection with "HVH2 / Ang." Synergism can also be determined in the active substances combined according to the invention in vivo by infection with "HVH2 / Ang." caused genital herpes in female guinea pigs at 72 hours after intravaginal infection (stage with clear symptoms) started treatment according to B. Lukas et al., Arch.

Ges. Virus research. Ai, 153-155 (1974) et al., 1-11 (1975) described methodology. The results of the corresponding double-blind experiments have been given in the following Table IV. f; 10 15 20 448 604 7 Table IV 'Preparation Effect on local symptoms b) No. HZ a)% animals with% symptoms- Para- Exitus IU% n more than 66% free animals lys% regression day 4 day 4 7 9 11 9 11 14 18 25 1. - F) 108 oos 6 z 3 5 915 39 24 2 - -d) 35 0 11 17 37 14 25 40 48 51 23 11 3 32 - 8 0 O 25 37 0 12 25 37 62 0 4 160 - - 7 36 0 11 39 61 30 30 41 58 66 3 5 - 0,5 10 0 0 10 10 0 10 30 50 70 20 0 6 - 1,0 18 0 5 5 17 0 17 28 33 33 11 5 7 32 0.5 10 10 10 30 40 0 10 50 40 40 20 0 8 32 1.0 10 0 0 30 20 20 20 30 50 80 0 0 9 160 0.5 19 0 5 84 94 53 72 94 94 94 10 5 10 160 1.0 46 6 37 63 87 43 74 78 87 90 4 0 H = heparin sodium salt, 160 IU / mg ("Biofac") Z = ZnSO4.7H2O. a) Number of animals> 10 is the total number of experimental animals of several experiments with 8 or 10 animals each time. b) Day 0 is the day of starting treatment 72 hours after the infection.

The treatment consisted of intravaginal application of 0.1 ml of the test preparation twice daily for 5 consecutive days. (c) Infected control animals which did not receive any treatment. d) Infected control animals, which were treated with the gel base used in all preparations, which contained 0.1% polyoxyethylene sorbitan monooleate.

The results of the comparative studies show that the administration of only heparin sodium salt or zinc ions as active substance with a suitable gel base only moderately enhances the beneficial effect of the latter, since after treatment with only one of the active substances mentioned during the study period end no more than 70% of the experimental animals are asymptomatic, while after treatment with the gel base alone, about 50% of the experimental animals are asymptomatic. In contrast, the combined administration of zinc ions and heparin sodium salt in the concentration range of the invention resulted in an almost complete cure of the experimental disease state, in that at the end of the study period 18 of 19 and 19, respectively. 42 of 46 experimental animals, ie. at least 90% were asymptomatic. In addition, even with the administration of heparin-sodium salt alone, the early onset of a curative effect after administration of the combination becomes even more pronounced, while the effect after administration of zinc ions alone only becomes apparent at a later time.

In addition, the synergism of the above active substances is enhanced when a preparation base, in particular a gel base, is used which contains one or more polyoxyethylene sorbitan fatty acid esters, such as polyoxyethylene sorbitan monostearate and / or in particular polyoxyethylene sorbitan monolaurate or in the first hand polyoxyethylene lens sorbitan monooleate.

The pharmaceutical preparations according to the invention contain the active ingredients defined above, preferably in combination with pharmaceutical carrier materials suitable for topical application.

As forms of preparation according to the invention, tinctures, solutions, creams, ointments and, above all, gels are particularly relevant.

Tinctures and solutions mostly show an aqueous-ethanolic resp. aqueous base, which is provided with b1.a. polyalcohols, such as propylene glycol or glycerol and / or low molecular weight polyethylene glycols, as humectants to reduce evaporation, and optionally re-greasing substances, such as fatty acid esters of low molecular weight polyethylene glycols, i.e. in aqueous-ethanolic mixture soluble, lipophilic substances, as a substitute for fatty substances extracted from the skin with ethanol, and possibly additional auxiliaries and additives, in addition to customary, as stated below, preservatives e.g. also already mentioned polyoxyethylene sorbitan fatty acid esters, such as polyoxyethylene sorbitan monolaurate or polyoxyethylene sorbitan monooleate.

Creams are oil-in-water emulsions, which contain more than g 50% water. for example lauryl, cetyl or stearyl alcohol, fatty acids, e.g. pal- As an oil base, fatty alcohols, mitic or stearic acid, liquid to solid waxes, e.g. isopropyl myristinate, wool wax or beeswax, and / or hydrocarbons, e.g. vasclin ("petrolatum") or paraffin oil. As emulsifiers, surfactants with preferably hydrophilic properties, such as corresponding nonionic emulsifiers, e.g. fatty acid esters of polyalcohols or ethylene oxide adducts thereof, such as polyglycerol fatty acid esters or polyoxyethylene sorbitan fatty acid esters ("Tweens"), further polyoxyethylene fatty alcohol ethers or fatty acid esters, or corresponding ionic emulsifiers, such as alkali metal alkaloids; sodium lauryl sulphate, sodium cetyl sulphate or sodium stearyl sulphate, which are commonly used in the presence of fatty alcohols, e.g. cetyl alcohol or stearyl alcohol. Additives to the water phase include agents which reduce the dehydration of the cream, e.g. polyalcohols, such as glycerol, sorbitol, propylene glycol and / or polyethylene glycols, further preservatives, odorants, etc.

Ointments are water-in-oil emulsions which contain up to 70%, but preferably from about 20% to about 50% water or aqueous phase. As the fat phase, hydrocarbons are primarily in question, e.g. Vaseline, paraffin oil and / or hard paraffins, which for the improvement of water holding capacity preferably contain useful hydroxy compounds, such as fatty alcohols, or esters thereof, e.g. cetyl alcohol or wool wax alcohols, resp. wool wax. sorbitan fatty acid esters ("Spans"), e.g. sorbitan oleate and / or sorbitan isostearate. Additives to water phases include moisture retention agents, such as polyalcohols, e.g. glycerol, Emulsifiers are corresponding lipophilic substances, such as Opropylene glycol, sorbitol and / or polyethylene glycol, as well as preservatives, odorants, etc.

Gels are in particular aqueous solutions of the active substances in which gel formers are dispersed and swollen, preferably those from the group of cellulose ethers, such as methylcellulose, hydroxiotylcellulose or carboxymethylcellulose, or from the group of vegetable hydrocolloids, such as sodium alginate, traction alginate, gummi arabicum. In addition, the gels preferably contain water-retaining agents from the group of poly-, alcohols, such as propylene glycol, glycerol and / or low molecular weight polyethylene glycols, as well as wetting agents, e.g. polyoxyethylene sorbitan fatty acid esters, such as polyoxyethylene sorbitan monostearate, monolaurate or monoolcate in concentrations of about 0.02-5%. As additional additives, the gels contain customary preservatives, e.g. benzyl alcohol, phenethyl alcohol, phenoxyethanol, p-hydroxybenzoic acid lower alkyl esters such as methyl and / or propyl esters, sorbic acid or organic mercury compounds such as merthiolate.

The compositions according to the invention may, in addition to customary preservatives, also contain additional biological, e.g. , antiflogistic or antimicrobial, such as antibacterial, antifungal or even antivirally active substances, such as "Flumethasone", neomycin, gentamycin, lactic acid or "Miconazole".

The preparations according to the invention are prepared in a manner known per se.

In particular, the present invention relates to topically useful antiviral compositions which contain pharmaceutically acceptable salts of acid sulfated polysaccharides or acid sulfated polymers, such as heparin, and zinc ions in a ratio of 1 mg to 0.18-18 mg, in in particular 1 mg to 0.18-4.5 mg, and optionally polyoxyethylene sorbitan monolaurate and / or monooleate.

For heparin salts, the above amounts refer to those of 160 IU / mg, and for salts of other heparin, the same IU amounts should be used. The zinc ions are added in the form of corresponding amounts of a dissociable zinc compound, e.g. 0.8-80 mg resp. 0.8-20 mg ZnSO 4 .7H 2 O. Correspondingly topically useful preparations, in particular gels, and furthermore tinctures, aqueous solutions, creams or ointments, contain, for example, per g or ml 0.1-5 mg, in particular 0.25-3 mg of a pharmaceutical preparation. absorbable salt of an acid sulphated polysaccharide or acid sulphated polymer, for example 16-800 IU, in particular 40-480 IU of a corresponding salt of heparin, and 0.18-18 mg of zinc ions, corresponding to e.g. about 0.8-80 mg of ZnSO4.7H2 O and optionally in addition 0.2-50 mg of polyoxyethylene sorbitan monolaurate and / or monooleate.

Particularly preferred are a content of 80-320 IU of a pharmaceutically acceptable salt of heparin, 0.45-4.5 mg of zinc ions and optionally in addition 0.5-10 mg of polyoxyethylene sorbitan monolaurate and / or monooleate per g or ml.

Instead of a pharmaceutically acceptable salt of heparin, an antiviral with respect to the action of an equivalent salt of another acid sulphuted polysaccharide or acid sulphated polymer, such as one of the following sude, in particular dextran sulphate resp. polyvinyl sulfate may be used, with a content of, per g or ml, 0.25-2 mg being especially preferred.

By acidic sulphated polysaccharides is meant those in which monovalent sulfuric acid residues -SO 2 -OH are attached to oxygen atoms and, as is the case with heparin, to nitrogen atoms. Such acid sulfated polysaccharides may be of natural origin, such as heparin, chondroitin sulfate (chondroitin sulfuric acid) or carrageenan, or prepared by sulfation of natural or partially degraded polysaccharides, such as sulfated amylopectins, sulfated dextrans, sulfated polyglucose or sulfated polyglucoses. These substances are used in the form of their pharmaceutically acceptable salts, such as potassium or in particular sodium salts.

As such, the sodium salt of heparin may be mentioned as a common commercial form of the latter, furthermore potassium and magnesium salts of heparin and sodium salts of sulphated dextrans with different molecular weights, e.g. 4, 8,104, 5,105 or 2,106. Acid sulphated polymers, such as acid sulphated polyvinyl alcohols (polyvinyl sulphates) with different molecular weights, which in turn are used in the form of pharmaceutically acceptable salts, such as sodium or especially potassium salts. In general, as pharmaceutically acceptable salts, sodium and potassium salts are of particular importance.

The zinc ions can, instead of in the form of zinc sulphate, also be added in the form of another dissociable zinc compound, such as zinc chloride, zinc acetate or zinc citrate, or as zinc salt of an acid or of another substance of an acidic nature and own biological e.g. . antibacterial or antiphlogistic properties, such as zinc-sudoxicam (zinc salt of 4-hydroxy-Z-methyl-N- (2-thiazolyl) -1,2-benzothiazine-3-carboxamide-1,1-dioxide). In this case, it is not necessary that the zinc salts added in the carrier medium used are completely dissociated, but it is sufficient that a content of zinc ions within the concentration data given above is present together with non-dissociated salt.

The compositions according to the invention are particularly suitable for the treatment of genital herpes, herpes dermatitis and labial herpes. For the treatment of both the former, gels according to the invention are applied as early as possible, e.g. by means of a tube or applicator, 2-3 times daily, and for the treatment of herpes labialis several times daily in the diseased places of the body until the symptoms subside resp. for healing. Aqueous solutions can e.g. be used for rinsing diseased body cavities, e.g. for the treatment of herpes gingivostomatitis, or for the treatment of herpes keratoconjunctivitis. The following examples describe the preparation of some typical application forms, of course without thereby limiting the scope of the invention in any way.

Example 1. To prepare 10.0 liters of gel, 200.0 g of highly viscous sodium carboxymethylcellulose and 50.0 g of polyoxyethylene sorbitan monostearate ("Tween 60") are mixed with 1000 g of glycerol and 6.5 liters of "Aqua conservans" and the mixture of sheep swell to a homogeneous mucus. Then a solution of 1.6,106 IU of heparin sodium salt (eg 10.0 g of "Heparin Biofac"), 50.0 g of zinc sulphate heptahydrate and 10.0 g of polyoxyethylene sorbitan monooleate ("Tween 80") is added. ") in 2 liters" Aqua conservans ". Finally, fill with "Aqua conservans" to 10 liters, mix thoroughly and fill the resulting gel into tubes.

By "Aqua conservans" is meant an aqueous solution of 0.07% p-hydroxybenzoic acid methyl ester ("Methylparaben") and 0.0§% p-hydroxybenzoic acid propyl ester ("Propylparaben"). "Tween 60" and "Tween 80" are trademarked trademarks of the above compounds from ICI of America Inc., Stamford, Connecticut 06904.

Instead of 50.0 g of ZnSO 4 .7H 2 O, 100.0 g can also be used and otherwise follow the above procedures.

Example 2. In analogy to Example 1, a gel is prepared using 5.0 g of sodium salt of dextran sulfate having a molecular weight of about 4.8.104 instead of heparin.

Example 3. In analogy to Example 1, a gel is prepared using 2.5 g of sodium salt of dextran sulfate having a molecular weight of about 2,106 instead of heparin.

Example 4. In analogy to Example 1, a gel is prepared using 2.5 g of potassium salt of polyvinyl sulfate having a molecular weight of about 8,104 instead of heparin.

Example 5. To prepare 10.0 liters of gel, 200.0 g of highly viscous sodium carboxymethylcellulose and 50.0 g of polyoxyethylene sorbitan monostearate ("Tween 60", compare Example 1) are mixed with 1000 g of glycerol and 6.5 liters. Aqua conservans "and the mixture is allowed to swell to a homogeneous mucus. Then a solution of 5.0 g of sodium salt of dextran sulfate with a molecular weight of about 5,105, 50.0 g of zinc sulfate heptahydrate and 10.0 g of polyoxyethylene sorbitan monooleate ("Tween 80", compare Example 1) in 2.0 liters is mixed. Aqua_conservancy "(compare Example 1). Finally, fill with "Aqua conservans" to 10.0 liters, mix thoroughly and fill the resulting gel into tubes. a) F! na

Claims (12)

l \\ 448 604 lb Eatentkrav Pharmaceutical preparations for topical use, in particular for topical treatment of virus infections, characterized by an antivirally active, synergistic combination of a pharmaceutically acceptable salt of heparin, dextran sulphate or polyvinyl sulphate and zinc ions in an amount ratio of 1 mg: 111 0, 18-la mg. Pharmaceutical preparations according to Claim 1, characterized in that they are in the form of gels, creams, ointments, tinctures or aqueous solutions. Pharmaceutical preparations according to Claim 1, characterized by an additional content of polyoxyethylene sorbitan monolaurate and / or monooleate. Pharmaceutical preparations according to Claim 1, characterized by a content of the pharmaceutically acceptable salts and zinc ions in an amount ratio of 1 mg to 0.18-4.5 mg. Pharmaceutical preparations according to claim 1, characterized in that a content, per g, of 0.1-5 mg of the pharmaceutically acceptable salts and 0.18 to 18 mg of zinc ions. Pharmaceutical preparations according to Claim 1, characterized in that a content, per g, of 0.25 to 3 mg of the pharmaceutically acceptable salts and 0.18 to 18 mg of zinc ions. 7. Pharmaceutical preparations according to claim 1, characterized in that a content, per g, of 80 to 320 IU of a pharmaceutically acceptable salt of heparin and 0.45 to 4.5 mg of zinc ions. Pharmaceutical preparations according to Claim 1, characterized in that a content, per g, of 0.25-3 mg of a pharmaceutically acceptable salt of dextran sulphate and 0.18 to 18 mg of zinc ions. Pharmaceutical preparations according to Claim 1, characterized in that a content, per g, of 0.25-3 mg of a pharmaceutically acceptable salt of polyvinyl sulphate and 0.18 to 18 mg of zinc ions. 10. Pharmaceutical preparations according to any one of claims 1 to 9, characterized in that they contain, as a pharmaceutically acceptable salt now mentioned, a sodium or potassium salt, _ _ ___., _.._... -..___-__ 448 604 14 Pharmaceutical preparations according to Claims 1 to 10, characterized in that an additional content, per g, of 0.2 to 50 mg of polyoxyethylene sorbetan monolaurate and / or monooleate. U Pharmaceutical preparations according to Claims 1 to 10, characterized in that an additional content, per g, of 0.5 to 10 mg of polyoxyethylene sorbitan monolaurate and / or monooleate. -73