DE102005004643A1 - Antiviral combination useful for prevention and therapy of viral diseases, comprising hyaluronic acid sulfate and/or carboxyalkylated derivatives and natural substance or its extract with antiinflammatory and antiviral characteristics - Google Patents

Abstract

Antiviral combinations (A) with mutually reinforcing antiviral activity against coated viruses comprise hyaluronic acid sulfate and/or carboxyalkylated derivatives (I) and a natural substance or its extract with antiinflammatory and antiviral characteristics. Antiviral combinations (A) with mutually reinforcing antiviral activity against coated viruses comprise hyaluronic acid sulfate and/or carboxyalkylated derivatives of formula (I) and a natural substance or its extract with antiinflammatory and antiviral characteristics. R : H, (CH 2) nCOOX or SO 3X; X : H or Na; and n : 1-2. [Image] ACTIVITY : Virucide. MECHANISM OF ACTION : None given.

Description

The The invention relates to antiviral combinations with each other reinforcing antiviral activity against enveloped viruses and their use.

By the development of highly selective nucleoside and nucleotide viruses such as Acyclovir, penciclovir, ganciclovir, brivudine and cidofovir for herpesviruses as well as Zidovudine, Diodanosine and lamivudine for HIV (De Clercq, Nature Rev. Drug Disc., 11, 2002: 13-25) has become a significant Progress in fighting life-threatening viral infections. Under the aspect the increasing resistance of viruses to these agents However, it regarded as urgently needed, novel antiviral effective To develop products for and the manufacture of cosmetic preparations, of microbicides or of To provide medicines. For long-term use of nucleoside and nucleotide viruses, for example, have often become resistant to development across from the said active substances (Arts et al., J. Virol. 1998: 4858-65; Morfin and Thouvenot, J. Clin. Virol. 26, 2003: 29-37). On Reason of the same or similar In principle, cross-resistance also occurs. To such cross resistance to overcome looking for new alternative agents that are antiviral Prophylaxis or therapy can be used. Another disadvantage The said compounds is that they also in the DNA metabolism The infected cells intervene and therefore the danger in itself cause mutagenic, teratogenic or oncogenic effects (Carr, Nature Rev. Drug Disc., 2, 2003: 624-634; Wutzler and Thust, Antivir. Res., 49, 2001: 55-74). Furthermore, none of the antivirals mentioned inhibits viral transmission. Substances or substance combinations that are already in a very early Stage intervene in the viral propagation cycle and attaching For example, to prevent the viruses from reaching the cells would be the palette the available expand antiviral agents. This could in addition to the virus multiplication also Virus spreading is blocked and new infections effectively avoided become.

polyanionic Compounds such as polysulfates, sulfonates or carboxylates are able to interact with viral glycoproteins or competitively the virus-receptor binding z. B. of herpesviruses or HIV (Norkin, Clin Microbiol Rev., 8, 1995: 293-315; Stone, Nature Rev. Drug Disc., 1, 2002: 977-85; Coen and Schaffer, Nature. Rev. Drug Disc., 2, 2003: 278-88). The size Molecular weight of these molecules, bad However, pharmacokinetics as well as inadequate effects in vivo contributed to help with this Substances have not yet been used.

It is known that sulfated polysaccharides are suitable for the treatment of viral diseases ( DE 39 21 761 . DE 40 21 066 . EP 464759 . EP 0940410 . US 5861383 or WO 2001013910).

The sulfation of polysaccharides or their substituted derivatives belongs to the prior art and can be done in different ways (such as in US 27 15 09 , WO 880 02 11, JP 719 67 02 . DE 39 21 761 or DE 698 03 362 disclosed). A preferred method of preparing hyaluronic acid sulfates is the sulfation of hyaluronic acid with sulfur trioxide / pyridine or sulfur trioxide / DMF complexes in an aprotic solvent such as DMF (See Möller, H.-P. Klöcking, P.-J. Müller, J. Med H. Ozegowski, DE 198 13 234, 1998 ). The extent of the reactions on the hydroxyl groups of hyaluronic acid is usually described by the average degree of substitution (DS), which indicates the number of functionalized hydroxyl groups relative to a disaccharide repeat unit of hyaluronic acid. Depending on the number of hydroxyl groups per disaccharide repeat unit of hyaluronic acid, DS values between 0 and 4.0 can be realized. The average degree of substitution (DS) on SO ₃ groups (DS _S ) is between 0.5 and 3.9 for the abovementioned sulfation processes.

Also known is the use of medical or pharmaceutical compositions for herpes treatment based on plant products. In WO 95 110 35 a cosmetic or pharmaceutical preparation with antiviral activity based on a St. John's Wort extract is described, in DE 44 189 76 a phytotherapeutic for the treatment of infections of herpes from extracts of sage, plantain, widespread and mistletoe.

US 65 090 42 describes an antiviral composition containing an extraction product of broad bean.

Also, the antiviral and anti-inflammatory effect of aqueous lemon balm extracts is the subject of patents (such as. HU 208 255 . DE 101 00 889 , WO 2002 055 056 and EP 13 495 61 ), which also mentions the treatment of cold sores.

The writings US 64 755 26 . US 2004 086 575 and US 67 303 29 have zinc-containing compounds in combination with phenolic antioxidants and plant extracts such as balm oil for antiviral use the subject.

A pharmaceutical composition for the treatment of herpes consisting of a quaternary ammonium compound (eg benzalkonium chloride), an antiviral active substance (eg acyclovir) and a plant extract (eg Aloe vera, lemon balm) describes, inter alia, the documents WO 96 243 67, DE 696 24 340 and EP 80 81 68 ,

Synergistic effects of different antiviral compounds have been poorly described. The DD 21 41 92 describes a method for increasing the local biological effect of known antiherpetics by adding phosphocholines to the pharmaceutical preparations containing a conventional antiviral agent, wherein due to increased viral efficacy, the dosage of the antiviral agent could be reduced. The added phosphocholines are themselves not or only very weakly antivirally effective and there are no known combinations of phosphocholines with polyionic compounds. EP 049 73 41 has a synergistic composition with antiviral activity, consisting of a fibroblast growth factor and sulfated polysaccharides such as heparin, dextran sulfate, pentosan polysulfate or dermatan sulfate to the object. A major disadvantage of these combinations is the fact that the provision of the fibroblast growth factors consuming and costly procedures are required, which severely limit the use of the combinations described for economic reasons.

Even though, As described above, a variety of substances or substance combinations with antiviral properties is known, due to the partially unsatisfactory In vivo efficacy of these compounds, their sometimes not safe toxic side effects and increased resistance of viruses known active ingredients a great Need for new antiviral compounds.

task Therefore, it is the object of the present invention to provide novel compositions ready to provide, even in the smallest concentrations already as antivirals for effective prophylaxis and therapy of viral infections are suitable and have no toxic side effects in their use demonstrate.

R = H, (CH₂)_nCOOX, SO₃X
X = H, Na
n = 1, 2
in der X identisch oder unabhängig voneinander H und/oder Na darstellen sowie R H, (CH₂)_nCOOX und/oder SO₃X sowie n = 1 oder 2 bedeuten, wobei, jeweils bezogen auf die in Formel 1 angegebene Disaccharid-Wiederholungseinheit der durchschnittliche Substitutionsgrad der (CH₂)_nCOOX-Gruppierung (DS_CA) zwischen 0,1 und 1,0 und der durchschnittliche Substitutionsgrad der SO₃X-Gruppierung (DS_S) zwischen 0,5 und 3,9 beträgt sowie der nichtsubstituierte Anteil R = H sich jeweils zu 4 – (DS_CA + DS_S) ergänzt mit einem antiviral wirkenden Pflanzenextrakt oder dessen pflanzlichen Inhaltsstoffen, vorzugsweise mit einem wässrigen Extrakt aus Melisse.The present invention is based on the surprising finding that any combination of antivirally active Hyaluronsäuresulfaten and / or their carboxyalkylierten derivatives with antiviral and additionally anti-inflammatory melissa extracts leads to a tremendous increase in the effect. Surprisingly, even concentrations which show no antiviral activity when used alone, inhibit the HSV-1-induced cytopathic effect very well when combined. The present invention accordingly relates to a combination of hyaluronic acid sulfates and / or carboxyalkylated hyaluronic acid sulfates of the general formula

R = H, (CH ₂ ) _n COOX, SO ₃ X
X = H, Na
n = 1, 2
in which X is identical or independent of each other, represent H and / or Na and also denote RH, (CH ₂ ) _n COOX and / or SO ₃ X and n = 1 or 2, where, in each case based on the disaccharide repeating unit indicated in formula 1 average degree of substitution of the (CH ₂ ) _n COOX grouping (DS _CA ) between 0.1 and 1.0 and the average degree of substitution of the SO ₃ X grouping (DS _S ) is between 0.5 and 3.9 and the unsubstituted portion R = H in each case to 4 - (DS _CA + DS _S ) supplemented with an antiviral plant extract or its vegetable ingredients, preferably with an aqueous extract of melissa.

To provide the preparation of the antiviral combination of the invention as a component the carboxyalkylated Hyaluronsäuresulfate carried by sulfation of carboxyalkylated hyaluronic acids, which according to a method of the prior art, for. B. after DE 39 21 761 , can be synthesized. The subsequent sulfation of the carboxyalkylated hyaluronic acids with the SO ₃ / DMF complex is preferably carried out, since in this case the average degree of substitution of sulfate groups per disaccharide repeat unit of hyaluronic acid (DS _S ) of the carboxyalkylated hyaluronic acid sulfate can be precisely adjusted via a suitable choice of the reaction conditions. A significant advantage of said production process is further the feasibility of the sulfation reaction and the product workup in a homogeneous phase, whereby a uniform sulfation with adjustable DS _S value is made possible.

The Substance combinations according to the invention show a high antiviral activity. They greatly inhibit the multiplication of shrouded Viruses, such as B. herpesviruses.

The Substance combinations according to the invention are therefore for the production of cosmetic dosage forms, microbicides or of medicaments for the prophylaxis and treatment of viral diseases suitable. You can z. B. in the form of an antiviral lip balm or Hydrogels or as microbiocidal vaginal gels find use and because of their composition for the prophylaxis and cure of Serve viral infections. The active substances are the hyaluronic acid derivatives in solid or liquid Form and / or hydrogel in combination with the also antiviral or antiinflammatory natural herbal medicines such as As melissa introduced into the above-mentioned dosage forms.

The combinations according to the invention are thus suitable for the development of antivirals, in comparison to the compounds mentioned in the prior art a completely different Possess mechanism of action with equally good or higher antiviral activity, and can due to these antiviral properties for the production of cosmetic Preparations, microbiocidal vaginal gels and medicines for Combat viral Serve infections. Special uses arise, for example for the prophylaxis and therapy of herpes infections. You can both alone as well as in combination with known antivirals as well as physiologically acceptable Auxiliaries and carriers be applied. Furthermore, you can they are used in topical use as prophylactics and virus transmission prevent.

The following embodiments should the invention in more detail explain, but in no way limit.

1. Synthesis of carboxymethylated hyaluronic acid

Example 1 (CH-1)

2 g (4.98 mmol hyaluronic acid, Na salt, molecular weight about 1 million) are dissolved at room temperature in a protective gas atmosphere in water. 3.5 ml (49.8 mmol) of 40% strength aqueous sodium hydroxide solution (14.3 mol) and, after 15 min, 2.36 g (24.9 mmol) of chloroacetic acid are added. The solution is warmed to 60 ° C and stirred for 2 h. Subsequently, the reaction solution is cooled to room temperature, neutralized, dialyzed and freeze-dried.
Yield: 80%.

Average degree of substitution of carboxymethyl groups (DS _CM , determined by acid-base titration): see Table 1.

2. Synthesis of carboxymethylated hyaluronic acid sulfate

Example 2 (CS-1, 2)

1.15 mmol carboxymethylated hyaluronic acid (DS _CM ~ 0.4) are placed under protective gas at room temperature in 50 ml of DMF and suspended after addition of 1.84 mmol of p-toluene-sulfonic acid for about 30 min. The suspension is cooled to 4 ° C. After addition of molecular sieve A3 and the corresponding amounts of SO ₃ / DMF complex (CS-1: OH / SO ₃ = 1: 3, CS-2: OH / SO ₃ = 1: 5), the reaction mixture is stirred for 1 h. Subsequently, the molecular sieve is filtered off, the polymer is precipitated in 400 ml of acetone and neutralized with sodium hydroxide solution. The precipitate is centrifuged off, washed, dialyzed against water, freeze-dried for 24 h and dried in vacuo at 40.degree.
Yield: 80-90%.

Sulfur content (determined by elemental analysis) and DS _s value: see Table 1.

Example 3 (HS-1, CHS-3-6)

1 g hyaluronic acid or their carboxymethylated derivative (Na salt) and 80 ml of water (bidest.) Are placed in a closed reaction vessel at room temperature until the complete solution the polymer is stirred. The clear solution gets to 0 ° C cooled, with 5 g of ion exchanger (Dowex WX 8, H-form) and 30 min touched. The centrifuged from the ion exchanger polymer solution is by adding 10% ethanolic tributylamine solution pH 9 is set. Subsequently becomes the solution extracted three times with diethyl ether, the solution is freeze-dried and at 40 ° C in oil pump vacuum dried.

500 mg of the ammonium salt of hyaluronic acid or its carboxymethylated derivative thus obtained are dissolved in 50 ml of absolute DMF under argon at room temperature, cooled to 0 ° C. and treated with appropriate amounts of SO ₃ / DMF complex (HS-1: OH / SO ₃ = 1:20, CHS-3, CHS-4: OH / SO ₃ = 1:20, CHS-5: OH / SO ₃ = 1: 5, CHS-6: OH / SO ₃ = 1: 3). It is stirred for 1 h at 0 ° C. The clear reaction solution is precipitated in about 500 ml of acetone and neutralized with ethanolic NaOH. The precipitate is washed several times with acetone, dissolved in water (bidist.), Dialyzed in water and then freeze-dried. The product obtained is dried at 50 ° C in vacuo to constant weight.
Yield: 80-90%.

Sulfur content (determined by elemental analysis) and DS _S value: see Table 1.

3. Examples of use

3.1. Cytotoxicity and antiviral Effect of carboxymethylated hyaluronic acid sulfates

By means of the substances listed in Table 1, the cytotoxic and antiviral effect of carboxylated hyaluronic acid sulfates is to be demonstrated by way of example. Table 1: Examples of synthesized hyaluronic acid derivatives and control substances.

3.2. cytotoxicity

test Description

The cytotoxicity of the test compounds is determined on two day old GMK cell monolayers (Schmidtke et al., J. Virol., Meth., 95: 2001, 133-143). For this purpose, 9 dilutions of the substance are added in 100 ul of test medium. Cells without substance serve as controls. After an incubation period of 72 h at 37 ° C with 5% CO ₂ , further processing takes place. After removal of the cell culture supernatant, the Cell monolayer washed 3 times with 300 μl of physiological saline to remove dead cells. The fixation and staining of the cells is carried out with 50 .mu.l of a 0.3% crystal violet solution for 10 min. After washing 6 times with 300 μl of water, the dyed monolayers are treated for 20 minutes with 100 μl of lysis buffer (0.8979 g of sodium citrate, 1.25 ml of 1N HCl in 98.05 ml of 47.5% ethanol) and the crystal violet is dissolved out , The optical density of the individual wells is determined at 550/630 nm. The optical density of the 6 controls without substance is set 100%. Based on the mean dose-response curve of 3 independent tests, the 50% cytotoxic concentration (CC ₅₀ ) and the maximum tolerated dose (MTD = CC ₁₀ ) are calculated.

test result

The investigated substances and melissa listed in Table 1 proved to be very well tolerated for GMK cells in the concentration range tested. Both CC ₅₀ and MTD were> 200 μg / ml.

3.3. Antiviral effect in the cytopathic effect (zpE) inhibition test

The zpE inhibition assay is performed on 2-day-old GMK cell lawns in 96-well microtiter plates (Schmidtke et al., J. Virol., Meth. 95, 2001: 133-143). After aspirating the cell growth medium, 50 μl of the test substance dissolved in the test medium and a specific virus concentration (HSV-1) in 50 μl of the test medium are added. 6 wells with no virus and 6 wells with virus but no test substance serve as cell or virus control on each plate. In each test, phosphonoformic acid (PFA), which had previously been determined in the plaque reduction assay as 50% and 100% inhibitory (IC ₅₀ = 12.5 μg / ml and IC ₁₀₀ = 125 μg / ml) control, was included , The plates are incubated at 37 ° C and 5% CO ₂ . The development of the cytopathic effect (zpE) is followed microscopically. About 48 hours after infection, when the virus controls show the maximum cytopathic effect and the reference compound (PFA) has about 50 and 100% inhibition respectively, the cells are fixed and stained with 0.3% crystal violet in ethanol / formalin solution. After washing three times and then dissolving out the dye with a lysis buffer, the optical density at 550/630 nm is determined and the antiviral activity is calculated. Based on the mean dose-response curves of 3 independent tests, the 50% inhibitory concentration is determined. Table 2: 50% inhibitory concentration (IC ₅₀ ) of the example substances

test result

The antiviral activity of caboxymethylated hyaluronic acid sulfates is closely related to their degree of sulfation (Table 2). The higher the sulfation, the stronger the demonstrated inhibition of HSV-1-induced zpE in GMK cells and the lower the 50% inhibitory concentration. For the compounds with a sulfur content of> 11% (CHS-3 and CHS-4), IC ₅₀ values of 0.7 μg / ml were determined. These values corresponded approximately to the 50% inhibitory concentration of hyaluronic acid sulfate (HS-1) and are many times lower than that of the control substance PFA. Unsulfated hyaluronic acid or their caboxymethylated derivative (CH-1) are not antivirally effective in the concentration range used up to 100 μg / ml.

3.4. Antiviral effect of HS-1 and CHS-4 in combination with lemon balm

Tabelle 4: Hemmung des HSV-1-induzierten zpE durch kombinierte Behandlung mit CHS-4 und Melisse (Mittelwert aus 3 unabhängigen Versuchen)

For the comparative studies on the antiviral effect of HS-1 and CHS-4 in combination with lemon balm, the zpE inhibition tests already described under point 2 were used. The concentrations used individually and in combination were 12.5, 25, 50, 100 and 200 μg / ml for melissa, 0.1, 0.2, 0.4, 0.8 and 0.6 for HS-1 and CHS-4 1.6 μg / ml. The antiviral effects achieved with HS-1 and melissa or CHS-4 and melissa are summarized in Tables 3 and 4, respectively. Table 3: Inhibition of HSV-1-induced PPE by combined treatment with HS-1 and melissa (mean of 3 independent experiments)

Table 4: Inhibition of HSV-1 induced zpE by combined treatment with CHS-4 and melissa (mean of 3 independent experiments)

Out The data will show that both the combination of hyaluronic acid sulfate with melissa (Table 3) as well as caboxymethylated hyaluronic acid sulfate (Table 4) with lemon balm to a strong increase in antiviral Effect led. Surprisingly even inhibited levels of single drug use did not show any antiviral effect, with combined use very good HSV-1-induced cytopathic effect (in bold).

All in the specification, tables and following claims Features can both individually and in any combination with each other invention essential be.

Claims (14)

Antiviral combinations with mutually enhancing antiviral activity over enveloped Vi consisting of Hyaluronsäuresulfaten and / or their carboxyalkylated derivatives of the general formula

R = H, (CH _{2) n} COOX, SO ₃ XX = H, Na n = 1.2 in which R is H, (CH _{2) n} COOX and / or SO ₃ X and X is H and / or Na, and n = 1 or 2 and a natural product or natural extract with anti-inflammatory and antiviral properties. Antiviral combinations according to claim 1, characterized characterized in that the Hyalurosäuresulfate substitution degrees on sulfate groups in the range of 0.5 to 4.0, based on a disaccharide repeating unit hyaluronic acid, exhibit. Antiviral combinations according to claim 1, characterized in that the carboxyalkylated hyaluronic acid sulfates average degrees of substitution of carboxyalkyl groups in the range from 0.1 to 1.0 and average degrees of substitution on sulfate groups in the range of 0.5 to 4.0, each based on a disaccharide repeating unit hyaluronic acid exhibit. Antiviral combinations according to claim 1, characterized characterized in that the natural product or the natural extract of lemon balm or an extract of lemon balm. Antiviral combinations according to claim 1, characterized in that the hyaluronic acid sulfates and / or their carboxyalkylated ones Derivatives as well as the natural substance or natural substance extract in any mixing ratio available. Antiviral combinations according to claim 1 or 5, characterized in that additionally further antiviral, antibacterial and / or antiinflammatory acting Compounds are included. Antiviral combinations according to claims 1 to 6, characterized in that additionally organic or inorganic Excipients are included. Antiviral combinations according to claims 1 to 7, characterized in that the antiviral combinations liquid, viscous, gel, pasty or particulate Have shape. Use of the antiviral combinations according to one or more of the preceding claims for prophylaxis and therapy of viral diseases. Use of the antiviral combinations according to one or more of the preceding claims for prophylaxis and therapy of viral diseases specifically caused by infection with the Herpes simplex virus are caused. Use of the antiviral combinations according to one or more of the preceding claims as pharmaceutical formulations for the prophylaxis and therapy of viral diseases. Use of the antiviral combinations according to one or more of the preceding claims as cosmetic preparations or cosmetic articles with antiviral properties. Use of the antiviral combinations according to claim 12, characterized in that in the cosmetic preparations or cosmetic articles additional Carrier- and adjuvants are used. Use of the antiviral combinations according to claims 12 or 13, characterized net, that the cosmetic preparations or cosmetic articles are lip balms, hydrogels, vaginal gels or care creams.