ANTIVIRAL COMPOSITION BASED ON EXTRACTS OF DURVILLAEA ANTARCTICA USEFUL FOR TREATING LESIONS CAUSED BY ALPHAHERPESVIRINAE VIRUS

Abstract

Composition with antiviral activity comprising water-soluble extracts of Durvillaea antarctica substantially free of alginates and a carrier or vehicle, and the method of obtaining it. Where this composition serves to prepare an antiviral drug of topical application, to prevent or treat skin or oral lesions caused by herpes viruses of the subfamily Alphahehrpesvirinae, such as herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella zoster virus (VZV), resistant or not to acyclovir (ACV).

Claims

1. A composition with antiviral activity wherein it comprises water-soluble extracts of Durvillaea antarctica substantially free of alginates and a carrier or vehicle.

2. The composition according to claim 1 wherein the water-soluble extract of Durvillaea antarctica substantially free of alginates is present in a concentration between 0.05 to 50 mg/mL.

3. The composition according to claim 2 wherein the water-soluble extract of Durvillaea antarctica substantially free of alginates, corresponds only to the protein fraction of the extract.

4. Process of preparation of the composition of the claim 1 wherein it includes the following steps: a) mixing ground biomass of Durvillaea antarctica with water in a ratio of 1 to 5% w/v, at a temperature between 15 and 45° C. and stirring for 2 to 24 hours; b) separating the residual biomass from the water-soluble supernatant; c) removing the alginates present in solution obtaining the water-soluble phase, which constitutes the extract which is optionally mixed with a carrier or vehicle.

5. Process according to claim 4 wherein in step b the biomass is separated by centrifugation or filtration.

6. Obtaining process according to claim 4 wherein in step c the alginates are removed by precipitation with absolute ethanol in a ratio of 1:1 ethanol to extract, and then centrifuged or filtered to separate the precipitated alginates.

7. Obtaining process according to claim 6 wherein ethanol is eliminated by evaporation.

8. Obtaining process according to claim 7 wherein optionally the alginate-free extract is freeze-dried.

9. Obtaining process according to claim 8 wherein the freeze-dried alginate-free extract is resuspended when used in water or buffer solution at pH 7.

10. Obtaining process according to claim 4 wherein additionally a fractionation is carried out, separating the protein fraction by using ammonium sultafo in a ratio of 1:1 weight/volume.

11. Use of the composition containing water-soluble extracts of Durvillaea antarctica substantially free of alginates, according to claim 1 or its protein fraction wherein it serves to prepare an antiviral drug.

12. Use according to claim 11 wherein the drug is for topical application.

13. Use according to claim 11 wherein the drug allows to prevent or treat skin or oral lesions caused by herpes virus of the subfamily Alphahehrpesvirinae.

14. Use according to claim 11 wherein the drug allows to improve the aesthetics of skin or oral lesions caused by herpes virus of the subfamily Alphahehrpesvirinae.

15. Use according to claim 13 wherein viruses of the subfamily Alphahehrpesvirinae are selected between herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella zoster virus (VZV), resistant or not to Aciclovir (ACV).

Description

DESCRIPTION OF THE FIGURES

[0016] FIG. 1: Cell viability assay. HeLa cells were continuously treated with water-soluble extract of alginate-free Durvillaea antarctica at concentrations 0.49-125 mg/mL for 24 hours and then incubated for one hour at 37° C. with AlamarBlue® (resazurin) and quantified by colorimetry on black wall plates and transparent background. The figure shows the percentage of viable cells for each extract concentration. As controls, cells treated with vehicle (saline phosphate, PBS) and cells treated with 75% ethanol (lethal) are incorporated. Significant cell death values were obtained for any concentration greater than 7.81 mg/ml by one-way ANOVA (*p<0.05; **p<0.01; ***p<0.001). The CC50 calculated by linear regression is 25 mg/ml.

[0017] FIG. 2: Expression of the green fluorescent protein gene (GFP reporter) in HeLa cells infected with A-B. HSV-1 in the presence of 1 mg/ml of alginate-free water-soluble extract of Durvillaea antarctica, or C. HSV-2 in the presence of different concentrations (0.01, 0.05; 0.1, 0.25 and 5 mg/m L) of water-soluble extract of Durvillaea antarctica free of alginates . In A. the percentage of positive GFP cells (HSV-1 positive) is shown and in B. the fluorescence intensity associated with GFP (HSV-1) 24 hours post-infection determined by flow cytometry. In C. the fluorescence intensity of GFP (VHS-2) per well determined with a multi-mode reader is shown. Controls: cells with infection but no treatment (TS), cells without infection and without treatment (SI), cells treated with 50 ug/ml of Acyclovir). Statistical analysis performed by one-way ANOVA (***p<0.001).

[0018] FIG. 3: Production of plaque forming units (PFUs) in HeLa cells incubated with different concentrations of water-soluble extract of Durvillaea antarctica free of alginates of the invention treated continuously to viral inoculation with a known amount of PFU of A. HSV-1 or B. VHS-2. The number of PFUs was quantified 24 hours after infection. A group without treatment but with infection (TS), a group without treatment without infection (SI) and treatment with Acyclovir (50 ug/ml) are included as controls. Significant values of antiviral effectiveness were obtained for any concentration greater than 0.05 mg/ml for HSV-1 and 0.25 mg/ml for HSV-2 by one-way ANOVA (*p<0.05; ***p<0.001). The EC50 calculated by linear regression is 0.13 mg/ml for HSV-1 and 0.15 mg/ml for HSV-2.

[0019] FIG. 4: Production of plaque forming units (PFUs) in oral gingival fibroblasts incubated with different concentrations (mg/ml) of water-soluble extract of Durvillaea antarctica free of alginates of the invention treated continuously to viral inoculation with a known amount of PFU of A. HSV-1 or B. VHS-2. The number of PFUs was quantified 24 hours after infection. Included as controls are a group without treatment but with infection (TS), a group without treatment without infection (SI) and treatment with Acyclovir (50 ug/ml). Significant values of antiviral effectiveness were obtained for any concentration greater than 0.05 mg/ml for HSV-1 and 0.1 mg/ml for HSV-2 by one-way ANOVA (*p<0.05; **p<0.01; ***p<0.001).

[0020] FIG. 5: Production of plaque forming units (PFU) in HeLa cells incubated with different concentrations (mg/ml) of water-soluble extract of Durvillaea antarctica free of alginates of the invention treated continuously to viral inoculation with a known amount of PFU of HSV-1 or HSV-2 resistant to Acyclovir (ACV.sup.R). The number of PFUs was quantified 24 hours after infection. Included as controls are a group without treatment but with infection (TS), a group without treatment without infection (SI) and treatment with Acyclovir (50 ug/ml). Significant values of antiviral effectiveness were obtained for any concentration greater than 0.25 mg/ml for HSV-1 and 0.05 mg/ml for HSV-2 by one-way ANOVA (*p<0.05; **p<0.01; ***p<0.001).

[0021] FIG. 6: Production of plaque forming units (PFUs) in heLa cells incubated with different concentrations (mg/ml) of proteins precipitated from water-soluble extract of Alginate-free Durvillaea antarctica which were continuously treated with the protein fraction related to viral inoculation, which was performed with a known amount of PFU of A. HSV-1 or B. HSV-2. The number of PFUs was quantified 24 hours after infection. Included as controls are a group without treatment but with infection (TS), a group without treatment without infection (SI) and treatment with Acyclovir (50 ug/ml). Significant values of antiviral effectiveness were obtained for any concentration greater than 0.001 mg/ml for HSV-1 and 0.0001 mg/ml for HSV-2 by one-way ANOVA (*p<0.05; **p<0.01; ***p<0.001).

[0022] FIG. 7: A. Evaluation of pathological progression associated with HSV-1 skin infection for 11 days. Clinical manifestations produced in the skin of animals (mice, mus musculus) infected with HSV-1 and treated 2 times daily with alginate free Durvillaeae antarctica extract of the invention formulated pharmaceutically with hydroxymethylcellulose in a concentration 5 mg/ml or vehicle were evaluated, only topically. Included as a control is a group treated with Acyclovir in 5% cream format (commercial, bioequivalent) after infection with HSV-1 and a group of animals that was prepared for infection (hair removal and skin erosion), but without infection (Mock). B. Quantification of the area under the curve (AUC) of the data shown in A (except Mock which has values tending to zero),” which integrates the severity and duration of the infection (score and time). One-way ANOVA (**p<0.01).

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides a composition comprising an extract prepared from Durvillaea antarctica, where the extract corresponds to the water-soluble fraction and is substantially free of the gelling agent alginates. This composition has antiviral properties, and is especially useful for the treatment and prevention of lesions caused by viruses of the subfamily Alphahehrpesvirinae (e.g. HSV-1, HSV-2 and VZV), both sensitive and resistant to Acyclovir.

[0024] The inventors have developed a composition based on water-soluble extracts of Durvillaea antarctica, where the extract obtained is substantially free of alginates, a compound with gelling properties. This extract can be used completely, or only its protein fraction, since this fraction maintains the highest activity. The proteins with antiviral activity have not yet been fully identified by the inventors.

[0025] It is important to note that the invention mainly targets the water-soluble or protein fraction, which differs from organic fractions, where high concentrations of organic polymers such as alginates, fucoidans and phenolic compounds, among others, are obtained.

[0026] Thus the invention points to a composition with antiviral activity comprising water-soluble extracts of Durvillaea antarctica substantially free of alginates and optionally a carrier or vehicle. Where the carrier or vehicle may simply be water, buffer, saline, ethanolic solution, or excipients for formulation of pharmaceutically acceptable hydrogels, creams or ointments, such as carboxymethylcellulose, hydroxymethylcellulose, glycerol, propylene glycol or others available in the technique, optionally together with preservatives or stabilizers.

[0027] Where the water-soluble extract of Durvillaea antarctica substantially free of alginates or their protein fraction is present in a concentration between 0.05 to 50 mg/mL. Especially between 0.1 to 25 mg/mL, and more especially preferred between 1 and 10 mg/mL.

[0028] In a second aspect, the invention refers to the process of the composition preparation, which comprises the following steps: [0029] (a) mixing ground biomass of Durvillaea antarctica with water in a ratio of 1 to 5% w/v, at a temperature between 15 and 45° C. and stiring for 2 to 24 hours; [0030] (b) separating the residual biomass from the water-soluble supernatant; [0031] c) removing the alginates present in solution, obtaining the water-soluble phase, which constitutes the extract which is optionally mixed with a carrier or vehicle.

[0032] Where in step (b) the biomass is separated by centrifugation or filtration, or any other available technique. And in step (c) the alginates are removed for example by precipitation with absolute ethanol in a ratio of 1:1 ethanol to extract, and then centrifuged or filtered to separate the precipitated alginates, or by any other technique available for this purpose.

[0033] In one embodiment the added ethanol can be removed by evaporation, especially by evaporation at reduced pressure, for example, at 175 mBar. Once the ethanol is removed, the extract of the invention is available and which can be used directly, or optionally can be frozen and freeze-dried, to then be resuspended in water or pH 7 buffer.

[0034] Optionally, a precipitation of the proteins in the extract is carried out, to obtain the protein fraction, for example, by precipitation with ammonium sulfate in a ratio of 1:1 ammonium sulfate to extract. Where this protein fraction maintains the greatest effectiveness of the extract of the invention. In this way the extract of the invention can be used in different degrees of purification or fractionation, maintaining its activity, where all these variants are part of the present invention.

[0035] Finally, the invention points to the use of the composition containing water-soluble extracts of Durvillaea antarctica substantially free of alginates or their protein fraction, to prepare an antiviral drug. Where in a preferred embodiment the drug is for topical application for skin and mucous membranes. This medicine helps prevent or treat skin lesions caused by viruses of the subfamily Alphaherpesvirinae (e.g. HSV-1, HSV-2 and VZV), sensitive or resistant to Acyclovir.

[0036] The composition of the invention can also be used as an additive with antiviral properties to prevent the transmission of viruses of the subfamily Alphahehrpesvirinae in compositions that are in contact with the skin or mucous membranes, such as lipsticks, cosmetics in general, toothpastes, mouthwashes, chewing gum or oral health candies (such as propolis candies). As well as in other hygiene and body care products such as soap, talcs, anti-wrinkle creams, moisturizers, bronzers, shampoo, conditioner, both for human and animal care. Or in any other article or composition that may come into contact with a body region infected with virus of the subfamily Alphahehrpesvirinae.

[0037] The antiviral composition of the invention can be used to relieve the symptoms of herpes virus infection and/or to improve the aesthetics of skin or oral lesions produced by herpes simplex virus or varicella zoster virus.

[0038] Surprisingly, the inventors have found that the extracts of Durvillaea antarctica, as described in this invention, decrease by approximately 96% and 94% the infection of HSV-1 and HSV-2 viruses, respectively on human cells (HeLa cells at 2.5 mg/ml) in in vitro experiments.

[0039] The inventors have performed tests to determine the selectivity index (SI) of the composition of the invention in vitro, based on tests of reduction in the number of plate-forming units (PFU) after viral infection and treatment. This index corresponds to the value obtained from the CC50/EC50 ratio. The higher the value of the CC50/EC50 ratio (>1), the greater the therapeutic potential of antiviral compounds, since it means that they have high antiviral activity, over adverse effects on the viability of host cells (substrate cells).

[0040] On the one hand, the CC50 value (cytotoxic concentration 50%) in culture is calculated, which corresponds to the concentration of algae extract that causes the death of 50% of host cells in the culture. On the other hand, the EC50 value (effective concentration 50%) is calculated to determine the concentration of extract that inhibits 50% of viral activity. These assays are shown in example 6, included below, and gave for the alginate-free durvillaea antarctica extract of the invention an approximate value of 192 for HSV-1 and 167 for HSV-2 (based on results of viability and effectiveness in HeLa cells). For reference, the selectivity index of Acyclovir, the most commonly used drug to treat herpes virus skin lesions, is 300-340.

[0041] The inventors also developed in vivo tests, where the composition of the invention significantly decreased the severity of the injury caused by HSV-1, and decreased the time and severity of infection with respect to the untreated control, which is exposed as the integration of these two parameters (area under the curve, AUC). It is significant to indicate that Acyclovir, although it reduced the injury time was less effective in decreasing its severity compared to the alginate-free Durvillaea antarctica extract of the invention. The AUC of Acyclovir is greater than the AUC of the alginate-free extract of the invention

[0042] For the expert in the technique it will be evident that the antiviral properties of the composition of the invention, although they have been demonstrated in the examples for the HSV-1 and HSV-2 viruses, are extrapolable to other viruses of the subfamily Alphahehrpesvirinae, for example, VZV and herpes viruses that affect the skin or mucous membranes of the human or animal body.

[0043] As demonstrated in the examples included below, the invention shows an equivalent effectiveness in vitro in inhibiting the production of infectious plaques of herpes simplex virus, sensitive or resistant to Acyclovir, making it especially useful for the control of the latter viruses.

[0044] Examples are described below to better understand the scope of the protected invention.

EXAMPLES

Example 1

Obtaining the Extract of D. antarctica

[0045] Specimens of the macroalgae Durvillaea antarctica collected in Caleta Chome (Biobío Region, Chile) were washed with drinking water for the elimination of mineral salts, sand and epiphytic organisms. Subsequently, the macroalgae were freeze-dried for 5 days at a temperature of −70° C.±1° C., ground and sieved in a 0.5 mm sieve. The sieved dry biomass powder was stored in vacuum-sealed plastic bags.

[0046] Subsequently, 3.00 g of freeze-dried biomass was measured and taken to an Erlenmeyer flask. 120 mL of deionized water was added and kept in agitation, in an orbital agitator, at 200 RPM at a temperature of 30° C. for 4 h.

[0047] It was then centrifuged at 4,000 RPM for 10 min and the decanted residual biomass was removed. Obtaining the water-soluble extract of the algae, which must be subjected to additional purifications to eliminate alginates and wall polysaccharides from the algae, which may be present.

[0048] Alginate precipitation was performed by adding absolute ethanol to the crude water-soluble extract of Durvillaea antarctica in a 1:1 ratio. Due to the change in polarity of the solution of extracts, alginates precipitated.

[0049] Subsequently, to achieve a good separation of the alginates, the dissolution of algal extracts with ethanol was centrifuged at 4,000 RPM for 10 min obtaining the water-soluble extract substantially free of alginates.

[0050] Finally, ethanol was removed from the water-soluble phase of the extract by evaporation of ethanol at reduced pressure (175 mBar).

Example 2

Cytotoxicity Test

[0051] To assess whether Durvillaea antarctica extract has toxic effects on human cells, a cell viability assessment was performed after being incubated in the presence of different concentrations of the alginate-free extract of Durvillaea antarctica obtained in Example 1.

[0052] Continuous treatments were performed on HeLa cells with the alginate-free Durvillaea antarctica extract at concentrations of 0.1-125 mg/mL for 24 hours. Subsequently, the cells were incubated for one hour at 37° C. with AlamarBlue® (reagent based on resazurin), and then evaluated by a colorimetric quantification assay the percentage of viable cells for each condition compared to the control without treating with the extract. The results are shown in FIG. 1, and show that there was incremental toxicity of the alginate-free extract of the invention on human cells at concentrations greater than 7.81 mg/mL.

Example 3

Evaluation of Antiviral Activity of Water-Soluble Extracts In Vitro by Evaluating the Expression of a Reporter Gene Encoded in HSV Genomes

[0053] To evaluate the antiviral activity of the alginate-free water-soluble extracts of the invention, obtained in example 1, HeLa cells (ATCC® CCL-2™, human cervix epithelial cells) were used as target cells to analyze HSV-1 and HSV-2 infection. These cells have the advantage of being human and therefore it is expected that the results obtained in this model resemble what would happen in a physiological context of natural infection.

[0054] HeLa cells were treated with water-soluble extract of alginate-free Durvillaea antarctica obtained in example 1, prior to infection with varying extract concentrations (mg/ml) and inoculated with known amounts of plaque-forming units (PFUs) of HSV-1 K26GFP or HSV-2 ZAG(333), viral strains that contain in their genome the gene that codes for green fluorescent protein (GFP).

[0055] After 24 hours post-infection, the number of green fluorescence-positive cells derived from GFP expression (HSV-1) or the green fluorescence intensity derived from GFP expression (HSV-2) were quantified by flow cytometry or in a multimode plate reader, respectively. FIG. 2 shows the results, where the data is shown, expressed as the number of green fluorescence positive cells derived from GFP expression (HSV-1) or the green fluorescence intensity derived from GFP expression (HSV-2) as quantified by flow cytometry or in a multimode plate reader, respectively. The extract of the invention resulted in a decrease of more than 95% of the fluorescence of HSV-1 GFP and more than 91% of the fluorescence of HSV-2 GFP for the condition of treatment with 1 mg/ml of water-soluble extract of alginate-free Durvillaea antarctica.

Example 4

Inhibition of Infectious Plaque Production

[0056] HeLa cells were treated with different concentrations (mg/mL) of water-soluble extract of alginate-free Durvillaea antarctica, obtained in example 1, continuously with respect to the challenge with HSV with a known amount of HSV-1 K26GFP or HSV-2 ZAG(333). Within 24 hours of infection, the number of plaque forming units (PFUs) present in each well was determined. The results are shown in FIG. 3, where it is seen that the treatment with water-soluble extract of alginate-free Durvillaea antarctica of the invention decreases the production of PFUs in the cultivation.

[0057] A trial similar to the previous one was performed, but on another cell type, healthy human oral gingival fibroblasts, to evaluate the response in the oral cavity, where herpes virus infections frequently occur. The results are shown in FIG. 4, where a similar result of the water-soluble extract of alginate-free Durvillaea antarctica obtained in example 1 on these human primary cells infected with HSV-1 or HSV-2 is observed.

[0058] On the other hand, a test was carried out in a similar way to what has already been described, on HeLa cells, which were incubated with different concentrations (mg/ml) of water-soluble extract of alginate-free Durvillaea antarctica obtained in example 1, continuously with respect to viral inoculation, with a known amount of PFU of HSV-1 or HSV-2 resistant to Acyclovir (ACV®).

[0059] The results show that the extract of the invention is able to decrease the production of PFUs in the cultivation, see FIG. 5, and therefore, the water-soluble extract of alginate-free Durvillaea antarctica of the invention is effective against HSV-1 or VHS-2 (ACV®). As in previous trials (FIGS. 3 and 4), the number of PFUs was quantified 24 hours after infection.

[0060] Treatments with Acyclovir (50 ug/ml), group without treatment but with infection (TS) and group without treatment without infection (SI) were included as controls in all these experiments.

Example 5

Selectivity Index (IS)

[0061] 5.1 Cytotoxic Concentration 50% (CC50)

[0062] To determine cell viability after the addition of increasing concentrations of alginate-free algae extracts, the cytotoxic concentration 50% (CC50) of the water-soluble extract of alginate-free Durvillaea antarctica was determined, obtained according to Example 1. The viability of HeLa cells was evaluated 24 hours after being treated with increasing concentrations of the extract of the invention between 0.49 to 31.25 mg/ml, as described in example 2. The results of FIG. 1 allow the calculation of the concentration of extract that induces cell viability 50% (CC50), this being 25 mg/mL.

[0063] 5.2 Effective Concentration 50% (EC50)

[0064] The effective concentration 50% (EC50) antiviral of the water-soluble extract of alginate-free Durvillaea antarctica of the invention corresponds to the formation of plaque-forming units (PFU) after the continuous addition of the extract with respect to the challenge with HSV and then a known amount of HSV-1 K26GFP or HSV-2 ZAG(333) 24 hours after infection, evaluated in example 4. The results shown in FIG. 3 allow the calculation of that treatment with 0.13 mg/ml and 0.15 mg/ml of water-soluble extracts of alginate-free Durvillaea antarctica decreases by 50% the production of HVS-1 and HSV-2 PFUs in the culture, respectively, these being the EC50 values for the extract with each type of virus.

[0065] 5.3 Selectivity Index (SI)

[0066] Based on the data obtained from CC50 and EC50 in HeLa cells described above for HSV-1 and HSV-2, it is possible to determine approximate selectivity index values (CC50/EC50) of 192 and 167 for HSV-1 and HSV-2, respectively with the water-soluble extract of Durvillaea antarctica of the invention.

Example 6

Antiviral Activity Against Herpes Simplex Virus in Fractions of the Water-Soluble, Alginate-Free Durvillaea antarctica Extract

[0067] To determine the nature of the antiviral activity in the water-soluble extracts of alginate-free Durvillaea antarctica of the invention a precipitation of proteins was made from the water-soluble, alginate-free Durvillaea antarctica extract obtained in example 1 with ammonium sulfate in a 1:1 weight/volume propotion and then these were resuspended in saline buffer solution (PBS) and their antiviral activity was evaluated.

[0068] As shown in FIG. 6, proteins precipitated from the water-soluble, alginate-free Durvillaea antarctica extract exhibit significant antiviral activity against HSV-1 and HSV-2, at a lower concentration than the alginate-free parent extract, indicating that the ability to inhibit the replication of herpes simplex virus in the extract is mainly due to its protein component.

Example 7

Formulation for Topical Application

[0069] The extract of the invention obtained in example 1 was incorporated at a final concentration of 5 mg/mL, in a pharmaceutical base of hydrogel composed of hydroxymethylcellulose. The resulting formulation can be used directly on a skin lesion to reduce the adverse effects of HSV-1 skin infection.

Example 8

Antiviral Activity In Vivo

[0070] For the ability of the water-soluble, alginate-free Durvillaea antarctica extract of the invention to inhibit the progression of pathological damage after skin infection by HSV-1, animals (mice) were infected with HSV-1 and followed for 11 days, being treated 2 times a day, 24 h after infection, either with the composition of the invention (formulation for topical application obtained in example 7, or Acyclovir cream 5% for clinical use (commercially available, bioequivalent). In FIG. 7, it is shown that the treatment with the formulation contained in the invention reduces the pathology significantly compared to the group treated with the vehicle when considering, both severity and duration of the pathology (integrated with the value of the area under the curve of the graphs, AUC) (FIGS. 7A and 7B). Treatment with the water-soluble extract of alginate-free Durvillaea antarctica of the invention gave a better in vivo result than treatment with 5% Acyclovir bioequivalent in cream.