EP2675462A2 - Sulfatierte arabinogalaktane, apiogalacturonane und sulfatierte heteroglycane zur behandlung von durch den influenza-virus verursachten erkrankungen - Google Patents

Sulfatierte arabinogalaktane, apiogalacturonane und sulfatierte heteroglycane zur behandlung von durch den influenza-virus verursachten erkrankungen

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Publication number
EP2675462A2
EP2675462A2 EP12702582.3A EP12702582A EP2675462A2 EP 2675462 A2 EP2675462 A2 EP 2675462A2 EP 12702582 A EP12702582 A EP 12702582A EP 2675462 A2 EP2675462 A2 EP 2675462A2
Authority
EP
European Patent Office
Prior art keywords
sulphated
extract
arabinogalactans
polysaccharides
apiogalacturonans
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP12702582.3A
Other languages
English (en)
French (fr)
Inventor
Bruno Lina
Olivier Ferraris
Ho Hong Hai VO
François-Loïc Cosset
Judit SZECSI
Alain Heyraud
Hugues Lortat-Jacob
Julia BARTOLI
Rabia Sadir
Thierry Livache
Benoît DARBLADE
Stéphane HAVET
Silvère BONNET
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Elicityl
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Original Assignee
Centre National de la Recherche Scientifique CNRS
Commissariat a lEnergie Atomique CEA
Institut National de la Sante et de la Recherche Medicale INSERM
Elicityl
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
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Application filed by Centre National de la Recherche Scientifique CNRS, Commissariat a lEnergie Atomique CEA, Institut National de la Sante et de la Recherche Medicale INSERM, Elicityl, Commissariat a lEnergie Atomique et aux Energies Alternatives CEA filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP2675462A2 publication Critical patent/EP2675462A2/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A61K36/05—Chlorophycota or chlorophyta (green algae), e.g. Chlorella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/888—Araceae (Arum family), e.g. caladium, calla lily or skunk cabbage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the present invention relates to the technical field of influenza viruses. More specifically, the invention relates to the use of certain polysaccharides as a medicament for the treatment, preventive or curative, of an influenza virus.
  • Influenza is a respiratory infection caused by influenza viruses. It is observed all over the world and reappears each year in winter epidemic waves. Today, it is the second leading cause of infectious mortality after pneumonia.
  • the influenza viruses responsible for human pathologies are influenza A and B viruses. While influenza B viruses circulate as lineages, influenza A viruses are classified into viral subtypes according to their properties. antigens of the two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA).
  • HA hemagglutinin
  • NA neuraminidase
  • the circulating viruses in humans and responsible for seasonal epidemics are A (H1N1) and A (H3N2) viruses.
  • Vaccination is, for now, the only effective way to protect populations from influenza viruses. Nevertheless, alternative solutions, in the form of anti-viral drugs are the subject of many investigations.
  • WO 2009/027057 describes the use of carrageenans in a pharmaceutical composition for the prophylaxis and treatment of respiratory viral diseases caused by orthomyxoviruses, and in particular by influenza A and B viruses.
  • heparin Polymer formed by the chain of disaccharide units composed of a sulphated iduronic acid and a sulphated galactosamine pentosane polysulfate: Polymer of sulphated D-xylan
  • mannan sulfate sulphated mannan polymer.
  • US patent application 2010/0015248 proposes a composition for the treatment of colds and influenza, which comprises a mixture of acetaminophen, diphenylhydramine, dextromethorphan, arabinogalactan, vitamin C, zinc, extract of olive oil, resveratrol and elderberry extract. It is stated in this document that arabinogalactan, vitamin C and zinc are present to stimulate the immune system. Olive oil extract, resveratrol and elderberry extract are used as anti-viral agents. Acetaminophen, diphenylhydramine and dextromethorphan are used to relieve influenza symptoms.
  • the document WO 98/11778 describes drug compositions for the treatment of various viral diseases comprising an inhibitor of the microbial infection at the origin of the disease to be treated, in the form of an isolate of different plants.
  • the influenza virus is cited among a list of different viral infections, but the invention is rather directed to the treatment of the herpes virus to which all the examples and results relate.
  • the present invention is based on the discovery by the inventors, and those totally unexpectedly, that certain particular polysaccharides exhibited anti-viral activity against influenza viruses.
  • the subject of the invention is thus polysaccharides chosen from sulphated arabinogalactans, apiogalacturonans and heteroglycan sulphates for their use as medicaments in the preventive or curative treatment of a disease caused by an influenza virus.
  • “Treatment” means any prophylactic or suppressive therapeutic measure of a disease caused by an influenza virus leading to a desirable clinical effect or any beneficial effect, including in particular the suppression or reduction of one or more symptoms, regression, slowing or stopping the progression of the disease associated with it.
  • Diseases caused by an influenza virus are usually called flu.
  • the invention relates to polysaccharides chosen from sulphated arabinogalactans, apiogalacturonans and sulphated heteroglycans for their use as an antiviral agent against the influenza virus in a medicament intended for the treatment or prevention of a virus. influenza.
  • the present invention relates to the use of a polysaccharide chosen from sulphated arabinogalactans, apiogalacturonans and heteroglycan sulphates, for the preparation of a medicament intended for the treatment, preventive or curative, of an influenza virus. .
  • the polysaccharide selected from sulfated arabinogalactans, apiogalacturonans and heteroglycan sulfates acts as an active ingredient, i.e., it is used for its anti-viral activity against influenza viruses.
  • the anti-viral activity against the influenza viruses is ensured by at least 70%, preferably at least 90%, and preferably at least 95% by the sulphated arabinogalactans, apiogalacturonans or heteroglycan sulphates present.
  • said drug or pharmaceutical composition containing at least one polysaccharide chosen from sulphated arabinogalactans, apiogalacturonans and heteroglycan sulphates contains no other compound, than sulphated arabinogalactans, apiogalacturonans and heteroglycans sulphates. , exhibiting significant anti-viral activity.
  • a combination of polysaccharides chosen from sulfated arabinogalactans, apiogalacturonans and heteroglycans sulfates By compound exhibiting a significant anti-viral activity is meant a compound which causes a significant inhibition of the replication of the target virus.
  • an inhibition of at least 25%, preferably at least 50%, and preferably at least 80% at a concentration of 250 g / ml, in a test for inhibiting the replication of the influenza virus target such as those described in paragraph 1 of the examples below.
  • the inventors have demonstrated that polysaccharides belonging to the family of sulphated arabinogalactans, apiogalacturonans or heteroglycans sulphates had an influence on the replication cycle of the influenza viruses and made it possible to inhibit the infection. of cells susceptible to infection. These sugars have also been shown to inhibit the entry of influenza viruses into viral pseudoparticles (VLPs).
  • VLPs viral pseudoparticles
  • Sulphated arabinogalactans are water-soluble sulphated polysaccharides composed of Galactose units linked together by glycoside bonds Beta 1-3. This main chain may have branches of Arabinose or Galactose units via Alpha 1-6 glycosidic linkages. These units may have several Arabinose and / or Galactose units linked together by glycoside bonds 1-3 or 1-6. Arabinoses or Galactoses can show sulfations on secondary alcohol groups.
  • the arabinose / galactose ratio preferably belongs to the range from 3/7 to 2/1, and corresponds, for example, to 1/1, 1/2 or 2/1.
  • sulfated arabinogalactans having a size belonging to the range of 1 000 to 2 ⁇ 10 6 g / mol, preferably in the range of 3000 to 1.1 ⁇ 10 6 g / mol and / or a corresponding average charge of 2 to 50%, preferably 5 to 40% of the sulfate groups.
  • Apiogalacturonans are polysaccharides which can be sulfated, soluble in water formed by a linear chain of Galacturonic Acid units linked together by alpha 1-4 glycosidic linkages. This main chain may have Apiose motifs linked by Beta 1-2 glycoside linkages. These motifs may have several Apiose motifs linked together by glycoside bonds Beta 1-5.
  • apiogalacturonans having a size in the range of 10 000 to 10 6 g / mol, preferably in the range of 50 000 to 700 000 g / mol and / or a corresponding average charge of 2 to 20%, preferably 4 to 12% of the sulfate groups.
  • the sulphated heteroglycans used in the context of the invention are water-soluble polysaccharides formed by a linear chain of alpha-1-3 and terminal-linked Galactose units, 1-4-linked xylose units and units. arabinose related in 1-4.
  • the Galactose units may have C 6 sulphates and the arabinose units C 3 sulphates.
  • sulfated heteroglycans having a size in the range of 500,000 to 5 ⁇ 10 6 g / mol and, for example, approximately 10 6 g / mol and / or a charge, will preferably be used. corresponding average of 5 to 30%, preferably 13 to 22% of the sulfate groups.
  • sulphated arabinogalactans, apiogalacturonans and sulphated heteroglycans used in the context of the invention may be prepared by chemical synthesis or will preferably be extracted from a natural source such as algae or plants. In particular, they can correspond to:
  • any extraction method well known to those skilled in the art may be used.
  • the methods for extracting complex sugars by aqueous or organic solvents, followed or not by purification by selective precipitation, ionic interactions, filtration, etc. can be implemented.
  • an extraction comprising a step of extraction with water and a precipitation step with an alcohol such as ethanol.
  • influenza virus is meant all influenza viruses, including influenza viruses human, avian, equine, porcine and feline.
  • influenza viruses can be selected from types A, B and C.
  • influenza virus can be of type A and in particular correspond to the subtype strains H1N1, H2N2, H3N2, H4N2, H4N6, H5N1, H5IM2, H7N1. , H7N7 and H9N2.
  • influenza virus is selected from type B viruses, A-H5N1, A-H7N1 and A-H3N2.
  • the present invention also relates to the pharmaceutical compositions can be administered to animals, particularly humans, comprising, in combination with at least one pharmaceutically acceptable excipient according to the European Pharmacopoeia in particular 7 th edition:
  • excipients present in the medicaments and pharmaceutical compositions according to the invention are chosen according to the pharmaceutical form and the desired mode of administration.
  • the polysaccharides may be administered in unit dosage forms. administration, in admixture with conventional pharmaceutical carriers, to animals and humans for the prophylaxis or treatment of the above diseases.
  • Suitable unit dosage forms include oral forms, such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual, oral, intratracheal, intranasal, subcutaneous, intramuscular, intracartilage or intravenous administration and forms of rectal administration.
  • oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual, oral, intratracheal, intranasal, subcutaneous, intramuscular, intracartilage or intravenous administration and forms of rectal administration.
  • the compounds according to the invention can be used in creams, ointments or lotions.
  • the drug or composition generally contains a therapeutically effective amount of polysaccharides selected from sulfated arabinogalactans, apiogalacturonans and heteroglycan sulfates.
  • therapeutically effective amount is meant any amount of a composition that improves one or more of the parameters characteristics of the condition being treated.
  • the dose of polysaccharide varies, for example, between 0.001 and 100 mg per kg of body weight per day. Nevertheless, in the context of the invention, the pharmaceutical compositions also include homeopathic compositions.
  • the main active ingredient is mixed with a pharmaceutical carrier, such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic or the like.
  • a pharmaceutical carrier such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic or the like.
  • the tablets can be coated with sucrose, a cellulose derivative, or other suitable materials or they can be treated in such a way that they have prolonged or delayed activity and continuously release a quantity predetermined active ingredient.
  • a preparation in capsules is obtained by mixing the active ingredient with a diluent and pouring the resulting mixture into soft or hard gelatin capsules.
  • compositions containing a polysaccharide according to the invention may also be in liquid form, for example, solutions, emulsions, suspensions or syrups, and in particular in a form suitable for oral or intranasal administration, for example.
  • suitable liquid carriers may be, for example, water, organic solvents such as glycerol or glycols, as well as their mixtures, in varying proportions, in water.
  • a preparation in the form of a syrup or elixir or for administration in the form of drops may contain the active ingredient together with a sweetener, preferably acaloric, methylparaben and propylparaben as antiseptic, as well as a flavoring agent. and a suitable dye.
  • a sweetener preferably acaloric, methylparaben and propylparaben as antiseptic, as well as a flavoring agent. and a suitable dye.
  • Water-dispersible powders or granules may contain the active ingredient in admixture with dispersants or wetting agents, or suspending agents, such as polyvinylpyrrolidone, as well as with sweeteners or scavengers disgust.
  • the drugs or compositions comprising at least one polysaccharide chosen from sulphated arabinogalactans, apiogalacturonans and heteroglycan sulphates are for human or veterinary use.
  • the polysaccharide may be in a food additive.
  • the antiviral drugs and antiviral compositions against the influenza virus according to the invention do not contain olive oil, resveratrol or elderberry extract, in particular when or they contain an arabinogalatane as previously defined.
  • the present invention also relates to a method for treating humans or animals, and in particular chickens, comprising administering the use of a polysaccharide chosen from sulphated arabinogalactans, apiogalacturonans and sulfated heteroglycans to fight, as a preventive or curative, a disease caused by an influenza virus.
  • a polysaccharide chosen from sulphated arabinogalactans, apiogalacturonans and sulfated heteroglycans
  • the present invention also relates to the use of a polysaccharide chosen from sulphated arabinogalactans, apiogalacturonans and heteroglycan sulphates in a food composition in which said polysaccharide is used for preventive or curative control, against a disease caused by an influenza virus.
  • the food composition comprises:
  • the food composition does not contain olive oil, resveratrol or elderberry extract.
  • Obtaining the Codium Fragile extracts presented in the invention consists, in a first step, in the depigmentation of the crushed algae by an organic solvent, in particular ethanol.
  • the depigmented residue is then extracted, at ambient temperature, in an aqueous medium for a few hours at 24 hours.
  • the extract thus obtained can be improved by using polysaccharide purification methods, such as selective precipitation with organic solvents such as ethanol and / or ionic interaction, and / or ultrafiltration dialysis.
  • the extract thus obtained mainly presents the described structure of sulphated arabinogalactan (Marina Ciancia et al., 2007 - International Journal of Macromolecules 41 (2007) 641-649), namely a linear chain of galactoses linked together by glycosidic bonds Beta 1-3.
  • This main chain can present branchings of Arabinose or Galactose units by glycoside bonds Alpha 1-6. These units may have several Arabinose and / or galactoses linked together by glycoside bonds 1-3 or 1-6.
  • Arabinoses or Galactoses can show sulfations on secondary alcohol groups.
  • the monosaccharide composition of the extracts obtained in the context of this invention is confirmed by assays of monosaccharides by the HPAEC PAD chromatography technique following total acid hydrolysis.
  • the extracts show Arabinose and Galactose (3/7 to 1/1).
  • the characteristic signals of the glycosidic bonds of arabinogalactan are evidenced by Nuclear Magnetic Resonance of Proton.
  • Assays of sulphate groups by the method of Barium Chloride make it possible to assess the rate of sulphation of the extracts between 18 and 30% by weight.
  • Size Exclusion Chromatography (SEC) of the polymers makes it possible to estimate the average molecular weight of sulfated arabinogalactan polysaccharides present in the extracts. This, depending on the samples, is in the range of from about 3,000 to about 1.103 ⁇ 4 / mol.
  • the seaweed can be depigmented.
  • 50 g of Codium Fragile in Powder can be depigmented by 4 successive washes with an organic solvent such as ethanol. Each wash consists of suspending the 50 g of algae powder in one liter of pure ethanol and shaking for 2 hours at 400 rpm with a temperature of 20 ° C. After each wash, the extraction residue is isolated by filtration on Whatman paper. This operation is renewed three times. Following these depigmentation steps, the extraction residue is dried overnight in an oven at 40 ° C.
  • the depigmentation residue can be extracted in an aqueous medium.
  • the residue corresponding to 50 g of depigmented Codium Fragile is resuspended in DI of deionized water, stirred for 16 h at 600 rpm and at room temperature. After 16h, the soluble extract is isolated by centrifugation (7000 rpm 4 ° C 40 minutes). This aqueous extract is called A aqueous extract.
  • the sample extract No. 98 is obtained by desalting the aqueous extract A by tangential ultrafiltration on a membrane of 10 kDa. Desalting is obtained after diafiltration with the passage of 6 volumes of deionized water, ie 6L. The ultrafiltration retentate corresponds to sample No. 98.
  • the extracted sample No. 79 is obtained by a new refining step of the sample No. 98 by tangential ultrafiltration membrane 0.2 ⁇ . This ultrafiltration is carried out at a concentration of 10 g / L with a diafiltration in deionized water of 10 volumes. The fraction retained corresponds to the extracted sample No. 79.
  • the aqueous extract A can also be treated by tangential ultrafiltration on a membrane of 0.2 ⁇ . This ultrafiltration is carried out at a concentration of 10 g / L with a diafiltration in deionized water of 10 volumes. The fraction retained corresponds to the sample extracted No. 141.
  • the algae can be directly extracted with an aqueous solvent.
  • 50 g of Codium Fragile in Powder are dissolved in 1 L of deionized water with stirring of 400 rpm, at room temperature overnight.
  • the soluble extract is isolated by centrifugation (8000 rpm, 4 ° C, 60 minutes). This aqueous extract is called aqueous extract B.
  • Extracted samples Nos. 109 and 238 are obtained by precipitation with an organic solvent, such as ethanol.
  • an organic solvent such as ethanol.
  • the aqueous extract B is precipitated with 50% v / v of pure ethanol.
  • the precipitate is isolated by centrifugation (5000 rpm, 4 ° C, 10 minutes).
  • the sample extract No. 294 is obtained by a new refining step of the sample No. 109 or 238 by ionic interactions.
  • This step purification by ionic interactions is carried out on an anion exchange support, in particular polysaccharide microbumbles formed of crosslinked chitosan.
  • the sample No. 109 or 238 is dissolved in a solution of pH 4-5 by acidification, in particular with hydrochloric acid.
  • the sample is then brought into contact with the ion exchange support, then rinsed with deionized water, preferably acidified, in particular with hydrochloric acid to reach a pH of 4-5.
  • the sugar of interest is released from the support with a basic buffer, in particular a bicarbonate / carbonate buffer, preferably adjusted to a pH of about 10.
  • a basic buffer in particular a bicarbonate / carbonate buffer, preferably adjusted to a pH of about 10.
  • the soluble fraction is isolated, preferentially by filtration.
  • This fraction is then desalted, in particular by precipitation with an organic solvent, such as ethanol.
  • the fraction is concentrated, preferably to a concentration of 1 to 10 g / l polysaccharides, then precipitated with 50% v / v of pure ethanol.
  • the precipitate is isolated by centrifugation (5000 rpm, 4 ° C., 10 minutes) and then oven-dried.
  • the fraction thus obtained is sample No. 294.
  • Extracted sample No. 242 is obtained by a new step of refining sample No. 109 or 238 by tangential ultrafiltration on a 0.2 ⁇ m membrane. This ultrafiltration is carried out at a concentration of 10 g / l with a diafiltration in deionized water of 10 volumes. The fraction retained corresponds to the extracted sample No. 242.
  • the sample extracts No. 152 is obtained by acid hydrolysis, in particular with hydrochloric acid, of the extracted samples No. 109 or 238 followed by a selection of the molecular mass on two ultrafiltration membranes 3 and 10 kDa.
  • the mass selection is carried out by a first tangential ultrafiltration on a 10 kDa membrane made at 10 g / L with a diafiltration of 10 volumes with deionized water.
  • the permeate is then passed through tangential ultrafiltration on a 3 kDa membrane, with a first concentration phase of a factor of 10, then a diafiltration of 10 volumes with deionized water.
  • the retentate of this second tangential ultrafiltration on a membrane of 3 kDa constitutes the sample extracted No. 152.
  • Extracted samples Nos. 237 and 239 are obtained by treating the aqueous extract B with bentonite followed by ethanol precipitation.
  • the aqueous extract B obtained from the extraction of 50 g of Codium Fragile in 1 L of water is treated by the addition of 10 g of bentonite previously resuspended in 100 ml of deionized water. The solution is stirred and then left to stand for about 1 hour at room temperature.
  • the soluble phase is isolated by centrifugation (8000 rpm 4 ° C 15 minutes).
  • the isolated soluble phase is precipitated with an organic solvent, in particular with final 50% v / v ethanol.
  • the precipitate is isolated by centrifugation (5000 rpm, 4 ° C., 10 minutes) and constitutes the extracted samples No. 237 and 239.
  • the extracted sample No. 240 is obtained by a new step of refining sample No. 237 or 239 by tangential ultrafiltration on a 0.2 ⁇ m membrane. This ultrafiltration is carried out at a concentration of 10 g / L with a diafiltration in deionized water of 10 volumes. The fraction retained corresponds to the extracted sample No. 240.
  • the extracts obtained present, for the most part, the described structure of sulphated arabinogalactan (Marina Ciancia et al., 2007 - International Journal of Biological Macromolecules 41 (2007) 641-649), namely a linear chain of galactoses linked together by glycosidic bonds Beta 1-3.
  • This main chain may have branches of Arabinose or Galactose units via Alpha 1-6 glycosidic linkages. These units may have several Arabinose and / or Galactose units linked together by glycoside bonds 1-3 or 1-6.
  • Arabinoses or Galactoses can show sulfations on secondary alcohol groups.
  • Obtaining the Zostera marina extracts presented in the invention consists in a first step in the depigmentation of the plant with an organic solvent, in particular ethanol.
  • the depigmented residue is then extracted under heat in an aqueous medium for a few hours at 24 hours.
  • the extraction residue undergoes a second extraction in an acidic medium, in particular in TFA medium.
  • the extraction polysaccharide is obtained by precipitation according to the pH of the filtrate obtained previously. This polysaccharide can be improved using polysaccharide purification methods such as selective precipitation with organic solvents such as ethanol, and / or ultrafiltration dialysis.
  • the extract thus obtained mainly presents the described structure of apiogalacturonan (Véronique BRUDIEUX- 2007 PhD thesis - University of Limoges - Extraction, enzymatic modification and chemical characterization of new pectic structures Application of the structure / activity relationship to dermocosmetics. ), namely a linear chain of Galacturonic Acids linked together by alpha 1-4 glycosidic bonds. This main chain may have branched Apiose motifs linked by Beta 1-2 glycoside bonds. These motifs may have several Apioses linked together by glycoside bonds Beta 1-5.
  • the monosaccharide composition of the extracts obtained in the context of this invention is confirmed by assays of monosaccharides by the HPAEC PAD chromatography technique following total acid hydrolysis and by gas chromatography technique after derivatization of the monosaccharides by acetylation.
  • the extracts present Galacturonic Acid and Apiose.
  • the characteristic signals of the glycosidic linkages of apiogalacturonan are evidenced by Nuclear Magnetic Resonance of Proton, in particular the chain of Galacturonic Acid in alpha 1-4 after partial acid hydrolysis of Apioses.
  • Assays of sulfate groups by Barium Chloride method make it possible to evaluate the sulfation rate of the extracts between 4 and 12% by weight.
  • the SEC of polymers makes it possible to estimate the average molecular mass of sulfated arabinogalactan polysaccharides present in the extracts, which varies from 50,000 to 700,000 g / mol.
  • the first method makes it possible to obtain the extracted sample No. 138.
  • the marine plant, Zostera marina powder is washed with water. 40 g of Zostera marina powder are dissolved in 1 L of deionized water, about 5 hours at 95 ° C with stirring of 500 rpm. The wash residue is isolated by centrifugation (8000 rpm, 4 ° C, 20 minutes).
  • the resulting pellet is then extracted in an acid medium, for example with TFA.
  • the wash residue is redissolved in 0.5 N TFA, stirred at 500 rpm for about 2 hours at 25 ° C.
  • the soluble fraction is isolated by centrifugation (8000 rpm, 4 ° C, 30 minutes).
  • the soluble fraction thus isolated is neutralized to pH 7.5, a precipitate appears.
  • the precipitate is isolated by centrifugation (8000 rpm, 4 ° C., 30 minutes).
  • This precipitate is returned to solution with IL of deionized water.
  • This solution is then precipitated with ethanol previously placed at -20 ° C., up to 60% final v / v.
  • the precipitate formed is isolated by centrifugation (7000 rpm, 4 ° C., 10 minutes).
  • the precipitate is dissolved again at a rate of 10 g / L to be desalted by tangential ultrafiltration on a 650 Da membrane. A diafiltration of 10 volumes is carried out with deionized water. The retentate obtained is lyophilized to give the extracted sample No. 138.
  • the pellet obtained during the first extraction with hot water can also be extracted in acidic medium with hydrochloric acid.
  • the washing residue is redissolved in IL of 0.5N HCl, stirred at 500 rpm. for about 2 hours at 25 ° C.
  • the soluble fraction is isolated by centrifugation (8000 rpm, 4 ° C, 30 minutes).
  • the soluble fraction thus isolated is neutralized to pH 7.5, a precipitate appears.
  • the precipitate is isolated by centrifugation (8000 rpm, 4 ° C., 30 minutes).
  • This precipitate is returned to solution with IL of deionized water.
  • This solution is then precipitated with ethanol previously placed at -20 ° C., up to 60% final v / v.
  • the precipitate formed is isolated by centrifugation (7000 rpm, 4 ° C., 10 minutes).
  • the oven-dried precipitate gives the extracted sample No. 296.
  • the second method is an extraction of Zostera marina with oxalate after depigmentation with an organic solvent such as ethanol.
  • 50 g of Zostera marina powder are suspended in IL pure ethanol. The mixture is brought to 78 ° C. with stirring at 500 rpm for approximately 5 hours.
  • the depigmenting residue is isolated by filtration on a Buchner filter of 20-25 ⁇ m and dried in an oven at 40 ° C. overnight.
  • the depigmented marine plant is then dissolved in 1% ammonium oxalate solution with stirring at 750 rpm at 70 ° C. for approximately 2 hours.
  • the soluble extract is isolated by centrifugation (8000 rpm, 4 ° C, 30 minutes).
  • the extracted fraction can be desalted by tangential ultrafiltration on 10 kDa membranes. Diafiltration of about 10 volumes is carried out with deionized water.
  • the retentate is lyophilized to give the extracted sample No. 158.
  • the third method is an extraction of Zostera marina with 3% sodium carbonate after depigmentation with an organic solvent such as ethanol.
  • 50 g of Zostera marina powder are suspended in IL pure ethanol. The mixture is brought to 78 ° C. with stirring at 500 rpm for approximately 5 hours.
  • the depigmenting residue is isolated by filtration on 20-25 ⁇ m Buchner filter and dried in an oven at 40 ° C overnight.
  • the depigmented marine plant is then redissolved in IL 3% sodium carbonate with stirring of 500 rpm at 70 ° C for about 2 hours.
  • the soluble extract is isolated by centrifugation (8000 rpm, 4 ° C, 30 minutes) and then neutralized with hydrochloric acid.
  • the product thus isolated is precipitated with ethanol previously placed in 20 ° C, up to 50% v / v final.
  • the precipitate is isolated by centrifugation (6000 rpm, 4 ° C, 5 minutes).
  • the precipitate can be washed several times, three times for example, with pure ethanol. It is then dried in an oven at 40 ° C overnight.
  • the dried precipitate can then be desalted by tangential ultrafiltration on 10 kDa membranes. Diafiltration of about 10 volumes is carried out with deionized water.
  • the retentate is lyophilized to give the extracted sample No. 160.
  • the retentate may also be precipitated by an organic solvent such as ethanol previously placed at -20 ° C, up to 50% v / v final.
  • the precipitate is isolated by centrifugation at 6000 rpm 4 ° C. for approximately 5 minutes.
  • the precipitate can be washed several times, three times for example, with pure ethanol. It is then dried in an oven at 40 ° C overnight.
  • the dried precipitate constitutes the extracted sample No. 161. Characterization of Zostera marina extracts:
  • the extracts thus obtained mainly present the described structure of apiogalacturonan (Véronique BRUDIEUX - 2007 PhD thesis - University of Limoges - Extraction, enzymatic modification and chemical characterization of new pectic structures Application of the structure / activity relationship to dermocosmetics), namely a linear chain of galacturonic acids linked together by alpha 1-4 glycosidic linkages.
  • This main chain may have apiose motifs linked by Beta 1-2 glycoside bonds. These motifs may have several Apioses linked together by glycoside bonds Beta 1-5.
  • Obtaining the Caulerpa racemosa extracts presented in the invention consists in a first step in the depigmentation of the crushed algae by an organic solvent, in particular ethanol.
  • the depigmented residue is then extracted under heat in an aqueous medium for a few hours at 24 hours.
  • the resulting extract can be improved using polysaccharide purification methods such as selective precipitation with organic solvents such as ethanol and / or ionic interaction, and / or ultrafiltration dialysis.
  • the extract thus obtained predominantly exhibits the described structure of the sulphated heteroglycan (CHATTOPADHYAY Kausik et al., 2006 - Carbohydrate polymers 2007, vol.68, no.3, pp. 407-415), namely galactoses linked in alpha 1-3 and terminal, 1-4-linked xyloses and 1-4 arabinoses.
  • Galactoses can have C 6 sulphates and C3 sulphate arabinoses.
  • the monosaccharide composition of the extracts obtained in the context of this invention is confirmed by assays of monosaccharides by the HPAEC PAD chromatography technique following total acid hydrolysis.
  • the extracts show Arabinose, Galactose and Xylose (10/10 / 0.5 to 13/13/1).
  • the characteristic signals of the xyloarabinogalactan glycoside bonds are evidenced by Proton Nuclear Magnetic Resonance.
  • Assays of sulphate groups by the method of Barium Chloride make it possible to evaluate the rate of sulphation of the extracts between 22 and 13% by mass.
  • the SEC of the polymers makes it possible to estimate the average molecular weight of sulphated arabinogalactan polysaccharides present in the extracts at approximately 10 6 g / mol.
  • Extracted samples 162 and 234 are obtained by cold precipitation of the aqueous extract with an organic solvent, such as ethanol.
  • an organic solvent such as ethanol.
  • the aqueous extract is precipitated with 50% v / v of pure ethanol previously put at -20 ° C.
  • the precipitate is isolated by centrifugation (4000 rpm, 4 ° C, 10 minutes).
  • the precipitate can be washed several times, three times for example, with pure ethanol. It is then dried in an oven at 40 ° C overnight.
  • Extracted sample No. 295 is obtained by a new refining step of sample No. 162 or 234 by ionic interactions.
  • This step purification by ionic interactions is carried out on an anion exchange support, especially polysaccharide microbeads formed of crosslinked chitosan.
  • the sample No. 162 or 234 is dissolved in a pH of 4-5 by acidification, in particular with hydrochloric acid.
  • the sample is then brought into contact with the ion exchange support, then rinsed with deionized water, preferably acidified, in particular with hydrochloric acid to reach a pH of 4-5.
  • the sugar of interest is released from the support with a basic buffer, in particular a bicarbonate / carbonate buffer, preferably adjusted to a pH of about 10.
  • a basic buffer in particular a bicarbonate / carbonate buffer, preferably adjusted to a pH of about 10.
  • the soluble fraction is isolated, preferentially by filtration.
  • This fraction is then desalted, in particular by precipitation with an organic solvent, such as ethanol.
  • the fraction is concentrated, preferably to a concentration of 1 to 10 g / l polysaccharides, then precipitated with 50% v / v of pure ethanol.
  • the precipitate is isolated by centrifugation (5000 rpm, 4 ° C., 10 minutes) and then oven-dried.
  • the fraction thus obtained is sample No. 295.
  • the aqueous extract may also be treated with bentonite to improve its purification and then precipitated with an organic solvent, especially ethanol.
  • the aqueous extract from the extraction of 70 g of Caulerpa racemosa in 1 L of water is treated by the addition of 6.5 g of bentonite previously resuspended in 65 ml of deionized water. The solution is stirred and then left to stand for about 1 hour at room temperature.
  • the soluble phase is isolated by centrifugation (8000 rpm, 4 ° C, 15 minutes).
  • the product thus isolated is precipitated with ethanol previously placed at -20 ° C., up to 50% final v / v.
  • the precipitate is isolated by centrifugation (5000 rpm, 4 ° C, 10 minutes).
  • the precipitate can be washed several times, three times for example, with pure ethanol. It is then dried in an oven at 40 ° C overnight.
  • the precipitation residue constitutes the extracted sample No. 233. Characterization
  • the extracts thus obtained predominantly have the described structure of the sulphated heteroglycan (CHATTOPADHYAY Kausik et al. Carbohydrate polymers 2007, vol. 68, no. 3, pp. 407-415), namely alpha and terminal linked galactoses, 1-4-linked xyloses and 1-4 arabinoses.
  • Galactoses can have C 6 sulphates and C3 sulphate arabinoses.
  • the signals observed correspond to the characteristic signals of the described structures of sulfated Heteroglycans.
  • the milled Ulva armoricana seaweed is treated by extraction in aqueous phase.
  • 70 g of Ulva armoricana are suspended in one liter of deionized water at 95 ° C. for approximately 12 hours with stirring of 850 rpm.
  • the soluble extract is isolated by centrifugation (8000 rpm, 4 ° C, 45 minutes).
  • the product thus isolated is precipitated with ethanol previously placed at -20 ° C., up to 60% final v / v.
  • the precipitate is isolated by centrifugation (4000 rpm, 4 ° C, 5 minutes).
  • the precipitate can be washed several times, three for example, with pure ethanol. It is then dried in an oven at 40 ° C overnight.
  • the precipitated extract is then purified on tangential ultrafiltration membranes. First, the precipitated extract is dissolved in 5 g / l and diafiltered with 10 volumes of deionized water on a 300 kDa membrane.
  • the permeate of this ultrafiltration is concentrated by 10 on a second tangential ultrafiltration membrane of 100 kDa, then diafiltered by 10 volumes of deionized water.
  • the retentate of this second ultrafiltration is lyophilized to give the extracted sample # 244.
  • the permeat is lyophilized after concentration to give the sample No. 245 sample.
  • the extract thus obtained has predominantly the described structure of ulvane (Marc Lahaye, Carbohydrate Research 1998), namely a chain formed of patterns among the following four reasons:
  • the crushed Agardhiella tenera seaweed is treated by extraction in aqueous phase after depigmentation with an organic solvent such as ethanol.
  • 200 g of powdered Agardhiella tenera are suspended in IL of pure ethanol.
  • the mixture is brought to 78 ° C. with stirring at 500 rpm for approximately 3 hours.
  • the depigmenting residue is isolated by filtration on a 20-25 ⁇ m Buchner filter and dried in an oven at 40 ° C. overnight.
  • the alga thus depigmented is then redissolved in 10 liters of deionized water at 37 ° C. for approximately 6 hours with stirring of 850 rpm.
  • the soluble extract is isolated by centrifugation (8000 rpm, 30 ° C, 30 minutes).
  • the product thus isolated is precipitated with ethanol previously placed at -20 ° C., up to 67% v / v final.
  • the precipitate is isolated by centrifugation (4000 rpm, 4 ° C, 5 minutes).
  • the precipitate can be washed several times, three times for example, with pure ethanol. It is then left to dry in an oven at 40 ° C., forming the extracted sample No. 164.
  • the soluble portion resulting from the first precipitation step is taken up with ethanol addition at -20 ° C. to 83% final v / v of ethanol.
  • the precipitate is isolated by centrifugation (4000 rpm, 4 ° C.).
  • the precipitate can be washed several times, for example, three times with pure ethanol, and is then dried in an oven at 40 ° C., forming the extracted sample No. 165.
  • ethanol is added at -20 ° C. to obtain 92% final v / v of ethanol.
  • the precipitate can be washed several times, for example three times, with pure ethanol. It is then dried in an oven at 40 ° C forming the sample extracted No. 166.
  • the identification of active samples relies on the evaluation of the ability of molecules to block the viral replication of the influenza virus on a cell system that is permissive to the latter.
  • the evaluation was conducted against influenza viruses type A and B, which can induce epidemics in the human population. i).
  • viruses A / Brisbane / 10/2007 H3N2, and B / Florida / 4/06 were used in the neutralization tests. These represent antigenically the circulating strains of the winter seasons 2008-2009 for the southern and northern hemisphere. They were selected to enter the vaccine composition of the corresponding winters.
  • concentrations of samples are made in the middle of infection (250 ⁇ g / ml; ; 2.5mg / ml). Each concentration is contacted with an equal volume of a standardized amount of virus. This mixture is deposited at the rate of 4 cups per concentration, in 96 well plates coated with MDCK cells. Negative controls are performed to verify the absence of cytopathic effect of uninfected MDCK cells. A control of the amount of virus used in the test is also deposited. After 48 hours of incubation, the% neutralization is determined by the detection of the virus in the culture supernatant by an enzymatic test.
  • the polysaccharides 79, 98, 109, 141, 237, 239, 240, and 242 belong to the family of sulfated arabinogalactans.
  • the polysaccharide 138 belongs to the family of apiogalacturonans.
  • Polysaccharides 233 and 234 belong to the family of sulfated heteroglycans.
  • Polysaccharides 164 to 166 belong to the family of carrageenans, and finally ulvans are represented by polysaccharides 244 and 245.
  • Polysaccharides with the exception of ulvans, have demonstrated a high biological activity with respect to type B viruses. All the molecules inhibit more than 70% of the replication at a concentration of 250 mg / ml. The concentration which makes it possible to inhibit 50% of viral replication is between 25 and 250 mg / ml for apiogalacturonan 138 and is less than 2.5 g / ml for all sulphated arabinogalactans and sulphated heteroglycans.
  • sulfated arabinogalactan polysaccharides and heteroglycan sulphates which exhibit such activity are more effective than control carrageenans.
  • the sulfated arabinogalactans 79, 98, 109, 141 inhibit between 25 and 75% of the viral replication when they are used at 250 ⁇ 9 / ⁇ .
  • Polysaccharide 152 belongs to the family of sulfated arabinogalactans, polysaccharides 158, 160 and 161 to the family of apiogalacturonans, and polysaccharide 162 to the family of sulfated heteroglycans. All of these polysaccharides exhibit a high activity with respect to the type A virus, since for a concentration of 250 g / ml, the percentage inhibition is between 75 and 100%. The concentration to inhibit 50% of viral replication is between 2.5 and 25 mg / ml. Polysaccharides 152 and 162 are also characterized by high activity against type B virus, with a concentration to inhibit 50% of viral replication less than 2.5 ⁇ g / ml.
  • glycoproteins capable of embedding on the surface of retroviral particles and forming functional VLPs are impressive (Sandrin V, et al., (2003) Targeting retroviral and lentiviral vectors. Curr Top Microbiol Immunol 281: 137-78) and includes many viruses from very distant families.
  • the concept of pseudotyping has led to several applications in the different sectors of biotherapeutic research, and more particularly, in the search for antivirals. Indeed, by mimicking the cellular input properties of parental viruses whose glycoproteins originate, PPVs allow numerous fundamental studies in low confinement conditions (L2 or even L1) even for class 4 viruses, due to fact that such hybrids are defective for replication.
  • PPV infection is limited to the single step of cell entry and integration of the recombinant genome, and induces the insertion of the genetic marker by virtue of the properties of the retroviral particle used (Negro D, et al (2000) Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells, Gene Ther., 7: 1613-23.).
  • SIVmac251 novel safe lentiviral vectors derived from simian immunodeficiency virus
  • the quantitative measurement of the consecutive expression of the GFP marker gene by means of cytofluorimetry technique makes it possible to very precisely determine the infectious capacity of the PPVs.
  • VLPs are therefore particularly suitable for identifying compounds that have an antiviral effect due to interference with the functions of surface glycoproteins, such as, for example, cell entry inhibitors.
  • influenza A H3N2 PPVs that incorporate on their surface glycoproteins from two human influenza virus strains that regularly circulate in the population have been developed: influenza A H3N2 and influenza B. This has allowed the identification of compounds that, in the future, can protect the human population in the event of an epidemic caused by these viruses. iii) Production of retroviral particles pseudotyped with influenza virus glycoproteins.
  • influenza virus glycoproteins are incorporated on the surface of replication-deficient retroviruses and are native to murine leukemia virus (MLV).
  • Flu-PPVs consist of MLV GagPol proteins and a defective genome with the GFP marker gene (Hatziiannou et al., 1998, Szécsi et al., 2006). Expression of GFP in cells infected with these PPVs accurately reflects the infectious capacity of glycoproteins on the surface of PPVs. This system is therefore a very suitable tool for studying cell entry and inhibition of influenza virus entry. On the surface of PPV haemagglutinin (HA) and neuraminidase (NA) of the various influenza viruses mentioned above have been incorporated. Flu-PPVs were produced by transient expression in producer cells, internal viral (GagPol, GFP) and surface (HA, NA) components. Flu-PPVs were harvested from the supernatant of the producer cells 48 h after transfection.
  • HA haemagglutinin
  • NA neuraminidase
  • the level of target cell infectivity by FACS was measured by taking advantage of GFP expression in infected cells (Bartosch B, et al., (2003) In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes, Proc Natl Acad Sci USA 100: 14199-204).
  • The% inhibition that reflects the inhibition of virus entry was determined as the decrease in infectious titer in the presence of sugar compared to the titre determined without sugar.
  • This inhibition test is particularly sensitive and robust, and the results are reproducible. This test can be easily adapted to the 96-well plate format that can be easily analyzed with our cytometer (HTS / CantoII, Becton-Dickinson), which has an adapter to pass high-throughput samples.
  • This inhibition test was miniaturized to increase the screening capacity: the inhibition test was performed in 96-well plates that allowed us to test 96 different samples at a time.
  • Figure 16 shows the inhibitory activity of sulfated arabinogalactans tested on the entry of PPV influenza A H3N2.
  • Figure 17 shows the inhibitory activity of sulfated arabinogalactans tested on the entry of PPV influenza B.
  • Figure 18 shows the inhibitory activity of sulfated arabinogalactans tested at the entry of influenza A H5N1 PPVs.
  • Figure 19 shows the inhibitory activity of sulfated arabinogalactans tested on the entry of PPV influenza A H7N1.
  • Sulfated arabinogalactans have been very effective in preventing viral entry, even at a low concentration (ie, 10 ⁇ g / ml).
  • the inhibition obtained with these sulphated arabinogalactan compounds was more effective than the inhibition by the control sugars, the carrageenans.
  • the sulphating of the arabinogalactans was necessary to obtain the anti-inflammatory activity. -grippale.
  • Figure 20 shows the% inhibition of entry of influenza A H5N1 PPV obtained with sulfated arabinogalactans extracted from Codium brittle (79, 98, 109, 141, 152, 237, 238, 239, 240, 242) in comparison with those obtained in the case of two arabinogalactans extracted from fragile Codium, but which have been desulfated (278, 279) and from an unsulfated arabinogalactan extracted from larch (206). It is clear that, in the absence of sulfation, no activity is observed.
  • Figure 21 shows the inhibitory activity of the apiogalacturonans tested on the entry of PPV influenza A H3N2. Both samples of apiogalacturonan 160 and 161 inhibited the entry of influenza A H3N2 PPV at concentrations of 500 and 100pg / ml. The effectiveness of the inhibition obtained with these sugars was between 50 and 60%.
  • Figure 22 shows the inhibitory activity of apiogalacturonans tested at the entry of influenza B PPV. All apiogalacturonan samples tested inhibited the entry of influenza B PPV at concentrations of 500 and 100 g / ml. The effectiveness of the inhibition obtained with these sugar compounds was between 40 and 65%.
  • Figure 23 shows the inhibitory activity of the apiogalacturonans tested on the entry of PPV influenza A H5N1. All apiogalacturonan samples tested inhibited entry of influenza A H5N1 PPVs to concentrations of 500 and 100 ⁇ g / ml. The effectiveness of the inhibition obtained with these sugar compounds was between 40 and 55%.
  • Figure 24 shows the inhibitory activity of sulfated heteroglycans tested on the entry of influenza A H3N2 PPV. All sulfated heteroglycans tested inhibited the entry of influenza A H3N2 PPV at concentrations of 500 and 100 ⁇ g / ml. The effectiveness of the inhibition obtained with these sugar compounds was between 50 and 80%. The inhibition obtained with these sulfated heteroglycan compounds was similar to that obtained with the control sugars, carrageenans.
  • Figure 25 shows the inhibitory activity of sulfated heteroglycans tested on the entry of influenza B PPV. All sulfated heteroglycans tested inhibited the entry of influenza B PPV at concentrations of 500 and 100 ⁇ g / ml. The effectiveness of the inhibition obtained with these sugar compounds was between 90 and 65%. The inhibition obtained with these sulfated heteroglycan compounds was similar to that obtained with the control sugars, the carrageenans.
  • Figure 26 shows the inhibitory activity of sulfated heteroglycans tested on the entry of influenza A H5N1 PPVs. All sulfated heteroglycans tested inhibited the entry of influenza A H5N1 PPV at concentrations of 500 and 100 ⁇ g / ml. The effectiveness of the inhibition obtained with these sugar compounds was between 55 and 80%. The inhibition obtained with these sulfated heteroglycan compounds was similar to that obtained with the control sugars, carrageenans.
  • Figure 27 shows the inhibitory activity of sulfated heteroglycans tested on the entry of influenza A H7N1 PPVs. All sulfated heteroglycans tested inhibited the entry of influenza A H7N1 PPVs to concentrations of 500 and 100 g / ml. The effectiveness of the inhibition obtained with these sugar compounds was between 60 and 80%. The inhibition obtained with these sulfated heteroglycan compounds was similar to that obtained by the control sugars, carrageenans.
  • Figure 28 shows the inhibitory activity of carrageenan tested on the entry of PPV influenza A H3N2. All carrageenans tested inhibited the entry of influenza A H3IM2 PPV at concentrations of 500 and 100 g / ml.
  • the effectiveness of the inhibition obtained with these sugar compounds was between 60 and 70%.
  • Figure 29 shows the inhibitory activity of carrageenans tested on the entry of influenza B PPV. All carrageenans tested inhibited the entry of influenza B PPV at concentrations of 500 and 100 ⁇ g / ml.
  • the effectiveness of the inhibition obtained with these sugar compounds was between 90 and 80%.
  • the inhibition obtained with these carrageenans was lower than that obtained with the sulfated arabinogalactans.
  • Figure 30 shows the inhibitory activity of carrageenans tested on the entry of influenza A H5N1 PPVs. All carrageenans tested inhibited the entry of influenza A H5N1 PPV at concentrations of 500 and 100 ⁇ g / ml. The effectiveness of the inhibition obtained with these sugar compounds was between 90 and 80%. The inhibition obtained with these carrageenan compounds was lower than that obtained with the sulphated arabinogalactans.
  • Figure 31 shows the inhibitory activity of carrageenans tested on the entry of PPV influenza A H7N1. All carrageenans tested inhibited the entry of influenza A H7N1 PPV at concentrations of 500 and 100 ⁇ g / ml. The effectiveness of the inhibition achieved with these sugar compounds has been between 60 and 80%. The inhibition obtained with these carrageenan compounds was lower than that obtained with the sulphated arabinogalactans.
  • sulfated heteroglycans (295) and apiogalacturonans (296) are evaluated in a mouse influenza infection model.
  • mice 6-8 weeks old and free of specified pathogens (16-20 g) were provided by Charles Rivers Laboratories (France). The mice had a period of acclimatization and observation of more than 48 h (before virus inoculation) to possibly remove animals with signs of disease and / or physical abnormalities. All animal care and experimental procedures have been approved by the Animal Ethics Committee of the University of Lyon.
  • Figure 32 shows the effect of treatment with polysaccharides of sulphated arabinogalactan 294, sulphated heteroglycan 295 and apiogalacturonan 296 on the survival of H1N1 influenza A / Puerto Rico / 8/34-infected mice (25). MLD50).
  • the polysaccharides were administered orally in a single application of 20 mg / kg 4 hours after inoculation of the virus and then, 24 hours after inoculation, twice daily by application of 10 mg / kg.
  • mice were anesthetized with isoflurane and exposed by intranasal instillation to 20 ⁇ l of different dilutions in phosphate buffered saline (PBS) 1/10 of influenza A (H1N1) A virus / Puerto Rico / 8/1934.
  • PBS phosphate buffered saline
  • influenza A H1N1
  • MLD50 50% lethal dose
  • mice Three groups of 5 mice were formed for the evaluation of the activity of each of the polysaccharides: 1) Uninfected group and treated with the polysaccharide, 2) Group infected and not treated with the polysaccharide, 3) Infected group and treated with the polysaccharide.
  • mice from the infected groups were obtained after nasal administration under isoflurane anesthesia of the corresponding MLD50 viral load.
  • mice of the polysaccharide-treated and infected groups received a single 20 mg / kg nasal or oral (oral) administration and then, 24 hours after infection with the polysaccharide. virus, 10mg / kg twice daily for 3 days.
  • the untreated groups received a corresponding administration of saline (vehicle polysaccharides).
  • influenza infection causes death of animals in a few days preceded by weight loss.
  • the antiviral activity is evaluated over a period of 14 days by measuring the following parameters: weight loss, reduction of mortality and / or an increase in the survival time (mean day to death: MDD).
  • Treatment with polysaccharides of sulfated arabinogalactan 294, sulfated heteroglycan 295 and apiogalacturonan 296 reduces the mortality of animals infected with influenza H1N1 A / Puerto Rico / 8/34. While the survival percentage is 0% in the untreated infected group, the percentage survival in the infected and treated groups by sulphated arabinogalactan, sulphated heteroglycan and apiogalacturonan polysaccharides is 20%. % and 20%, respectively.
  • Treatment with polysaccharides of sulfated arabinogalactan and apiogalacturonan is also effective in preventing weight loss of infected animals and increases the MDD by 1 day.
  • the MDD of the untreated infected group is 6 days whereas MDD groups infected and treated with sulphated arabinogalactan polysaccharides and apiogalacturonan are 7 days, as shown in Figure 32. 3.
  • This technology - requiring a prior chemical functionalization of sugars before their immobilization - coupled with a Surface Plasmon Resonance (SPRi) imaging detection method, is a relevant method for studying biomolecules in real time and without labeling. between the immobilized polysaccharides and proteins in solution.
  • SPRi Surface Plasmon Resonance
  • the chosen ligand is the hemagglutinin HA envelope glycoprotein, more precisely the soluble subunit HA1, necessary for the attachment of the Influenza virus to the host cell.
  • the pyranose form of the sugar at the reducing end is in equilibrium with a minority open tautomeric form, having an aldehyde function. It is this function which reacts initially with the hydrazide of adipate dihydrazide in an acid medium. This step, limited by the ring opening rate of monosaccharides (1% open form), is decisive. During the reaction, an equilibrium towards the cyclic form of the sugar is favored, which makes it possible to recover the native form of the ose.
  • This reaction must take place in a 50% PBS / 50% DMSO v / v buffer, in order to promote the solubility of the Pyrrole-NHS, without precipitating the polysaccharide.
  • the sugars functionalized with adipate dihydrazide are taken up in PBS at 20 mg / ml. 100 ⁇ l of sugar, 60 ⁇ l of DMSO, and 40 ⁇ l of a stock solution of Pyrrole-NHS at 50 mM (in DMSO) are mixed. The reaction is carried out for 2 hours at room temperature.
  • Control conditions, with unmodified sugars are also performed.
  • the reaction volume is supplemented at 500 ⁇ with 50% PBS / 50% DMSO v / v buffer, and a dialysis step against this buffer makes it possible to eliminate the remaining Pyrrole-NHS.
  • the salts are then removed by dialysis against H 2 0.
  • the sugars are finally lyophilized, and weighed precisely. They are taken up to 20 mg / mL in water.
  • a colorimetric assay makes it possible to obtain an estimation of the coupling efficiency of the two reactions, and thus of being able to graft onto the surface the different polysaccharides of interest at the same sugar-pyrrolyl concentration.
  • This method using the TNBSA reagent (P2297-10 mL, Sigma-Aldrich), is based on the detection of primary amines and hydrazines. It allows after the first coupling reaction, to determine the proportion of sugars coupled with adipate dihydrazide, and after the second reaction, to estimate the amount of sugar-pyrrolylated, by measuring a decrease of hydrazines in the solution.
  • the detection is at 450 nm (Victor 1420 Multilabel Counter), and the quantification is estimated by means of a standard range made with different solutions of adipate dihydrazide.
  • 10 pL of sample (sugar modified or not) or reference range are added to 40 of buffer ⁇ _ 0.1M 2 C03 / NaHC0 3 pH 9.6.
  • 10 ⁇ of TNBSA reagent diluted to l / 10th in this buffer are added to each well.
  • the covalent immobilization of the "polysaccharide-pyrrole” molecules is carried out by electro-copolymerization with pyrrole monomers. This process, called electrospotting, allows the very rapid grafting of a biomolecule onto a gold surface via a polypyrrole film (Livache T, et al., Synth Met, 2001, 121 (2-3): 1443-1444). .
  • the protocol described by Mercey et al (Methods Mol Biol 2007 385: 159-75), consists in preparing reaction mixtures containing pyrrole at 20 mM and pyrololated sugars at different concentrations (5 and 50 ⁇ l) in buffer electrocopolymerization (50 mM NaH 2 PO 4 , 50 mM IMaCl, 10% (w / v) glycerol pH 6.8).
  • the construction of the chip is then performed using a robot (Genomic solutions) and a U12 acquisition card (LabJack), driven by Labview software.
  • the electrochemical system consists of a ceramic needle (X-Tend Pin, Genoptics) containing a platinum wire of 200 ⁇ , capable of taking the pyrrolylated sugar solutions contained in a 96-well plate and depositing them at a specific location on the prism.
  • a voltage pulse of 2.4 V for 100 ms between the needle (against electrode) and the gold surface (working electrode) causes the synthesis of the polypyrrole film and its deposition on the surface.
  • the prism is rinsed with water, dried and stored at 4 ° C.
  • FIG. 34 An illustration of the diagram of the arrangement of the pads on the golden surface of a prism, is given in Figure 34. To the left of Figure 34 is presented the differential image obtained in SPR, during the injection of HA ⁇ (H1N1) to 600 nM.
  • Family A Apiogalacturonans
  • Family B Sulphated Arabinogalactans
  • Family C Sulphated Heteroglycans
  • Family D Carrageenans
  • Family E Glucomannans
  • Hp 15 kDa heparin
  • PPy polypyrrole.
  • Haemagglutinin HA is a 220 kDa homotrimer, present on the surface of Influenza viruses, and responsible for their attachment to target cells via its binding to sialic acids attached to host glycoproteins and glycolipids (Skehel JJ, Wiley DC. 2000) Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin Annu Rev Biochem 69: 531-69). Each monomer is composed of two subunits, linked together by a disulfide bridge: HA1 and HA2. HA1, which is the soluble part of the protein, contains the receptor binding site. The hemagglutinins included in these studies come from two influenza viruses extremely pathogenic avian: H5N1 and H7N1.
  • haemagglutinin HA1 of the pandemic H1N1 virus strain was included in this study. All haemagglutinins were expressed as soluble proteins merged with a 6xHis tag, allowing their purification. Proteins were produced by transient transfection in these 293T human kidney cells. Soluble proteins were purified from the supernatant of the producer cells 48 hours after transfection. The purification of the different HAI by their 6xHis tag is carried out by means of a Nickel-NTA column (Quiagen), then by a gel filtration column (Superdex 75, GE Healthcare), in order to eliminate any contaminants.
  • the purity of the protein was verified by migration on an SDS PAGE gel followed by staining of the Coomassie blue proteins, as well as by Western Blot, by revelation with an anti-His-Tag monoclonal antibody (Sigma) or an anti-human antibody. - HA ⁇ . Evaluation of the amount of protein collected is carried out using a colorimetric assay (QuantiPro BCA assay kit, Sigma-Aldrich), and the proteins are stored at 4 ° C.
  • the interactions take place within a 7.2 ⁇ L PEEK interaction cell, connected to a syringe pump, in the rinse buffer (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, pH 7.4) delivered at a flow rate of 70 ⁇ l / min. All the buffers used for these experiments are previously filtered and degassed. Before each interaction study, the prism is saturated with an injection of rinse buffer containing 1% BSA (w / v). After injection of the protein for 7 minutes, the surface is rinsed with rinse buffer to remove unbound molecules, and dissociate the complexes. Regeneration is performed with rinse buffer containing 1M NaCl, or 2M Guanidine-HCl, for 5 minutes.
  • the proteins are injected at increasing concentrations, ranging from 50 nM to 800 nM.
  • the image on the left represents the prism seen in SPR imaging, when injecting the HA1 protein (H1N1) at 600 nM.
  • the values of the percentage of reflectivity of each species - sugar or pyrrole, which is the negative control of our experiments - are recorded at the end of the injection of the protein.
  • results are symbolized in the form of histograms, representing the percentage of reflectivity of the sugar (deposited at 50 ⁇ ) relative to the percentage of reflectivity of the pyrrole (recorded at the end of injection), for the concentration of 800 nM protein.
  • These standardized responses are used to compare the interactions obtained for each of the polysaccharides studied. The higher the value, the greater the observed interaction.
  • Figure 35 shows the interaction curves of sulfated arabinogalactans tested with HA1 protein. All sulfated arabinogalactans tested demonstrated significant interaction with purified haemagglutinins from the three virus strains studied. It can be noted that, when the three proteins could be tested, HA1 (H5N1) is the one with the strongest interaction. The interactions observed are much stronger than those obtained between the HAI proteins and the carrageenans, patented polysaccharides for their antiviral activity.
  • Figure 36 shows the association and dissociation curves between arabinogalactan 79 and ⁇ 1 of the three influenza viruses, at different concentrations
  • Figure 37 shows the association and dissociation curves between pyrrole (negative control) and HAI from three Influenza viruses, at different concentrations.
  • Figure 38 shows the interaction curves of sulfated heteroglycans tested with HA1 protein. Sulphated heteroglycans active in biological tests also interact specifically with the purified HA1 proteins of the three virus strains studied, and these interactions are superior to the carrageenans.
  • Figure 39 shows the association and dissociation curves between sulfated heteroglycan 233 and HA1 of the three Influenza viruses at different concentrations. As illustrated with the 233 polysaccharide, the observed interactions are specific and dependent on the concentration of injected protein (control: pyrrole). iii. Interaction studies with carrageenans
  • Figure 40 shows the interaction curves of the carrageenans tested with HAI protein.
  • Carrageenan polysaccharides already described for their antiviral activity, serve as efficacy controls for the biological tests developed.
  • the polysaccharides 164, 165 and 166 from this family did not interact or very little with the purified haemagglutinins.
  • the interactions obtained are indeed weak, while the antiviral activities are similar to those of other polysaccharides. It can therefore be assumed that the antiviral effect of these polysaccharides is not caused by the binding to the HA glycoprotein of the virus envelope, but manifests itself vis-à-vis another target.
  • Figure 41 shows the association and dissociation curves between carrageenan 164 and ⁇ 1 of the three influenza viruses at different concentrations.
  • b Example of MOA during the viral cycle
  • the modalities 1 to 4 allow to highlight an activity during the early phases of the infection.
  • the modality 5 allows the study of an antiviral activity on the late phase of the infection.
  • Figures 43 and 44 show the results of inhibition of viral replication according to the five modes of infection.
  • the inhibition of replication is mainly observable in modality No. 5, both in the presence of A / Brisbane / 10/07 virus and B / Florida / 04/06 virus, showing that the molecule 152 mainly inhibits the end of the cycle. replicative of the virus.

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EP12702582.3A 2011-01-04 2012-01-03 Sulfatierte arabinogalaktane, apiogalacturonane und sulfatierte heteroglycane zur behandlung von durch den influenza-virus verursachten erkrankungen Withdrawn EP2675462A2 (de)

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