WO2017013568A1 - Composition anti-herpétique et formulation pharmaceutique anti-herpétique - Google Patents

Composition anti-herpétique et formulation pharmaceutique anti-herpétique Download PDF

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WO2017013568A1
WO2017013568A1 PCT/IB2016/054262 IB2016054262W WO2017013568A1 WO 2017013568 A1 WO2017013568 A1 WO 2017013568A1 IB 2016054262 W IB2016054262 W IB 2016054262W WO 2017013568 A1 WO2017013568 A1 WO 2017013568A1
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extract
glycolic
dry
dry extract
graecum
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PCT/IB2016/054262
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Manuel DARIO
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Harven S.A.S Di Dario Manuel & C.
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Priority to EP16763312.2A priority Critical patent/EP3324989A1/fr
Publication of WO2017013568A1 publication Critical patent/WO2017013568A1/fr

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    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/488—Pueraria (kudzu)
    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/49—Fagaceae (Beech family), e.g. oak or chestnut
    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/61—Myrtaceae (Myrtle family), e.g. teatree or eucalyptus
    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A61K36/736—Prunus, e.g. plum, cherry, peach, apricot or almond
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses

Definitions

  • Anti-herpes composition and anti-herpes pharmaceutical formulation are provided.
  • the subject-matter of the present invention is a composition comprising the following extracts: Fenugreek - Trigonella foenum-graecum; manjakani - Quercus infectoria gall; Pueraria mirifica; Hamamelis virginiana. Also claimed is a pharmaceutical formulation comprising said composition for use in the topical treatment of herpes infections.
  • composition of the invention and the pharmaceutical formulation of the invention are particularly but not exclusively suitable for treating herpes simplex 1 (HSV-1) and especially herpes simplex 2 (HSV-2).
  • Fenugreek Trigonella foenum-graecum, a plant of the Fabaceae family, contains trigonelline as a characteristic component within its seeds. The clinical efficacy of this has been demonstrated for treating diabetes, since it is capable of rapidly reducing blood sugar levels. It also has a demonstrated galactagogue action.
  • Quercus infectoria a tree belonging to the Fagaceae family, is host to a parasite, of the hymenoptera order and Cynipidae family, Cynips tinctoria, which infects the tree.
  • the female insect bores into the buds and lays its eggs there. This puncture leads to the formation of the galls, inside which the eggs hatch and from which the larvae emerge before becoming adult insects.
  • the wall of the gall is very strong and contains a large amount of tannins. Because of this, galls are used as astringents and haemostatics.
  • Pueraria mirifica is a herb with tuberous roots. These have a high phytoestrogen content and are traditionally used in traditional Thai medicine, with what are considered to be anti-aging effects.
  • Hamamelis virginiana is a plant of the Hamamelidaceae family.
  • the healing properties of a decoction of Hamamelis have long been known to native Americans.
  • the tannin content of the leaves and bark mean that it can be used as a decongestant, astringent and haemostatic.
  • Herpes simplex 1 (HSV-1) and herpes simplex 2 (HSV-2) viruses are two members of the Herpesviridae family. HSV generally infects fibroblasts or epithelial cells, causing their lysis, as well as neurons, in latent form in the latter case. Pathological manifestations are herpes labialis, typically caused by HSV-1 , and genital herpes, associated with HSV-2. The first, extremely widespread form is responsible for the occurrence of characteristic febrile blisters that normally involve the skin of the face (lips, nose).
  • HSV-1 The infection caused by HSV-1 can readily recur because the virus survives throughout life within the cells of the trigeminal nerve ganglion and, under specific conditions (stress, lowered immune defences etc.) can be reactivated, migrating via the nerves to the skin.
  • the second form is a genital infection, also known as herpes genitalis. Both are contracted through physical and sexual contact.
  • HSV-2 Genital herpes
  • Genital herpes can have a significant impact on the interpersonal relationships of patients, as well as considerable public health implications because of the possibility of simultaneous transmission of other sexually transmitted infections. For example it has been seen that anyone suffering from HSV-2 runs a three times greater risk of contagion from the HIV virus.
  • antiviral drugs are antiviral drugs, though these have to be administered systemically, for long periods and at a high dosage.
  • the antiviral drugs most frequently used at present are based on aciclovir or its derivatives such as valaciclovir and famciclovir.
  • Another object of the invention is to provide a composition for treating herpes simplex 1 and/or 2 that can be effectively applied locally, i.e. administered topically.
  • Another objective of the invention is to provide a composition for treating herpes simplex 1 and/or herpes simplex 2 that is both effective in treating the virus and not cytotoxic.
  • Another objective of the invention is to provide a composition for treating herpes simplex 1 and/or herpes simplex 2 that produces its effects in a short time and therefore requires shorter administration times.
  • composition comprising the following extracts: Fenugreek - Trigonella foenum-graecum; manjakani - Quercus infectoria gall; Pueraria mirifica; Hamamelis virginiana.
  • a pharmaceutical formulation that comprises the above-mentioned composition for use in treating herpes infections.
  • a pharmaceutical formulation containing at least the composition of the invention as an active ingredient, in a quantity such as to produce a significant therapeutic effect, together with one or more pharmaceutically acceptable carriers and/or excipients.
  • This pharmaceutical formulation can be prepared by conventional technical methods in common practice in the pharmaceutical industry, such as those illustrated in Remington's Pharmaceutical Sciences handbook, Mack Pub. N.Y. - latest edition.
  • the formulations according to the present invention contain, together with the active composition, at least one pharmaceutically acceptable carrier or excipient. These can be adjuvants with particularly useful formulations, for example solubilising agents, dispersants, suspending agents and emulsifiers.
  • the formulations of the present invention are administered in a "therapeutically effective quantity".
  • the quantity of formulation actually administered will typically be determined by a doctor, having regard to the relevant circumstances, including the condition to be treated, the chosen route of administration, the formulation actually administered, any combination of drugs, the age-related body weight, and the response of the individual patient, the severity of the patient's symptoms and so on.
  • the therapeutically effective dose can initially be estimated either in cell culture assays or on animal models, usually mice, rats, guinea-pigs, rabbits, dogs or pigs. The animal model can also be used for determining the appropriate concentration range and route of administration. This information can then be used for determining doses and routes to be used for administration in humans.
  • HED human equivalent dose
  • the formulation can also contain a pharmaceutically acceptable carrier for administration of a therapeutic agent.
  • compositions in therapeutic compositions can also contain liquids such as water, saline solution, glycerol and ethanol.
  • additional substances such as wetting agents or emulsifiers, substances for buffering pH and the like, can be present in these formulations.
  • the pharmaceutical formulations for the invention are preferably formulations for topical use, advantageously in the form of a cream or gel, or also lipstick.
  • composition of the invention containing the combination of the four extracts fenugreek - Trigonella foenum-graecum; manjakani - Quercus infectoria gall; Pueraria mirifica; Hamamelis virginiana has a high antiviral potency against the HSV-2 virus and, although to a slightly lesser extent, on HSV-1.
  • composition containing the combination of the four extracts fenugreek - Trigonella foenum-graecum; manjakani - Quercus infectoria gall; Pueraria mirifica; Hamamelis virginiana has a virucidal effect on the HSV-2 virus and, although to a slightly lesser extent, on HSV-1.
  • the above-mentioned composition produces its antiviral and virucidal effect in the absence of cytotoxicity.
  • the synergistic effect observed from combining the four above-mentioned extracts is surprising: whereas the four extracts taken individually and tested under the same experimental conditions lead to a reduction of around 15% in the virus titre, a combination of all four surprisingly leads to a reduction in virus titre of around 93%.
  • the tests carried out and reported below have demonstrated that, while the four extracts taken individually do have some cytotoxicity, the sample comprising all four extracts tested under the same experimental conditions has no cytotoxicity.
  • the extracts are present as dry extract or glycolic extract.
  • the composition comprises (the percentages, where not otherwise indicated, as expressed as weight/volume ratios):
  • said composition comprises:
  • said composition comprises: dry extract or glycolic extract of fenugreek (Trigonella foenum-graecum) 0.01 to 10%;
  • said composition comprises:
  • said composition comprises:
  • said composition comprises:
  • dry extract or glycolic extract of fenugreek (Trigonella foenum-graecum) 0.5%; dry extract or glycolic extract of Quercus infectoria gall 3%;
  • said extracts are dry extracts.
  • said composition comprises other active substances.
  • said composition also comprises pharmaceutically accepted excipients.
  • the pharmaceutical formulation is in cream form.
  • said cream is an oil-in-water emulsion comprising: extract of Trigonella foenum-graecum, extract of Quercus infectoria gall, Pueraria mirifica root extract, extract of Hamamelis virginiana and, preferably, one or more emollients, one or more sunscreens, emulsifiers, preservatives, humectants, stabilisers, perfumes.
  • said cream also comprises one or more further active substances, preferably selected from the group comprising: Prunus amygdalus dulcis oil, Simmondsia chinensis seed oil, Persea gratissima oil, phenoxyethanol, Panax ginseng root extract, Melaleuca alternifolia oil, extract of Echinacea angusti folia.
  • further active substances preferably selected from the group comprising: Prunus amygdalus dulcis oil, Simmondsia chinensis seed oil, Persea gratissima oil, phenoxyethanol, Panax ginseng root extract, Melaleuca alternifolia oil, extract of Echinacea angusti folia.
  • said cream comprises: extract of Trigonella foenum-graecum, extract of Quercus infectoria gall, Pueraria mirifica root extract, extract of Hamamelis virginiana, coco-caprylate, aqueous extract of Hamamelis virginiana leaves, water, glycerol, Prunus amygdalus dulcis oil, Simmondsia chinensis seed oil, sodium stearoyl lactylate, tocopheryl acetate, titanium dioxide, caprylic/capric glycerides, ethylhexyl methoxycinnamate, maltodextrin, arginine HCI, caprylic/capric triglycerides, Persea gratissima oil, phenoxyethanol, Panax ginseng root extract, sodium polyacrylate, xanthan gum, sodium hyaluronate, Melaleuca alternifolia oil, extract of Echinacea angusti folia.
  • said cream comprises (% weight/volume): extract of Trigonella foenum- graecum 0.5%, extract of Quercus infectoria gall 3%, Pueraria mirifica root extract 1.8%, extract of Hamamelis virginiana 0.6%, caprylic/capric triglycerides 1 %, ethylhexyl methoxycinnamate 1.25%, Prunus amygdalus dulcis oil 2%, tocopheryl acetate 2%, coco-caprylate 38%, Simmondsia chinensis seed oil 2%, sodium stearoyl lactylate 2%, Persea gratissima oil 1 %, aqueous extract of Hamamelis virginiana leaves 24%, glycerol 3%, xanthan gum 0.5%, arginine HCI 1 %, Panax ginseng root extract 1 %, extract of Echinacea angustifolia 0.5%, Melaleuca alternifolia oil 0.
  • the cream formulation described above further improves the antiviral and virucidal potency observed with the composition of the present invention comprising fenugreek - Trigonella foenum- graecum; manjakani - Quercus infectoria gall; Pueraria mirifica; Hamamelis virginiana, showing virucidal activity, i.e. a reduction of some 99.99% with respect to the HSV-2 virus strain.
  • Another subject-matter of the present invention is formed by a composition comprising fenugreek - Trigonella foenum-graecum; manjakani - Quercus infectoria gall; Pueraria mirifica; Hamamelis virginiana, for use in treating HSV-1 and/or HSV-2 infections.
  • composition and/or said pharmaceutical formulation is a preferred aspect of the present invention for use in treating HSV-2 infections.
  • Another aspect of the present invention is formed by a pharmaceutical form comprising said composition and the use thereof in treating HSV-1 and/or HSV-2 infections.
  • the pharmaceutical formulation is for topical use.
  • the pharmaceutical formulation is in gel form.
  • the Vero cell line is kept at 37°C, 5% C0 2 in Dulbecco's modified minimum essential medium (MEM) (BioWhittaker Europe) with the addition of 10% fetal bovine serum (FBS), 1 % L-glutamine, 1 % penicillin-streptomycin.
  • MEM Dulbecco's modified minimum essential medium
  • PBS phosphate-buffered saline
  • PBS is a Tris-buffered saline solution (TBS) (25 mM Tris): 8 g NaCI, 0.2 g KCI and 3 g Tris base are dissolved in 800 ml_ distilled H 2 0. The pH is brought to 7.4 ⁇ 0.2 by adding HCI. Distilled H 2 0 is added to make up to a litre. The solution is divided into aliquots and sterilised in an autoclave at 121 °C for 20-30 minutes.
  • TSS Tris-buffered saline solution
  • Each virus suspension was prepared and amplified on a large scale in monolayer cell cultures. After infection and multiplication of the virus, the cell debris was removed by means of double centrifugation at low speed (2550 rpm for 10 min) and the supernatant containing the virus was taken off so as to determine its virus titre. The supernatant was then subdivided into 2-mL aliquots of a known titre and stored at a temperature of -80°C in a freezer.
  • 0.1 ml_ of each dilution was transferred into a 96-well plate containing the cell monolayer at confluence (>90%) after drawing off the culture medium.
  • Each dilution of the virus suspension was plated in sextuple. Twelve control wells were left untreated. After 1 hour's incubation at 37°C with rocking agitation for the time to viral adsorption, 100 ⁇ _ MEM + 10% FBS was added to the inoculum.
  • the infections were incubated in 5% C0 2 at 37°C ⁇ 1 for 6 days and observed with an inverted microscope to detect the formation of lysis plaques, caused by the cytopathic effect (CPE) of the virus suspension.
  • CPE cytopathic effect
  • the CPE results (quantitative test) of each dilution are expressed as a percentage of positive results between 100% and 0% and recorded as 0 for no CPE and from 1
  • the virus titre was calculated by using the Spearman-Karber method (estimation of
  • the applicant has previously tested the activity of samples containing the four extracts separately and then the activity of a sample containing the four extracts together.
  • A- dry extract of fenugreek (Trigonella foenum-graecum) 0.5%
  • the percentages indicated refer to the weight of the dry extract diluted in the volume of serum-free MEM.
  • the plant extracts at the above-mentioned tested concentrations were prepared (w/v) by diluting them in serum-free MEM.
  • a fifth sample, F was obtained by mixing the four extracts A, B, C, E at the above- mentioned concentrations, obtaining a sample F with the following composition: F- dry extract of fenugreek (Trigonella foenum-graecum) 0.5% + dry extract of Quercus infectoria gall 0.3% + dry extract of Pueraria mirifica 1.8% + dry extract of Hamamelis virginiana 0.6%. Subsequently the samples of the four extracts A, B, C, E were then diluted 1 :2, still in MEM, giving the following samples:
  • A- dry extract of fenugreek (Trigonella foenum-graecum) 0.25%
  • F- dry extract of fenugreek (Trigonella foenum-graecum) 0.25% + dry extract of Quercus infectoria gall 0.15% + dry extract of Pueraria mirifica 0.8% + dry extract of Hamamelis virginiana 0.3%.
  • test concentrations of the above-mentioned samples were obtained by dilution using serum-free MEM as a diluent, which had absolutely no influence on the results, being the culture medium of the cells.
  • sample G An O/W pharmaceutical preparation, called sample G, was also prepared, comprising: extract of Trigonella foenum-graecum 0.5%, extract of Quercus infectoria gall 3%, Pueraria mirifica root extract 1.8%, extract of Hamamelis virginiana 0.6%, caprylic/capric triglycerides 1 %, ethylhexyl methoxycinnamate 1.25%, Prunus amygdalus dulcis oil 2%, tocopheryl acetate 2%, coco-caprylate 38%, Simmondsia chinensis seed oil 2%, sodium stearoyl 25 lactylate 2%, Persea gratissima oil 1 %, aqueous extract of Hamamelis virginiana leaves 24%, glycerol 3%, xanthan gum 0.5%, arginine HCI 1 %, Panax ginseng root extract 1 %, extract of Echinacea angustifolia 0.5%, Melale
  • Samples A, B, C, E, F and G are tested to check cytotoxicity and then antiviral potency and virucidal efficacy.
  • Tests were first conducted to check the cytotoxicity effect of the samples previously prepared.
  • a cytotoxicity test was performed by mixing 1 mL serum- free MEM with 8 mL of each sample, A to F, at the following concentrations: 10 mg, 1 mg, 100 ⁇ g, 10 ⁇ g, 1 ⁇ g/mL. For each one, serial dilutions from 10 "2 to 10 "6 (1 :10) were prepared, taking 0.2 mL of the mixture obtained and combining it with 1.8 mL serum-free MEM.
  • 0.1 mL (100 ⁇ ) of each dilution was plated in sextuple in the monolayer cell cultures at confluence (>90%). The mixture was not added to 6 wells, which served as controls for the cell line. After 1 hour at 37°C 100 ⁇ MEM + 10% FBS was added and the cell culture was placed in an incubator with 5% C0 2 at 37°C ⁇ 1 and observed with an inverted microscope constantly for the next 6 days, to detect any cytopathic effect (CPE), caused by the cytotoxic action of the test substance.
  • CPE cytopathic effect
  • each sample and PBS were removed and the viral inoculum was performed in the 0.1-mL volume of each dilution obtained by preparing dilutions from 10 "2 to 10 "9 of the mixture obtained by taking 0.5 ml_ of virus suspension and adding to it 4.5 ml_ MEM + 2% FBS.
  • the infections were incubated in 5% C0 2 at 37°C ⁇ 1 for 6 days and observed with an inverted microscope to detect the formation of lysis plaques, caused by the cytopathic effect (CPE) of the virus suspension.
  • CPE cytopathic effect
  • the infection of the cells was assisted by putting them at 37°C for 1 hour with rocking movement. 100 ⁇ _ MEM + 10% FBS was then added to each well and the cultures placed in an incubator with 5% C0 2 at 37°C + 1 for 6 days and observed microscopically.
  • the CPE results of each dilution are expressed as a percentage of positive results depending on the degree of cell damage.
  • the data express the logarithm of plaque forming units (PFU) relative to 1 mL of test viral suspension.
  • sample A at 0.5% concentration is cytotoxic.
  • results obtained demonstrate that sample A has an inhibitory potential against the herpes simplex virus type 2 at 0.25% concentration.
  • sample A has an antiviral inhibitory effect that takes the form of preventing the replication and cellular propagation of the virus.
  • Sample A at 0.25% concentration has an antiviral effect of approximately 16%.
  • sample B at 3% concentration is cytotoxic.
  • results obtained demonstrate that sample B has an inhibitory potential against the herpes simplex virus type 2 at 1.5% concentration. At this 1.5% concentration a slight antiviral effect, approximately 1 1 %, is evident.
  • Sample C at 1.8% concentration is cytotoxic, but demonstrates an inhibitory potential against the herpes simplex virus type 2 at 0.9% concentration.
  • Sample C at 0.9% concentration has an inhibitory effect of approximately 11 %.
  • Sample E at 1.8% and 0.9% concentration Table 4
  • the data express the logarithm of plaque forming units (PFU) relative to 1 mL of test viral suspension.
  • sample E is similar to those obtained for sample A.
  • the results obtained demonstrate that extract E at 0.6% concentration is cytotoxic.
  • This sample has an inhibitory potential against HSV-2 at 0.3% concentration.
  • the 0.3% concentration slight antiviral activity is detected; in particular, after treatment, the viral plaques obtained are much smaller than those formed by the viral control (untreated cells).
  • Sample E at 0.3% concentration has an inhibitory effect of approximately 16%.
  • Sample F at 1 mg and 10 mg concentration Table 5
  • the data express the logarithm of plaque forming units (PFU) relative to 1 mL of test viral suspension.
  • sample F is a great deal less than that observed for the individual extracts.
  • sample F demonstrate excellent inhibitory capacity of the mixture against the herpes simplex virus type 2, amounting to a mean reduction of
  • Quantitative test in suspension to assess virucidal activity against the herpes simplex type 2 virus Quantitative test in suspension to assess virucidal activity against the herpes simplex type 2 virus.
  • test was conducted on the sample G formulation according to the Italian standard UNI EN 14476:2007: Quantitative test in suspension to assess the virucidal activity of chemical disinfectants and antiseptics used in human medicine.
  • 3 g of formulation G was added to 7 mL serum-free MEM.
  • Serial dilutions up to 10 "3 (dilution factor 1 : 10) were prepared, taking 1 mL of the mixture obtained and adding it to 9 mL serum-free MEM.
  • CPE cytopathic effect
  • test product modifies cell sensitivity to viral infection
  • the following procedure was carried out: 0.1 mL of the proven non-cytotoxic test concentration of the test product was seeded into 12 wells of the microplates containing the cell culture at confluence and the other 12 wells were treated with 0.1 mL PBS. After 1 hour's incubation at 37°C ⁇ 1°C the solution of the test substance and PBS were removed and the viral inoculum was performed in the 0.1 -mL volume of each dilution obtained by preparing dilutions from 10 "2 to 10 "9 (1 :10) of the mixture obtained by taking 0.5 mL of 5 test virus suspension + 4.5 mL MEM + 2% FBS. After incubation with 5% C0 2 at 37°C ⁇ 1 °C it was observed with an inverted microscope constantly for the next 6 days, to detect any CPE.
  • virus suspension (herpes simplex type 1) was mixed with 8 mL PBS and 10 mL 1.4% (w/v) formaldehyde solution to check the validity of the system.
  • 0.2 mL of this solution was mixed with 1.8 mL MEM + 2% FBS on ice.
  • Serial dilutions from 10 "2 to 10 "6 (1 : 10) were made with PBS + 2% FBS kept on ice.
  • 100 was distributed among 6 wells of a 24-well microplate and this was placed in an incubator at 37°C for 1 hour. At the end, 100 MEM + 10% FBS was added.
  • the cell culture was placed in an incubator with 5% C0 2 at 37°C ⁇ 1 °C for 6 days, and observed with an inverted microscope to detect any CPE.
  • the plaques present in the wells at the countable dilution were counted after fixation and staining of the cell monolayer with crystal violet solution in methanol.
  • the CPE results of each dilution are expressed as a percentage of positive results between 100% and 0% and recorded as "0" for no CPE and from "1" (25% CPE) to "4" (100% CPE) depending on the degree of cell damage.
  • the virus titre was calculated by using the Spearman-Karber method (estimation of ID 50 ).
  • the infectivity of the test virus suspension must be determined at a contact time of
  • the virucidal test was performed at 20°C
  • the test mixture containing the virus suspension and the test product was prepared as follows: 1 mL virus suspension; 8 mL dilution of the test product (separately, all the dilutions of the sample product previously prepared).
  • Test temperature 20°C ⁇ 1 °C
  • Herpes simplex virus type 2 (ATCC VR-734)
  • Herpes simplex virus type 1 KOS strain
  • Each virus suspension was prepared and amplified on a large scale in monolayer cell cultures. After infection and multiplication of the virus, the cell debris was removed by means of double centrifugation at low speed (2500 rpm for 10 min) and the supernatant, containing the virus, was taken off in order to determine its virus titre.
  • test contact time Immediately after the test contact time, checked with a stopwatch, 0.5 ml_ of the test mixture was taken after agitation and added to 4.5 ml_ MEM + 2% FBS, and held on ice, in order to inactivate the activity of the test solution.
  • Virus quantification was performed by preparing serial dilutions from 10 "2 to 10 "9 (1 :10). At each dilution, 6 wells were inoculated in 24-well microplates containing the cell culture at confluence (>90%). The test mixture was not added to 6 wells, but just the virus suspension, and another 6 wells of the plate did not receive the inoculum but served as controls for the cell line.
  • the infection of the cells was assisted by putting them at 37°C for 1 hour with rocking movement. 100 ⁇ _ MEM + 10% FBS was added to each well. After incubation in a temperature-controlled incubator with 5% C0 2 at 37°C ⁇ 1 °C the culture was observed with an inverted microscope constantly for the next 6 days, to detect any CPE.
  • the test product is considered VIRUCIDAL when, at 20°C, after the contact time used for the test, it demonstrates a reduction in viability of at least 10 4 , corresponding to a 4 log reduction (99.99%) against the test viral strain, based on the method and acceptability criteria of Italian standard UNI EN 14476:2007.
  • the virucidal activity test is valid when the following parameters are detected in the preliminary tests:
  • Herpes simplex virus type 2 ATCC 734
  • Herpes simplex virus type 1 KOS strain
  • Herpes simplex virus type 1 Herpes simplex virus type 1 :
  • Example G is VIRUCIDAL against the herpes simplex virus type 2 (ATCC VR 734), after 60 min. contact, at the 0.25 g/mL concentration, demonstrating a reduction in viability of more than 10 4 (equal to a 99.99% reduction), when conducted according to the test methods and requirements of Italian standard UNI EN 14476:2007 - Phase 2 / Stage 1.
  • herpes simplex virus type 1 KOS strain, after 60 min. contact, a 3.83 log reduction in viability from the initial titre was shown, from which it can be inferred that the test formulation has inhibitory action also against herpes simplex virus type 1 , though less than the results for the type 2 strain.
  • test sample G i.e. in the pharmaceutical formulation of the invention.

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Abstract

Cette invention concerne une composition comprenant : de 0,01 à 90 % d'extrait de fénugrec (Trigonella foenum-graecum); de 0,1 à 90 % d'extrait de galle du chêne Quercus infectoria; de 0,1 à 90 % d'extrait de Pueraria mirifica; et de 0,01 à 90 % d'extrait d'Hamamelis virginiana.
PCT/IB2016/054262 2015-07-17 2016-07-18 Composition anti-herpétique et formulation pharmaceutique anti-herpétique WO2017013568A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
US10966948B2 (en) 2019-07-23 2021-04-06 Johnson & Johnson Surgical Vision, Inc. Compositions and methods for treating the eye
US11197841B2 (en) 2019-07-23 2021-12-14 Johnson & Johnson Surgical Vision, Inc. Compositions and methods for treating the eye
US11931331B2 (en) 2018-07-27 2024-03-19 Johnson & Johnson Surgical Vision, Inc. Compositions and methods for treating the eye
US11969454B2 (en) 2019-11-19 2024-04-30 Johnson & Johnson Surgical Vision, Inc. Compositions and methods for treating the eye

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11806327B2 (en) 2018-07-27 2023-11-07 Johnson & Johnson Surgical Vision, Inc. Compositions and methods for treating the eye
US11931331B2 (en) 2018-07-27 2024-03-19 Johnson & Johnson Surgical Vision, Inc. Compositions and methods for treating the eye
US10966948B2 (en) 2019-07-23 2021-04-06 Johnson & Johnson Surgical Vision, Inc. Compositions and methods for treating the eye
US11197841B2 (en) 2019-07-23 2021-12-14 Johnson & Johnson Surgical Vision, Inc. Compositions and methods for treating the eye
US11969454B2 (en) 2019-11-19 2024-04-30 Johnson & Johnson Surgical Vision, Inc. Compositions and methods for treating the eye

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