Classifications

• • 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)
  • A61K36/37—Celastraceae (Staff-tree or Bittersweet family), e.g. tripterygium or spindletree
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/365—Lactones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
  • A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/04—Antipruritics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
  • C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings
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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
  • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons

Definitions

• the invention relates to a method for producing triptolide from a suspension cell culture of Tripterygium sp., For example Tripterygium wilfordii.
• Triptolide a diterpene triepoxide
• Tripterygium wilfordii This plant has been used for more than 4 centuries in traditional Chinese medicine in the treatment of autoimmune diseases, inflammatory diseases and in particular in rheumatoid arthritis. Recently, the potent anti-cancer activity of Triptolide has also been discovered. Anti-proliferative and pro-apototic activities have been demonstrated in different types of cancer cells in vitro and in vivo. Clinical trials have been conducted in the treatment of rheumatoid arthritis and in cancer patients, such as advanced blood leukemia. A recent publication (Brinker AM et al, Phytochemistry 68 (2007) 732-766) summarizes the pharmacological properties of triptolide and its derivatives derived from Tripterygium wilfordii.
• the chemical synthesis of Triptolide is very difficult because it requires the implementation of a process containing about 20 steps.
• triptolide is provided by the company PHARMAGENESIS. It is produced by extraction of Tripterigium wilfordii roots and purification by 2 chromatographic steps. This process is complex and time consuming.
• the extraction / purification yield, for example, from the roots is 0.0005%.
• Kutney JP et al. disclose a method for culturing Tripterygium wilfordii leaf cell suspension and a method of separating triptolide and its derivatives from this culture (Can J Chem 58 (1981): 2677-2683). However, the production yields are even lower than those of the conventional process.
• CN101358180A published Feb. 4, 2009, discloses a method of suspension culture of stem cells. These cells were generated from the roots of Tripterygium wilfordii. The reported triptolide productivity is 0.027 mg / L per day.
• the authors of this patent describe, inter alia, a process consisting of a cell propagation medium, a triptolide production medium and a total alkaloid production medium.
• an abiotic elicitor as described according to the present invention precursors of terpenes biosynthesis, or hormonal withdrawal step.
• the present invention provides a method for producing triptolide from trefoil stem cells of Tripterygium wilfordii having a high yield suitable for industrial production.
• the present invention also provides the culture media that have achieved this particularly interesting yield.
• triptolide yield of 3 mg / L of culture per day. This yield is 1 10 times greater than the best overall yield described in the application CN101358180A; 73 times higher than the best yield in the supernatant and 127 times higher than the best percentage yield of triptolide relative to the weight percentage of the dry biomass.
• triptolide enriched extract obtained by this method inhibits the activation of the NF- ⁇ transcription factor by TNF ⁇ , in a manner equivalent to pure triptolide.
• triptolide-enriched extract obtained according to the method of the invention inhibits the influx of intracellular calcium induced following the specific stimulation of PAR-2 receptors by trypsin.
• RNA chips PCR array
• the invention therefore relates to a method for producing triptolide from a culture of cells in suspension of aerial parts of Tripterygium sp., For example Tripterygium wilfordii, Tripterygium regeli, or Tripterygium hypoglaucum or plants of the Famile of Celastracea.
• the method of producing triptolide is made from a culture of cells suspended from above-ground parts, for example stems, petioles, leaves, and / or inflorescences.
• the present invention relates to a method for producing triptolide in the culture medium from a cell culture of Tripterygium species comprising the following steps:
• step (ii) a phase of hormonal weaning of the cell cultures obtained in step (i) in a weaning medium substantially free of auxins
• step (iii) an elicitation phase by addition of an elicitation cocktail to the cells of step (ii) in the weaning medium
• step (iv) preparing an extract enriched with triptolide from the culture medium at the end of step (iii).
• the elicitation phase consists in adding the elicitation cocktail to the weaning medium containing the weaned biomass produced in step (ii) and culturing; triptolide production takes place in said culture medium.
• the elicitation phase within the meaning of the present invention therefore corresponds to the production phase of the triptolides.
• a) at least one cell differentiating factor of plant cells for example a cytokinin, for example chosen from benzylaminopurine (BAP), abscisic acid, kinetin, thidiazuron, 6-YY-dimethylallylaminopurine, zeatin; isopentenyladenine, or gibberellin;
• BAP benzylaminopurine
• abscisic acid for example chosen from benzylaminopurine (BAP), abscisic acid, kinetin, thidiazuron, 6-YY-dimethylallylaminopurine, zeatin; isopentenyladenine, or gibberellin
• BAP benzylaminopurine
• abscisic acid for example chosen from benzylaminopurine (BAP)
• kinetin thidiazuron
• 6-YY-dimethylallylaminopurine for example chosen from benzylaminopurine (BAP
• terpenes synthesis route for example triptolide synthesis, for example geraniol, farnesol, including their pyrophosphate forms, sodium acetate, pyruvic acid, or mevalonic acid.
• step (i) of the method of the invention is preceded by the following steps:
• arterial parts are meant those parts of the plant above ground, for example, leaves, stems, petioles and / or inflorescences.
• said method can also be applied to any other part of the plant such as seeds and roots.
• Step (a) of the process of the invention consists in the production of calli from a tissue explant, for example an explant of aerial part (s) of Tripterygium wilfordii, for example a piece of a sheet approximately 1 cm 2 in size, cultured on an agar medium comprising dedifferentiation inducers.
• a tissue explant for example an explant of aerial part (s) of Tripterygium wilfordii, for example a piece of a sheet approximately 1 cm 2 in size, cultured on an agar medium comprising dedifferentiation inducers.
• the aerial part (s) of Tripterygium wilfordii are leaves, stems, petioles and / or inflorescences.
• the term "callus” refers to a mass of dedifferentiated cells also called stem cells.
• the dedifferentiation medium is for example a medium comprising:
• At least one macroelement for example chosen from NH NO 3 , KNO 3 , CaCl 2 .2H 2 O, MgSO 4 .7H 2 O, KH 2 PO 4 , for example at a concentration of up to 6000 mg / L,
• At least one microelement for example Kl, H 3 BO 3 , MnSO 4 .4H 2 O, ZnSO 4 .H 2 O, Na 2 MoO 4 .2H 2 O, CuSO 4 .5H 2 O, CoCl 2 .6H 2 O, FeSO 4 7H 2 O, Na 2 EDTA 2 H 2 O, for example at a total microelement concentration in said medium of up to 200 mg / L of culture medium, - at least one vitamin, for example myo-lnositol , the acid
• Nicotinic, Pyridoxine-HCl, Thiamine-HCl for example at a concentration of up to 3 g / L of culture medium
• At least one amino acid for example at a concentration in the culture medium of up to 3 g / l, for example glycine, at least one carbon source, for example sucrose, for example at a concentration of from 20 to 70 g / l of culture medium, for example at 30 g / l,
• At least one preferably plant hormone or preferably plant growth factor or preferably plant growth regulator for example, kinetin, 2,4-dichlorophenoxyacetic acid (2,4D), naphthalene acetic acid (NAA) for example at a concentration of 0.001 to 10 mg / L of culture medium, for example 0.1 to 3 mg / L of culture medium.
• kinetin 2,4-dichlorophenoxyacetic acid (2,4D)
• NAA naphthalene acetic acid
• the pH of said medium is adjusted, for example to pH 6 ⁇ 0.5, and autoclaved at 121 ° C. for a minimum of 20 minutes or by filtration over 0.2 ⁇ m.
• the incubation can take place in the dark, for example at a temperature of about 25-30 ° C, for example at 27 ° C or 28 ° C.
• the dedifferentiation medium is, for example, a solid medium, for example gelled by addition of 8 to 12 g / l of agar, for example 8 g / l.
• Step ( ⁇ ) of the process of the invention consists in suspending dedifferentiated cells derived from calli obtained by the first step in a liquid culture medium and propagation of the cells of the suspension.
• the cultivation takes place for a period of 10 to 30 days, for example 15 to 20 days, for example at a temperature of about 27 ° C.
• the culture takes place in the dark and with agitation.
• the culture medium of this step ( ⁇ ) is, for example, the cell propagation medium, for example adjusted to pH 6 and sterilized by autoclaving at 121 ° C. for a minimum of 20 minutes or by sterilizing filtration on a 0.2 ⁇ m filter:
• the cell propagation medium is a medium comprising:
• At least one macroelement for example chosen from NH NO 3 , KNO 3 , CaCl 2 .2H 2 O, MgSO 4 .7H 2 O, KH 2 PO 4 , for example at a concentration of up to 6000 mg / L,
• At least one microelement for example Kl, H 3 BO 3 , MnSO 4 .4H 2 O, ZnSO 4 .H 2 O, Na 2 MoO 4 .2H 2 O, CuSO 4 .5H 2 O, CoCl 2 .6H 2 O, FeSO 4 7H 2 O, Na 2 EDTA 2 H 2 O, for example at a total microelement concentration in said medium of up to 200 mg / L of culture medium,
• At least one vitamin for example myo-lnositol, Nicotinic acid, Pyridoxine-HCl, thiamine-HCl, for example at a concentration of up to 3 g / L of culture medium,
• At least one amino acid for example at a concentration in the culture medium of up to 3 g / l, for example glycine,
• At least one carbon source for example sucrose, for example at a concentration of 30 g / l,
• At least one preferably plant hormone or preferably plant growth factor or preferably plant growth regulator for example, kinetin, 2,4-dichlorophenoxyacetic acid (2,4D), naphthalene acetic acid (NAA) , for example at a concentration of 0.001 to 10 mg / L, for example from 0.1 to 3 mg / L of culture medium.
• kinetin 2,4-dichlorophenoxyacetic acid (2,4D)
• NAA naphthalene acetic acid
• a propagation medium is for example the medium of Example 2.
• the dedifferentiation (a) and / or propagation ( ⁇ ) steps may both be performed in a propagation medium or a dedifferentiation medium.
• Step (i) of the process of the invention consists of the production of biomass from dedifferentiated cells, for example cells of the suspension obtained in step ( ⁇ ) in a suitable nutrient medium, for example the medium of propagation described above. It lasts from 10 to 30 days. It is preferably carried out at 27-28 ° C.
• the cells are regularly "transplanted” or propagated, for example every 7 to 10 days.
• Transplanting consists in diluting part of the cell culture in new medium. For example, 1/5 of the culture is resuspended in a volume of medium corresponding to the new volume of the initial culture II allows maintenance of the cell line in liquid medium.
• Step (ii) of the process of the invention consists of a hormonal weaning step, for example of a duration of 5 to 15 days, for example of approximately 7 days.
• the aim of hormonal weaning is to eliminate auxin or auxins, such as 2,4-D growth hormone and / or NAA, present in the culture or propagation medium. This step makes it possible to obtain a metabolic synchronization of the cells, that is to say, the derepression of the terpenes biosynthetic pathway.
• the hormonal weaning medium is a non-auxin-containing medium, for example, not containing 2,4D, NAA, or a substantially auxin-free medium, for example a medium in which the 2,4D and the NAA are present respectively at a concentration of less than 0.01 mg / L of culture medium.
• the weaning medium has the following composition:
• At least one macroelement for example selected from NH NO 3 , KNO 3 , CaCl 2 .2H 2 O, MgSO 4 .7H 2 O, KH 2 PO 4 , and sodim pyruvate, for example at a concentration that can be as high as at 7000 mg / L,
• At least one microelement for example Kl, H 3 BO 3 , MnSO 4 .4H 2 O,
• At least one vitamin for example myo-lnositol, Nicotinic acid, Pyridoxine-HCl, Thiamine-HCl, Glycine, for example at a concentration of up to 60 mg / L of culture medium,
• At least one carbon source for example sucrose, for example at a concentration of 20 to 70 g / l, for example at 30 g / l,
• At least one preferably plant hormone or preferably plant growth factor or preferably plant growth regulator for example, kinetin, indole butyric acid (IBA), for example at a concentration of 0.001 to 10 mg / ml; L of culture medium, for example from 1 to 3 mg / L of culture medium.
• IBA indole butyric acid
• a weaning medium according to the invention is for example the weaning medium whose composition is indicated in Example 3.
• the pH of the medium is adjusted, for example to pH 6 ⁇ 0.5 and sterilized by an appropriate means.
• the elicitation phase of step (iii) of the process of the invention makes it possible to induce the production of triptolide from the weaned cell culture.
• the elicitation phase is also the production phase of triptolide. It lasts from 15 to 35 days, for example from 20 to 25 days.
• the inventors have indeed observed that the cell division and the production of triptolide are not concomitant. Surprisingly, they are even incompatible. To solve this problem, the inventors have developed a hormonal weaning step and a cocktail of eliciting agents that stops cell division, induces a cellular stress that activates biochemical defense pathways causing the production of triptolide and provides precursors of the terpenes biosynthesis pathway.
• this cocktail does not contain auxins, for example it does not contain 2,4D, or no NAA, or neither product.
• this cocktail comprises:
• At least one cell differentiation factor of the plant cells for example a cytokinin, or a gibberillin, for example benzylaminopurine (BAP),
• 5-Chloro SA 5-chloro-salicylic acid
• ASA acetylsalicylic acid
• MeJA methyl-jasmonate
• At least one precursor used in the synthesis of terpenes for example Farnesol, Geraniol, sodium acetate, pyruvic acid or mevalonic acid.
• a cytokinin in the sense of the present invention is for example absicissic acid, benzylamino purine, zeatin, kinetin, thidiazuron, isopentenyladenine, 6-Y-Y-dimethylallylaminopurine, gibberellin.
• the BAP is for example used at a concentration of between 0.01 and 5 mg / l, for example between 0.5 and 5 mg / l of culture medium.
• 5-chloro-salicylic acid is used at a concentration of between 0.1 and 15 mg / L of culture medium.
• Salicylic acid is for example used at a concentration of between 0.1 and 100 mg / l of culture medium, for example between 20 and 60 mg / l, for example 45 mg / l of culture medium.
• Farnesol is present at a concentration of 1 and 100 mg / l, for example from 15 to 30 mg / l, for example at 30 mg / l of culture medium.
• Geraniol is present at a concentration of 1 and 100 mg / L, for example 20 to 30 mg / L of culture medium.
• Methyl-jasmonate is present at a concentration of 1 and 100 mg / L.
• This elicitor cocktail has a triple action: it redirects the cells towards a cellular differentiation, for example the roots; it generates cellular stress activating genes involved in the production of chemical defense reaction products, for example triptolides and / or their derivatives, and provides plant cells with precursors for the synthesis of terpenes.
• composition of the elicitation cocktail is for example the following: benzylaminopurine (BAP) of 0.5 to 5 mg / L, for example 0.5 to 3 mg / l, for example 0.7 to 3 mg / l, 5-Chloro-Salicylic Acid (5-Chloro SA) of 2 to 6 mg / L, for example 3 to 5 mg / L, for example 3 or 5 mg / L, Acetyl-Salicylic Acid (ASA) and / or acid salicylic acid of 20 to 60 mg / L, for example 30 to 50 mg / L, for example 33 or 45 mg / L, Methyl-Jasmonate (MeJA) at 22.4 mg / L, Farnesol (F-OH) at 19 at 40 mg / L and Geraniol at 20 to 30 mg / L, the amount in mg / L corresponding to mg / L of culture medium. It's not about the concentrations of the different products in a stock solution.
• BAP benzy
• the elicitation cocktail is introduced, for example using concentrated stock solutions made in dimethyl sulfoxide, in the culture medium.
• the elicitation phase (iii) is conducted for 3 to 30 days, for example for 10 to 30 days, for example for 21 to 24 days.
• the elicitation phase (Ni) is conducted in the dark.
• the elicitation phase is conducted at about 27 ° C.
• the elicitation phase is conducted with stirring.
• the cells in suspension are cultured in containers of a volume of approximately 250 ml, for example in Erlenmeyer flask, or culture bottle.
• the suspended cells are cultured in a stirred bioreactor and supplied with air enriched with pure oxygen.
• the culture device comprises, for example, two bioreactors that can communicate with each other. It is a binary culture device.
• a bioreactor may be of the tub or pocket type.
• the first bioreactor of the binary device is the propagation bioreactor.
• the second is the production bioreactor. Biomass transfer can be carried out between the first and the second reactor.
• the first reactor in which the propagation phase takes place feeds the second biomass bioreactor for the production phase.
• the first propagation bioreactor retains a residue of cell suspension to restart a propagation step with fresh propagation medium. This is the culture technique in the bottom of the tank.
• This first bioreactor may be preceded by smaller bioreactors to provide the necessary precultures, in the case of production on an industrial scale.
• the second production bioreactor that has received the biomass of the first bioreactor is supplemented with nutrient medium for the hormonal bonding, optionally or directly the production medium of the secondary metabolite. Then the elicitation cocktail is introduced into the production bioreactor.
• An oxygen sensor is calibrated by air saturation and informs in real time a computerized device for regulating the activated pO 2 so as to maintain the p 2 to 80% by injecting sterile pure oxygen into the aeration device.
• This bioreactor is also equipped with an on-line CO 2 measurement device for the head space which provides a real-time computerized pC ⁇ 2 control device in order to maintain the pCO 2 at 6%. This is done by injecting sterile atmospheric air into the aeration device mixed with oxygen.
• the bioreactor is also equipped with a marine blade stirring system rotating at sufficiently constant speed to stir the cell suspension and prevent it from sedimentation.
• the productivity of the culture is greater than 2.75 mg of triptolide per liter per day
• the triptolide content of the culture supernatant after step (iii) is greater than 50 mg / L
• the triptolide content in% w / w of the dry biomass is approximately 0.385%.
• the reference CN 101358180 A describes a volume productivity of 0.041 mg of triptolide per liter per day (0.82 mg / L in 20 days of culture).
• the method of the present invention provides in about 6:30 that the method of CN101358180 gets several days.
• Step (iv) of the process of the invention consists in extracting the triptolide from the culture medium in which it is produced.
• the tripolide can be extracted from the culture medium according to methods that are well known to a person skilled in the art, for example by liquid / liquid extraction. This extraction results in either pure triptolide or an extract enriched with triptolide.
• step (iv) is a liquid / liquid extraction with isopropyl acetate.
• Another object of the invention is a dedifferentiation medium as described above.
• Another object of the invention is the propagation medium for the production of biomass.
• Another subject of the invention is the hormonal weaning medium.
• Another object of the invention is the elicitation cocktail as described above.
• Another subject of the invention is the use of the elicitation cocktail as described above for the cell culture of the Tripterygium species.
• Another subject of the invention relates to an extract enriched in triptolide that can be obtained by extraction of the culture medium from an in vitro culture of dedifferentiated cells of the Tripterygium species, in particular Tripterygium wilfordii.
• said triptolide enriched extract is obtainable by the process for producing triptolide in the culture medium from a cell culture of the Tripterygium species according to the invention.
• Another subject of the invention relates to a dermocosmetic or dermatological composition
• a dermocosmetic or dermatological composition comprising triptolide or an extract enriched with triptolide as active principle and one or more excipients dermocosmically and / or dermatologically acceptable.
• the dermocosmetically and / or dermatologically acceptable excipients may be any excipient among those known to those skilled in the art in order to obtain a composition for topical application in the form of a cream, a lotion, a gel, an ointment, an emulsion, a microemulsion, a spray, etc.
• the dermocosmetic or dermatological composition according to the invention may in particular contain additives and formulation aids, such as emulsifiers, thickeners, gelling agents, water binders, spreading agents, stabilizers, dyes, perfumes and preservatives.
• Another subject of the invention relates to a dermatological composition
• a dermatological composition comprising triptolide or a triptolide-enriched extract as active principle and one or more cosmetically and / or pharmaceutically acceptable excipients, for its use in the treatment of inflammatory skin disorders, preferentially the pruritus, eczema, atopic dermatitis and psoriasis.
• Another subject of the invention relates to the use of a dermatological composition
• a dermatological composition comprising triptolide or a triptolide-enriched extract as active ingredient and one or more dermocosmetic and / or dermatologically acceptable excipients for the manufacture of a medicament intended to to the treatment of inflammatory skin disorders, preferentially pruritus, eczema, atopic dermatitis and psoriasis.
• Another subject of the invention relates to an extract enriched with triptolide for its use as a medicament.
• Another subject of the invention relates to triptolide or an extract enriched with triptolide for its use in the treatment of inflammatory skin disorders, preferentially pruritus, eczema, atopic dermatitis and psoriasis.
• Another subject of the invention relates to the use of triptolide or an extract enriched with triptolide for the manufacture of a medicament for the treatment of inflammatory skin disorders, preferentially pruritus, eczema, atopic dermatitis and psoriasis. .
• Another subject of the invention relates to the dermocosmetic use of triptolide or an extract enriched with triptolide.
• the following figures and examples illustrate the invention without limiting its scope.
• Figure 1 Example of culture in bioreactors (binary system). Propagation of biomass in fermenter A, transfer of the medium to the fermenter of production B, weaning, elicitation, and biomass harvest a few weeks later.
• Figure 2 Example of production kinetics of triptolide (PG 490) by elicitation in the fermenter B.
• FIG. 3 Wave Single-Use Bioreactor (GE Healthcare)
• Figure 4 Production kinetics of triptolide production in single-use bioreactor type Wave. Monitoring of physicochemical parameters: pH, dissolved oxygen partial pressure pO 2 , sucrose consumption, evolution of dry biomass (DW) and triptolide content in the culture supernatant.
• FIG. 5 HPLC assay makes it possible to determine the concentration of triptolide in the extract
• the calli are obtained from leaf explants of Tripterygium wilfordii.
• the explants are sterilized with 70% ethanol and then with hypochlorite of Na with 2.5% of active chlorine, then rinsed with sterile demineralized water.
• a wash with 7% hydrogen peroxide before rinsing with sterile demineralized water is possible
• the leaves are cut into pieces, for example in squares of about 8-10 mm.
• the leaf explants are deposited on an agar medium of dedifferentiation induction (MSO medium) and subculture.
• MSO medium dedifferentiation induction
• composition of the dedifferentiation medium is as follows:
• Macroelements NH 4 NO 3 at 1650 mg / L, KNO 3 at 1900 mg / L, CaCl 2 .2H 2 O at 440 mg / L, MgSO 4 .7H 2 O at 370 mg / L, KH 2 PO 4 at 170 mg / L, Microelements: KI at 0.83 mg / L, H 3 BO 3 at 6.2 mg / L, MnSO 4 .4H 2 O at
• Vitamins myo-lnositol 100 mg / L, Nicotinic acid 0.5 mg / L, Pyridoxine-HCl 0.5 mg / L, Thiamine-HCl 0.5 mg / L, Glycine 2g / L
• Carbon source Sucrose at 30 g / L
• Kinetin 0.1 mg / L, 2-4 dichlorophenoxyacetic acid (2,4D) at 0.5 mg / L, Naphthalene acetic acid (NAA) at 1 mg / L
• the dedifferentiation medium is gelled by addition of agar at 8-12 g / L, its pH is adjusted to pH 6 ⁇ 0.5 before a 20-minute autodavage at 121 ° C.
• the petri dishes containing the explants are incubated in the dark at 27-28 ° C.
• the calluses obtained are detached from the leaf explant and deposited on new dedifferentiation plates.
• the transplanting of the calluses is carried out every month on the same agar medium.
• Example 2 Formulation of the Culture and Propagation Media
• friable calluses are obtained, after a few months of transplanting, they are transferred into the liquid culture medium, optimized for the propagation of the cell suspension.
• the cell suspension is carried out by depositing about 40 g of friable callus in a 200 ml Erlen containing the propagation medium, incubation for one week on a stirring table at 100 RPM in the dark at 27-28 ° C. .
• the cell supernatant is collected by pipette leaving residual callus clusters.
• the cell suspension obtained is cultured for 15 days then propagated by dilution 1/5 in th new medium every 15 days.
• Cell suspensions cultured on TW2H6 propagation media gave rise to the NS line.
• the propagation medium has, for example, the composition indicated below:
• Macroelements NH 4 NO 3 at 1650 mg / L, KNO 3 at 2500 mg / L, CaCl 2 .2H 2 O at 440 mg / L, MgSO 4 .7H 2 O at 370 mg / L, KH 2 PO 4 at 130 mg / L
• Microelements KI at 0.41 mg / L, H 3 BO 3 at 6.2 mg / L, MnSO 4 .4H 2 O at 22.3 mg / L, ZnSO 4 .H 2 O at 7.5 mg / L , Na 2 MoO 4 .2H 2 O at 0.25 mg / L, CuSO 4 .5H 2 O at 0.025 mg / L, CoCl 2 .6H 2 O at 0.025 mg / L, FeSO 4 .7H 2 O at 19, 85 mg / L, Na 2 EDTA 2 H 2 O at 26.64 mg / L,
• Vitamins myo-lnositol 50 mg / L, Nicotinic acid 0.25 mg / L, Pyridoxine-HCl 0.25 mg / L Thiamine-HCl 0.25 mg / L.
• Hormones Kinetic at 0.083 mg / L, 2-4 dichlorophenoxyacetic acid
• Sources of C Sucrose at 30 g / L.
• the pH of the medium is adjusted to pH 6 ⁇ 0.5 before a suitable sterilization treatment, for example, autoclave at 121 ° C for a minimum of 20 minutes or by sterilizing filtration on 0.2 ⁇ .
• a suitable sterilization treatment for example, autoclave at 121 ° C for a minimum of 20 minutes or by sterilizing filtration on 0.2 ⁇ .
• the filling rate of the Erlenmeyer flasks is 20-40% and the rate of inoculation by cell suspension transfer is 20-25% of the volume, ie approximately 50-100 g / L env. fresh biomass.
• the culture is thus conducted for 15 days in the dark at 27-28 ° C with orbital agitation of 1 10-120 RPM (rotation per minute). At this stage the biomass is present at a concentration of up to 320-350 g / L of fresh biomass.
• the propagation can also take place in a dedifferentiation environment.
• the composition of the hormonal withdrawal medium is as follows:
• Macroelements NH 4 NO 3 at 2 g / L, KNO 3 at 3 g / L, CaCl 2 .2H 2 O at 440 mg / L, MgSO 4 .7H 2 O at 370 mg / L, KH 2 PO 4 at 43 mg / L, sodium pyruvate 2 g / L.
• Vitamins myo-lnositol at 50 mg / L, Nicotinic acid at 0.25 mg / L,
• Hormones 1mg / L Kinetin, 2mg / L Indole-Butyric Acid (IBA).
• the pH is adjusted to pH 6 ⁇ 0.5 before autoclaving for 20 minutes at
• the weaning is thus conducted for 7 days in the dark at 27-28 ° C with orbital shaking of 1 10-120 RPM.
• the biomass is drained by filtration on a beaker and inoculated into a new hormonal weaning medium at a rate of 100 to 200 g / l of fresh biomass.
• the elicitation cocktail is introduced, for example using stock solutions made in dimethylsulfoxide, in the culture medium.
• the composition of the elicitor cocktail is as follows: Abscissic Acid (ABA) at 1.25 mg / L, benzyl-aminopurine (BAP) at 0.7 or 3 mg / L, 5-Chloro Salicylic Acid (5-Chloro SA) at 3 or 5 mg / L, Acetyl-Salicylic Acid (ASA) at 33 or 45 mg / L, Methyl-Jasmonate (MeJA) at
• triptolide is thus conducted for 10 to 21 days in the dark at 27-28 ° C with orbital shaking of 120 RPM.
• the medium is filtered to recover the dark colored clear supernatant which contains the majority of triptolide.
• the concentration of triptolide in the culture supernatant is between 50 and 70 mg / liter, for example between 45 and 65 mg / L of culture medium.
• Example 4 Production of triptolide in stirred tank type bioreactors
• triptolides The production of triptolides in a bioreactor is divided into three phases:
• Inoculum of 2 L is poured into the propagation bioreactor
• This bioreactor contains 8 L of propagation medium at 27.5 ° C.
• An oxygen sensor is calibrated by air saturation and informs in real time a computerized device for regulating the activated pO 2 so as to maintain the pO 2 at 50 - 80%.
• This bioreactor is also equipped with an in-line CO 2 measurement device for the head space which provides a real-time computerized pCO2 control device in order to maintain pCO 2 at 6 - 8%.
• the bioreactor is also equipped with a marine blade stirring system rotating at 75 RPM so as to avoid sedimentation of the cells at the bottom of the reactor.
• the culture is maintained under these physico-chemical conditions for 15 days so as to reach a cell density of the order of 320 g / l of fresh biomass.
• the propagation bioreactor A retains a residue of 2 L of suspension to which is added a volume of 8 L of spilled propagation medium so as to restart cell propagation.
• the production bioreactor B is supplemented with weaning medium so as to reach a volume of 10 L and a cell density of 170 g / l.
• the culture in the production bioreactor is maintained as it is for 4 to 6 days so as to completely wipe out the trace biomass of the auxin-like growth hormones such as 2,4D present in the propagation medium.
• the eliciation cocktail is introduced into the production bioreactor B.
• the production of triptolide is thus conducted for 15 to 21 days.
• the culture supernatant is recovered by a pre-filtration step (15-20 ⁇ ) followed by filtration at 0.2 ⁇ . A clear solution is obtained.
• the concentration of triptolide obtained is 20 to 35 mg / l in the extracellular medium.
• This type of binary culture can be performed at larger fermenter scales (> 100 L in volume).
• the Wave A bioreactor (10L) placed on its support is filled with the nutrient medium by in-line sterilizing filtration and inflated with air. It is then inoculated thanks to a pre-culture performed in Erlenmeyer flask stirred for 15 days in propagation medium, in a stirred incubator.
• the bioreactor is incubated according to the following conditions:
• a volume of about 500 ml of culture of bag A is transferred to bag B (10 L) placed next to bag A of the tray.
• the culture in the elicitation phase is followed by the measurement of certain parameters, see FIG. 4.
• the maximum concentration of triptolide is 55 mg per liter of culture supernatant, after 19 days of incubation.
• the production rate of triptolide is about 2.87 mg / L of acellular supernatant and per day of culture.
• the kinetics of triptolide production begin to decline shortly after consuming almost all of the sucrose available.
• a clear colored solution (yellow or pink mauve) is obtained.
• Incubation temperature + 27 ° C for all phases (cell growth and elicitation in vials and bioreactor).
• the 500 ml inoculum is transferred to the single-use bioreactor installed and filled with sterile nutrient media, using sterile connection. This one is incubated on the rocker according to the following conditions:
• Wave Bioreactor Elicitation a volume of 2 liters of this culture is elicited by dilution in 2 liters of weaning medium containing the concentrated elicitation cocktail 2 times, the incubation is continued under the following conditions:
• the dry mass in suspension DW it increases from 5g / liter to 13g / liter in 10 days, stagnates for 10 days, then decreases slightly to 10g / I itre in 10 days, probably due to early cell lysis.
• the pH is fairly stable for 20 days (value around 5.5), then increases gradually to 7 over the last 10 days of culture. This rise in pH could be attributed to cell lysis, and release of the cytoplasmic contents.
• Example 8 Production of an extract enriched with triptolide of IBO.18.130 roots:
• the roots of Trypterigium wilfordii are peeled, dried and crushed. They are then extracted with 90% ethanol. Once concentrated, the extract is subjected to liquid / liquid extraction with 1,2-dichloroethane. The chlorinated phase is washed with a basic solution (NaOH), concentrated and adsorbed on silica. This crude silica extract is stored at -20 ° C.
• the NF- ⁇ transcription factor controls the expression of a large number of genes involved in the regulation of inflammatory response.
• Some pro-inflammatory stimuli such as TNF-cc (Tumor Necrosis Factor a) lead to the activation of NF- ⁇ that is to say to its nuclear translocation. Therefore, NF- ⁇ will induce the transcription of pro-inflammatory genes encoding cytokines, chemokines, adhesion molecules, growth factors and inducible enzymes such as cyclooxygenase 2 (COX2) and nitric oxide synthase (COX2). iNOS). NF- ⁇ plays a key role in the initiation and amplification of the inflammatory response.
• Some chronic inflammatory diseases of the skin such as atopic dermatitis or psoriasis, are characterized by a deregulation of the level of expression of mediators of inflammation expressed by keratinocytes.
• the anti-inflammatory activity of pure TRP compared with that of plant cell culture extracts (IBO 18.134) at equivalent doses of TRP is evaluated.
• Figure 6 shows the inhibition of different extracts on NF- ⁇ activation following TNF- ⁇ stimulation in HaCat keratinocyte cells.
• Dexamethasone (2 ⁇ ) which inhibits NF- ⁇ activation by 36%, was used.
• Pure Thptolide dose-dependently inhibits 8nM at 83nM.
• the objective of this study was to compare the anti-inflammatory activity of the CCV extract (IB-134) with that of pure thptolide.
• RAW264.7 cells murine macrophages
• RAW264.7 cells were seeded at 1, 4 x 10 5 cells / cm 2 . After 24 hours, the cells were incubated for 1 hour with different concentrations of the test products and then stimulated for 24 hours with 1 ⁇ g / ml of LPS.
• the NO2 was estimated in the culture supernatants using the Griess reagent.
• the comparison of IC50 shows that the CCV is greater than the TRP.
• the CCV extract is more active than the pure triptolide for inhibiting NO 2 ".
• PAR-2 Protease-Activated Receptor-2
• PAR-2 belongs to the superfamily of G-protein coupled transmembrane domain receptors, but has a unique activation pathway.
• PAR-2 is activated by serine proteases such as trypsin, tryptase, Xa and Vlla factors.
• serine proteases such as trypsin, tryptase, Xa and Vlla factors.
• the cleavage by these proteases of the extracellular part of the receptor exposes a new amino-terminal domain (SLIGKV) which acts as a ligand "attached" to the receptor: it binds to itself at the level of the extracellular loop 2 and auto-active.
• PAR-2 is expressed by the different cell types of the skin:
• keratinocytes myoepithelial cells of the sweat glands, hair follicle, dendritic-like cells of the dermis and the endothelial cells of the lamina intestinal and dermis.
• Melanocytes do not express this receptor although PAR-2 plays an important role in pigmentation by promoting melanin transfer from melanocytes to keratinocytes.
• serine proteases generated by the epidermis exert chemotactic effects inducing the recruitment of leucocytes into the skin. They are also involved in the regulation of epidermal homeostasis, mitogenesis and differentiation and modulate the barrier function of the skin. In addition, serine proteases contribute to the pathophysiology of cutaneous diseases related to inflammation, host defense, carcinogenesis, fibrosis and nerve stimulation.
• PAR-2 receptors are over-expressed in the epidermis, dermis and vessels of inflammatory skin diseases such as atopic dermatitis, lichen planus and psoriasis. PAR-2 receptors would also play a role in the development of pruritus in patients with atopic dermatitis.
• IL-8 chemokine-attractive chemokine for leukocytes, has been shown to allow the infiltration of neutrophils into the epidermis of patients with Psoriasis vulgaris.
• Intracellular signaling of the PAR-2 receptor is partially underpinned by intracellular calcium mobilization.
• the extracts were solubilized in DMSO to a stock solution of 200 mg / ml expressed as a concentration of pure triptolide. This concentration is imposed by the solubility of the root extract which requires special attention (dissolution at room temperature with gentle stirring).
• the compounds were solubilized extemporaneously for the pharmacological tests which were carried out for the 2 extracts at 2.5 ng / ml in equivalence concentration of pure triptolide.
• NHEK Normal human epidermal keratinocytes
• the NHEK cells are inoculated and cultured in Keratinocyte-SFM culture medium.
• the culture medium is replaced with medium containing or not containing (control) the extracts under test.
• the mixture (Polyl: C, IL4, IL13, TNF) is added in the case of Atopic Dermatitis.
• the mixture (IL17, Oncostatin M, TNF) is added.
• a control without inductor and without compound is also carried out in parallel, allowing us to validate the induced model (NHEK vs NHEK + inductors).
• RNA is extracted from the cells after a 24 hour incubation with the inducer +/- mixture at 2.5 ng / ml pure triptolide equivalents.
• Root extract (IBO.18.130) The root extract has a moderate anti-inflammatory effect on this model by reversing the effects of the pro-inflammatory mixture. Indeed the expression of the innate immunity marker S1 00A7, ch imiokines (CCL3, CCL5 and IL8) and the oxidative stress marker (HMOX1) was repressed while the expression of the marker involved in the differentiation of keratinocytes, KRT1 0, was stimulated (Table 1).
• the plant cell culture extract (CCV) has a stronger anti-inflammatory effect than the root extract by repressing the expression of the innate immunity markers S100A7 and RNase7, the cytokines TSLP and IL1A, the chemokines (CCL3, CCL5 and IL8) and the oxidative stress marker (HMOX1) ( Figure 9).
• Interleukin 18 Interferon-gamma-inducing factor
• HMOX1 Heme oxygenase (decycling) 1
• the cytokine mixture induces an increase in the expression of genes coding for anti-microbial or innate immunity peptides (CAMP, DEFB103A, DEFB4, PI3, S100A7, S100A7A, SPLI and TLR2) in chemotaxis (CCL5, CXCL5, CXCL10 and IL8), inflammation (IL1B), extracellular matrix degradation (MMP1 and MMP3) and cell defense against oxidative stress (HMOX1).
• CAMP anti-microbial or innate immunity peptides
• the root extract has an anti-inflammatory effect on this model. Indeed, a dozen genes induced by the cytokine cocktail are repressed by the extract. A dose effect on certain genes can also be observed (Figure 10).
• the plant cell culture extract also has an anti-inflammatory effect on this model. Indeed, thirteen genes induced by the cytokine cocktail are repressed by a factor of> 2 by the extract. The anti-inflammatory effect on the psoriasis model is more marked with the plant culture extract than the root extract ( Figure 10).
• Table 2 Genes tested after induction of a psoriasis phenotype
• S100A7A S100 calcium binding protein A7A
• CEACAM1 Carcinoembryonic antigen-related cell adhesion molecule 1
• Matrix metallopeptidase 3 (stromelysin 1, progelatinase)
• TRP TRP (MP0001 128), root extract (IBO 18.130) and CCV extract (IBO 18.134) with identical concentrations of TRP.
• the NHEK cells are seeded in 96 well microplates, black and incubated at 37 ° C., 5% CO 2 for 24 hours.
• the 96-well microplates are centrifuged for 10 minutes at 1200 RPM
• NHEK cells are contacted with increasing doses of the different compounds.

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