EP1015600A1 - Fermentative herstellung von statin - Google Patents

Fermentative herstellung von statin

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Publication number
EP1015600A1
EP1015600A1 EP98945280A EP98945280A EP1015600A1 EP 1015600 A1 EP1015600 A1 EP 1015600A1 EP 98945280 A EP98945280 A EP 98945280A EP 98945280 A EP98945280 A EP 98945280A EP 1015600 A1 EP1015600 A1 EP 1015600A1
Authority
EP
European Patent Office
Prior art keywords
host cell
formula
polynucleotide
compound
fungal
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Withdrawn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
EP98945280A
Other languages
English (en)
French (fr)
Inventor
Adriaantje Ykema
Hugo Streekstra
Rudolf Gijsbertus Marie Luiten
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.)
Koninklijke DSM NV
Original Assignee
DSM NV
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by DSM NV filed Critical DSM NV
Priority to EP98945280A priority Critical patent/EP1015600A1/de
Publication of EP1015600A1 publication Critical patent/EP1015600A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • 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
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the present invention relates to fungal cells that comprise a foreign polynucleotide that includes a hydroxylase enzyme.
  • the invention also relates to the production, by such cells, of hydroxy-containing compound (s) , that may be useful for reducing cholesterol levels, by culturing these cells .
  • hydroxy-containing compound (s) that may be useful for reducing cholesterol levels, by culturing these cells .
  • the "statins” are a family of compounds that are usually inhibitors of 3-hydroxy-3-methylbutyryl coenzyme A(HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis. As HMG-CoA reductase inhibitors, the statins are able to reduce plasma cholesterol levels in various mammalian species, including man, and are therefore effective in the treatment of hypercholesterolemia.
  • statins known today only two are producable by fermentation de novo . These are lovastatin
  • statins are produced by chemically or enzymatically derivatising lovastatin or compactin.
  • pravastatin also referred to as CS-514 in the art
  • lovastatin or compactin 1 is a more potent HMG-CoA reductase inhibitor than lovastatin or compactin 1 .
  • pravastatin or other similar hydroxy- containing statin-like compounds
  • This reduces yields, and increases costs.
  • One factor that has, until now, hindered progress towards a more efficient biosynthesis is the fact that compactin has mild anti- fungal properties which means that in the second hydroxylating stage manufacturers of pravastatin have used bacteria, such as of the genus Streptomyces . Indeed, many fungi have a low tolerance of
  • compactin and therefore, today, the conversion of compactin to pravastatin is preferably effected using bacteria 3 .
  • the present invention seeks to solve, or at least mitigate, some of the problems encountered in prior art processes, and seeks to provide a more efficient process for preparing various hydroxy-containing statins, in particular pravastatin. Certainly, it would be desirable to be able to produce a hydroxy-containing statin by a more efficient metho .
  • each of R 1 and R- independently represent a hydrogen atom, a hydroxy, C ⁇ _ 10 alkyl, C ⁇ o alkoxy, C 3 _ 10 cycloalkyl or C 6 _ 10 aryl group or a C 7 -u aralkyl group optionally containing one or more hetero atoms ,-
  • R 3 represents R x C0- or R fOlO-; each of R 4 and R D independently represent a hydrogen atom, -COOR 1 (except that then R 1 is not a hydroxy or alkoxy group) , -OR 1 or -COR 1 or, when combined complete a six-membered carbon ring having one or two oxygen heteroatoms ; wherein each of the alkyl, alkoxy, cycloalkyl, aryl and/or aralkyl groups can be optionally substituted with one or more halogen atoms, trifluoromethyl, hydroxy or C ⁇ alkoxy groups; and w, x, y and z represent none, 1 or when taken together up to 2 double bonds ; or a salt and/or isomer thereof; the process comprising culturing a fungal host cell that expresses a foreign hydroxylase enzyme under conditions that allow biosynthesis to take place.
  • alkyl and alkoxy groups can be either straight or branched.
  • Alkyl groups are preferably Cj_ 4 alkyl groups, for example methyl or ethyl.
  • Alkoxy groups are preferably methoxy or ethoxy groups .
  • Preferred cycloalkyl groups are pentyl or hexyl groups .
  • Suitable aryl groups include phenyl or naphthyl .
  • R 1 represents a hydrogen atom or a methyl group.
  • R 2 represents a hydroxy group.
  • R preferably represents -OC(0)R 1 , where R 1 is a C ⁇ K , alkyl group, for example a C ⁇ alkyl group.
  • R J represents a 2 -methyl butyrate group (-OC(0)CH(CH 3 )C 2 H 5 ) .
  • R 4 preferably represents a hydroxy group and/or R 5 preferably represents -COOH.
  • R 4 and R 5 are not combined the compound of formula (I) is in the acid form: this can be converted to the lactone form by ring closure, so that R ⁇ and R D when combined together can form a 6-membered ring (of carbon atoms) containing one or two oxygen heteroatoms .
  • R 4 and R 5 combined preferably represent -C(0)0-.
  • double bonds are present: in this situation, generally double bonds will be in the locations w and y or x and z .
  • Compactin, of formula (I) is the same as pravastatin as shown in Table 1 above but does not possess the hydroxy (HO-) group at the 6 -position.
  • the various isomers of compounds of formula (I) are included within the invention. This of course includes stereoisomers .
  • Salts include acidic and/or basic salts formed with inorganic and/or organic bases. Non-toxic, pharmaceutically acceptable salts are preferred. These include ammonium salts, as well as alkali metals (e.g. sodium, potassium and lithium) or alkaline earth metal salts (calcium and/or magnesium) .
  • Preferred compounds of formula (I) are HMG-CoA reductase inhibitors . Preferably they are hydroxylated derivatives of compounds of formula (II) .
  • Suitable compounds of formula (II) are polyketides. Polyketides are compounds that are biosynthesised internally by microorganisms, usually de novo, using low molecular weight precursors, for example
  • acetyl moieties 5 The acetyl moieties in turn can be obtained by the fungus from carbon sources such as starch, glucose, glycerol, etc.
  • the biosynthesis of compounds of formula (I) can be in the invention a one-step process, as opposed to a two-step process in the prior art.
  • the host cell employed is not only able to produce compounds of formula (II) , but it is also capable of converting, by hydroxylation, the compound of formula (II) to the compound of formula (I) . Both these processes may take place inside the cell and the latter can be achieved by the foreign hydroxylase enzyme. In this manner, the process of the invention can be significantly cheaper and more efficient than the 2-step processes used in the prior art.
  • the fungal host cell can preferably biosynthesise the compound of formula (I) de novo, that is to say from
  • the fungal host cell does not need to be fed or supplied with a compound of formula (II) , in order to be able to biosynthesise compounds of formula (I) , as it will usually be able to biosynthesise compounds of formula (II) itself.
  • the hydroxylase enzyme is "foreign" to the cell in the sense that it is heterologous to that cell. In other words, the cell would not normally express that hydroxylase enzyme.
  • the naturally occurring or wild-type version of the fungal host cell will not express that hydroxylase enzyme (or at least not to a significant extent) .
  • that fungal host cell will not normally, such as in its native state, express such a hydroxylase enzyme at all, although preferably it will not express a hydroxylase enzyme that is capable of hydroxylating compounds of formula (II) to form compounds of formula (I) .
  • the fungal host cell is preferably capable of biosynthesising a compound of formula (II), and by expression of the foreign hydroxylase enzyme can convert that compound into a compound of formula (I) •
  • the fungal host cell can be any suitable fungus .
  • the fungus is of the genus Paecilomyces
  • Preferred fungi are of the species Penicillium
  • Penicillium hrevicompac turn ci trinum or Penicillium hrevicompac turn .
  • Other suitable strains that can produce compactin are additionally contemplated 4 .
  • the most preferred fungus is Penicillium
  • the fungal host cell can be cultured in any suitable culture medium, and under conditions known to a person skilled in the art, that will allow the biosynthesis of compounds of formula (I) .
  • the cell will usually be able to biosynthesise compounds of formula (II) , such compounds are absent from this medium.
  • culture takes place under aerobic conditions, suitably in a culture medium that provides the fungus with all the ingredients it needs to biosynthesise compounds of formula (I) .
  • a culture (e.g. liquid growth) medium that includes malt extract, glucose, peptone is usually sufficient.
  • Agar can be used if the medium is solid.
  • a culture medium containing 2% malt extract, 2% glucose, 1% peptone (and 2% agar if solid) may be used.
  • the culture medium can contain any and all of the known nutrient materials for the fungus in order to allow biosynthesis to take place.
  • an assimilable carbon source and nitrogen source are preferably present .
  • a carbon source one can include glucose, glycerol, maltose, dextrin, dextrose, starch, lactose, sucrose, molasses, soybean oil, cotton seed oil etc. Glucose, dextrose and glycerol are preferred.
  • soybean meal As a nitrogen source, one can mention soybean meal, peanut meal, cotton seed meal, fish meal, corn steep liquor, peptone, rice bran, meat extract, yeast, yeast extract, sodium nitrate, ammonium nitrate, ammonia and ammonium sulphate.
  • Yeast extract is preferred.
  • Certain inorganic salts may additionally be present, for example sodium chloride, phosphate ions, calcium carbonate, iron (II) sulphate as can vitamins and/or amino acids . A minor amount of other metal salts may be added, if necessary.
  • the carbon and/or nitrogen source can be supplied at the beginning of biosynthesis, for example as in a batch process, or alternatively (and preferably) the carbon and/or nitrogen source is administered continuously and/or continually, as described in International Patent Application No. PCT/EP98/01123 filed on 20 February 1998.
  • this may be conducted with aeration and/or agitation.
  • the temperature of the culture medium is preferably from 20 to 30°C, such as from 24 to 26°C.
  • the fungal host cell can be cultured from 20 to 240 hours, preferably from 48 to 216 hours.
  • the compound of formula (I) can then be extracted by using a solvent and/or chromatography .
  • an organic solvent is used, for example ether, benzene, ethanol, ethyl acetate, chloroform, acetone or acetic acid.
  • the solvent is a combination of ethanol and ethyl acetate, or methanol, and column chromatography is employed.
  • the hydroxylase enzyme can be a naturally occurring enzyme, or can be a synthetic enzyme, for example a mutant or variant of a natural enzyme. Mutants and variants can be prepared either by truncation, or by addition, deletion or substitution of one or more amino acids as is known in the ar .
  • Preferred hydroxylase enzymes are from the species Amycolata autotrophica such as ATCC 35204, Streptomyces
  • ATCC 21411 Saccharothrix australensis
  • ATCC 21411 Saccharothrix australensis
  • Saccharopolyspora hirsuta such as ATCC 27875, 27876
  • Saccharopolyspora erythraea such as ATCC 11635
  • Streptomyces carbophilus such as FERM-BP1145 and Streptomyces
  • flavovirens such as SANK 63684. However this can exclude a
  • the enzyme is suitably not capable of converting deacetoxycepholosporin C (DAOC) to diacetyl cephalosporin C (DAC) .
  • the biosynthesis in the process of the first aspect can be accomplished by using host fungal cells which form the second aspect .
  • the second aspect of the invention therefore relates to a fungal host cell comprising a polynucleotide that encodes a foreign hydroxylase enzyme.
  • the host cell is thus suitably able to express the foreign hydroxylase enzyme which can convert compounds of formula (II) (e.g. biosynthesised within the host cell) into compounds of formula (I) .
  • the host cell will be one that, in the absence of the foreign hydroxylase gene, produces compactin.
  • a third aspect of the present invention relates to a vector comprising a polynucleotide comprising a sequence encoding a hydroxylase enzyme which is operably linked to a fungal expression signal and/or fungal promoter.
  • the vector is thus one that is effective in a fungal host cell.
  • the polynucleotide can therefore be adapted so that, when inside a host fungal cell, it allows the enzyme to be expressed by that cell.
  • the promoter or expression signal (s) can be from any fungus as described for the first aspect. However, it does not necessarily have to be from the same genus or species as the host cell.
  • Preferred promoters are from the genus Aspergillus , such as from the species Aspergillus terreus or Aspergillus nidulans , or from Penicillium, such as from the
  • the vector can be a plasmid or of bacteriophage origin. It may additionally comprise a terminator region: this may also be from a fungus .
  • the fungal expression signals (which include promoter and/or terminator) region are from a (e.g. highly or constitutively) expressed gene, such as a glycolytic gene, for example 3-phosphoglycerate kinase ipgk) or glucose-6 -phosphate
  • the terminator is from the
  • the vector may further contain one or more additional sequences in order to optimise the production of compounds of formula (I) .
  • sequences may include, for example, a P450 reductase.
  • the vector will suitably also contain a selective or selectable marker or, if two or more vectors are used (such as in a co-transformation procedure) the marker can be in another vector.
  • This marker may confer resistance to one or more inhibitory substances (e.g. antibiotics) or the ability to utilise certain nutrients. For example, it may confer resistance to hygromycin or phleomycin.
  • a marker-free system can be employed 14 .
  • the vector can therefore be used to transform or transfect a fungal cell in order to produce the cell of the second aspect. Transformation and/or transfection can be accomplished using known techniques .
  • a fourth aspect of the invention therefore relates to a process for transfectmg or transforming a host cell (such as to produce a cell of the second aspect) , the process comprising transforming or transf ⁇ cting a fungal cell with a vector of the third aspect.
  • a fifth aspect of the present invention relates to polynucleotide comprising a sequence encoding a hydroxylase enzyme operably linked to a fungal expression signal and/or fungal promoter.
  • a sixth aspect relates to a compound of formula (I) when produced by the process of the first aspect or a host cell of the second aspect .
  • a seventh aspect of the invention relates to a pharmaceutical composition comprising the compound of the sixth aspect and a pharmaceutically acceptable carrier or excipient .
  • FERM-BP1145 6 ' 7 was grown at 28°C for 72 hrs in a liquid medium containing: yeast extract lg/1 casar ⁇ ino acids 4g/l glycerol 4g/l
  • CaCl 2 O.lg/l trace elements solution (0.1% ZnS0 4 .7H 2 0, 0.1% FeS0 4 .7H 2 0, 0.1% MnCl 2 .4H 2 0) 2ml/l glycine (to a final concentration of 0.5%).
  • the mycelium was then harvested by centrifugation, washed once with 0.7% NaCl solution, and subjected to a total DNA isolation procedure 8 .
  • oligonucleotide primers were prepared based on the published sequence 9 (with minor adaptations to facilitate cloning) : primer 8804 5 ' -CACCATGGCCGAGATGACAGAGAAAGCC-3 ' primer 8805 5 ' -CAGGATCCCGCTCGGTCACCAGGTGACC-3 * Oligon cieoti ⁇ e comoination 8804/8805 was used to amplify the hydroxylase gene from the S .
  • a DNA fragment about 1.3 kb long was obtained containing the hydroxylase gene.
  • the 1.3 kb blunt-end PCR fragment was inserted into the general cloning vector pCR- blunt ⁇ tratagene, La Jolla, USA) resulting m plasmi ⁇ pCRP450a.
  • a set of transformation vectors was constructed. These vectors were based on several general funga expression vectors of the pAN series'", particularly pAN7-l and pAN8-l.
  • pAN8-l was digested with Ncol and Smal and into it ligate ⁇ the 1.3 kb fragment encoding the hydroxylase gene, obtaine ⁇ by ⁇ igestion of pCRP450a by £coRV and partially by Ncol.
  • the ligation mixture was mtro ⁇ uced m E. coll strain XLl-Blue (electrocompetent cells, Stratagene, La Jolla, USA) using tr.e tecnnique recommended by the supplier.
  • plasmid pANP45Ca was created and isolated, derived from insertion of the PC? fragment obtained from primer combination 8804/8805.
  • the identity of the pias id constructs of E . con transformants was confirmed by restriction enzyme analysis and DNA sequencing.
  • Protoplast formation, transformation with piasmid DNA, and regeneration of P. ci trinum was carried out similar to known methods for transforming filamentous fungi. lx
  • P. ci trinum ATCC 38065 was incubated on plates containing PDA (potato dextrose agar,D ⁇ fco) at 26°C for 10 days.
  • PDA potato dextrose agar,D ⁇ fco
  • YGG medium 6.6 g/1 Yeast Nitrogen Base (Difco)
  • 1.5 g/1 citric acid 1.5 g/1 citric acid.
  • conidiospores were added to a final concentration of 10 7 spores/ml.
  • Protoplasts were separated from the mycelium by filtration through sterile MyraclothTM.
  • the protoplast suspension was diluted (1:1) in STC buffer consisting of 1.2 M sorbitol, 10 mM Tris-HCl (pH 7.5), and 50 mM CaCl , and incubated on ice for 5-10 mm.
  • Protoplasts were collected by centrifugation (3000 rpm, 10 mm, 0°C) and wasned twice with STC. Finally, the protoplasts were resuspended at a final concentration of 10 7 - lOVml in STC.
  • Transformation of P. ci trinum was carried out by cotransformation cf plasmids pANP450a and pAN7-l as follows: 40 ⁇ l of a solution containing 40 ⁇ g of piasmid DNA 20 ⁇ g of each piasmid) m the presence of 200 mM aunn tricarboxyiic acid was carefully mixed with 100 ⁇ l of 20% PEG 4000 m STC, followed by 200 ⁇ l protoplast—suspension and incubated for 20 mm at 0°C. Next, 100 ⁇ l of 60% PEG 4000 m STC was added, followed by a 20 minute incubation at room temperature.
  • a batch of culture medium with the following composition was prepared:
  • Glucose 75 Og Yeast extract (Difco) 5.0g
  • the transformant strain numbers PRA201 to PRA208 were maintained on agar slants, and allowed to sporulate.
  • the spores were collected from the agar medium, suspended in sterile water, and used to inoculate 100ml shake flasks, containing 25ml of the culture medium.
  • the production cultures were incubated at 25°C and 250rpm on an orbital shaker. After 5 days of incubation, the cultures were sampled for statin production.
  • One ml of culture broth was transferred to a plastic tube, and diluted with 1ml methanol.
  • the tube was stoppered, and subsequently shaken for 30 minutes on a Vortex shaker. Subsequently the tube was centrifuged to remove the precipitate.
  • the supernatant fraction was analysed using HPLC .
  • the production of pravastatin was observed by the appearance of a peak at the characteristic retention time.
  • the identity of this peak was confirmed by the addition of a pravastatin standard to the sample, which caused an increase of this peak, and by analysis of the UV spectrum, which was identical to that of a genuine pravastatin standard.
  • the production levels (m mg/1) of the individual transformants is as follows:
  • PRA 205 1.78 / 1.12 PRA 206 5.00 / 5.92

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EP98945280A 1997-08-22 1998-08-24 Fermentative herstellung von statin Withdrawn EP1015600A1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP98945280A EP1015600A1 (de) 1997-08-22 1998-08-24 Fermentative herstellung von statin

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP97306462 1997-08-22
EP97306462 1997-08-22
PCT/EP1998/005362 WO1999010499A1 (en) 1997-08-22 1998-08-24 Statin production by fermentation
EP98945280A EP1015600A1 (de) 1997-08-22 1998-08-24 Fermentative herstellung von statin

Publications (1)

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EP1015600A1 true EP1015600A1 (de) 2000-07-05

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EP (1) EP1015600A1 (de)
AU (1) AU9264598A (de)
WO (1) WO1999010499A1 (de)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2949324A1 (de) 2014-05-27 2015-12-02 Consorci Institut Catala de Ciencies Cardiovasculars Prävention und/oder Behandlung von Ischämie-/Reperfusionsschäden
EP3381452A1 (de) 2017-03-31 2018-10-03 Instituto Catalán de Ciencias Cardiovasculares (ICCC), Hospital de la Santa Creu i Sant Pau, Avda. Statin zur verhinderung/verringerung von beschädigung im zusammenhang mit ischämie
WO2018178275A1 (en) 2017-03-31 2018-10-04 Instituto Catalán De Ciencias Cardiovasculares (Iccc), Hospital De La Santa Creu I Sant Pau, Avda. Statin for prevention/reduction of ischemia-related damage

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CZ20012728A3 (cs) 1999-01-29 2002-02-13 Kyowa Hakko Kogyo Co., Ltd. Způsob výroby inhibitorů HMG-CoA reduktasy
JP3881240B2 (ja) * 1999-11-30 2007-02-14 テバ ジョジセルジャール ザ−トケルエン ムケド レ−スベニュタ−ルシャシャ−グ 醗酵ブロスからスタチン化合物を回収するための方法
SK8312002A3 (en) 1999-12-14 2003-05-02 Biogal Gyogyszergyar Novel forms of pravastatin sodium
IL161079A0 (en) * 2001-09-27 2004-08-31 Biocon Ltd A strain of streptomyces flavidovirens and process for the manufacture of pravastatin utilizing the same
CA2546377A1 (en) 2003-11-24 2005-06-09 Teva Gyogyszergyar Zartkoruen Mukodo Reszvenytarsasag Method of purifying pravastatin
US20100048938A1 (en) * 2006-06-22 2010-02-25 Marco Alexander Van Den Berg Fermentation of pravastatin
EP1953233A1 (de) * 2007-02-02 2008-08-06 LEK Pharmaceuticals d.d. Fermentationsverfahren zur Herstellung von Pravastatin

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EP2949324A1 (de) 2014-05-27 2015-12-02 Consorci Institut Catala de Ciencies Cardiovasculars Prävention und/oder Behandlung von Ischämie-/Reperfusionsschäden
EP3381452A1 (de) 2017-03-31 2018-10-03 Instituto Catalán de Ciencias Cardiovasculares (ICCC), Hospital de la Santa Creu i Sant Pau, Avda. Statin zur verhinderung/verringerung von beschädigung im zusammenhang mit ischämie
WO2018178275A1 (en) 2017-03-31 2018-10-04 Instituto Catalán De Ciencias Cardiovasculares (Iccc), Hospital De La Santa Creu I Sant Pau, Avda. Statin for prevention/reduction of ischemia-related damage

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