CN104698108B - A kind of method utilizing immobilised enzymes screening I type 17 beta hydroxysteroid dehydrogenase inhibitors - Google Patents
A kind of method utilizing immobilised enzymes screening I type 17 beta hydroxysteroid dehydrogenase inhibitors Download PDFInfo
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Abstract
The invention provides a kind of method utilizing immobilised enzymes screening I type 17 beta hydroxysteroid dehydrogenase inhibitors, belong to zymetology and technical field of enzyme engineering.I type 17 11-beta-hydroxysteroid dehydrogenase type (17 β HSD1) plays a significant role in treatment hormone-dependent diseases.At present, study 17 β HSD1 activity main with substrate radioactive label method based on, but owing to 17 β HSD1 resolvases easily inactivate, and 17 β HSD1 preparation be difficult to, each new medicament screen experiment is required to fresh human placenta to prepare enzyme source, raw material obtain and are difficult to, expensive, bring difficulty to new medicament screen work.The present invention, uses amido modified silicon ball to be carrier, utilizes Euplotes woodruffi, the fixing 17 β HSD1 extracting from placenta, set up external immobilization 17 β HSD1 catalator, with androstenedione as substrate, use high performance liquid chromatography detection product, screen 17 β HSD1 potential inhibitors.The method, overcomes the unstability of resolvase, and simple to operate, cost of manufacture is cheap, repeatable utilization.
Description
Technical field
The present invention relates to a kind of method utilizing immobilised enzymes screening I type 17 beta hydroxysteroid dehydrogenase inhibitors, especially one utilizes immobilised enzymes, and the method setting up external drug screening belongs to zymetology and technical field of enzyme engineering.
Background technology
I type 17 11-beta-hydroxysteroid dehydrogenase type (17 β-HSD1) is the enzyme that a class has oxidation restoring function, participate in the key enzyme of estrogen and male hormone metabolism, regulating and controlling effect is played to the redox reaction between the ketone group on its C17 position and alcohol radical, conversion between mutual conversion between catalysis high activity hormone and low activity hormone, i.e. testosterone, estradiol and androstenedione, oestrone.17 β-HSD1 are the catalyzing enzymes of final step in sex hormone building-up process, in close relations with breast cancer, carcinoma of endometrium, endometriosis etc., therefore play a significant role in treatment hormone-dependent diseases.(Design and validation of specific inhibitors of 17b-hydroxysteroid dehydrogenases for therapeutic application in breast and prostate cancer, and in endometriosis [J] .Day JM, Tutill HJ, Purohit A, Reed MJ, Endocr Relat Cancer, 2008,15:665-692.)
17 β-HSD1 are widely distributed, are primarily present in the tissues such as placenta, liver, testis, prostate.Nineteen fifty-seven Langer and Engel (Human placental estradiol-17beta dehydrogenase 1concentration, characterization and assay [J] .Langer LJ, Engel LL, JBiol Chem, 1958,233:583-588.) it is partially separated and purified placenta 17 β-HSD1.1962, (Purification of a 17 β-hydroxysteroid dehydrogenase of human placenta and studies on its transhydrogenase function [J] the .Jarabak J such as Jarabak, Adams JA, Williams-Ashman HG, Talalay P, JBiol Chem, 1962,237:345-357.) separate from human placenta, purified 17 β-HSD1, and the biological characteristics of this enzyme is described in detail.This is the 17 β-HSD1 finding the earliest.17 β-HSD1 can affect the metabolism of estrogen, reductase is expressed too much in the tissue, or Oxidase Expression is very few, all can cause tissue generates too much estrogen, cause the hyperplasia of local organization, cause the generation of disease, the especially disease of estrogen-dependent, breast cancer as higher in the current incidence of disease, carcinoma of endometrium etc..If effectively suppressing 17 β-HSD1, being possible not only to the synthesis of estrogen, can also blocking the effect of stimulin in the level before acceptor, therefore, the research of 17 beta-HSD 1 inhibitors, the therapeutic potential for estrogen-dependent diseases is great.At present, for 17 β-HSD1, oneself filters out some specific inhibitor, such as flavonoids, coumarin kind compound etc., and its curative effect of preliminary identification in some specific animal disease model.
nullAt present，Measure the method that 17 β-HSD1 enzymes are lived，Frequently with substrate radioactive label method (Insulin-like growth factor type I and insulin-like growth factor type II stimulate oestradiol-17 β hydroxysteroid dehydrogenase (reductive) activity in breast cancer cells [J] .Singh A，Reed MJ，JEndocrinol，1991，129:R5-R8.)，But this method employs radioactive material，Bring hidden danger to post processing and personnel protection.In addition to the above drawbacks, there is also following open defect with free aromatizing enzyme for model: (1) enzyme source is hard-earned, owing to there is no the aromatizing enzyme supply of commercialization, each screening test is required to find Freshman placenta to prepare microsome as the enzyme source of aromatizing enzyme, and raw material obtain difficulty；(2) the artificial resolvase preservation condition extracted is harsh, easy in inactivation；(3) aromatizing enzyme that dissociates does not dissolves in organic solvent, and many micromolecular compounds to be screened water insoluble, it is necessary to add organic solvent or emulsifying agent, thus affect enzymatic activity；(4) aromatizing enzyme that dissociates easily is assembled in course of reaction, makes enzyme molecule can not be fully contacted with substrate molecule, and catalytic efficiency is low, causes screening test failure；(5) activity of aromatizing enzyme that dissociates is unstable, and easily by environmental factor such as temperature, acid-base value etc. affects and inactivates.(6) reacted enzyme separates difficulty with product, it is impossible to recycling.Due to above reason, bring challenges usually to the screening study of 173-HSD1 potential inhibitor.
Enzymic catalytic reaction has the advantages such as efficient, single-minded and reaction condition is gentle, but the actual application of enzymic catalytic reaction is still subject to perhaps multifactorial restriction, as unstable in enzyme, easily inactivate, it is difficult to repeat recycling etc..For overcoming disadvantage mentioned above, people have developed the method for immobilised enzymes (Immobilized enzyme, IE).At present, enzyme immobilization technology is own is used widely in industries such as food industry, fine chemicals industry, medicine, its theory of development is to be to work intracellular based on endocellular enzyme, being similar to the immobilised enzymes made by embedding method, therefore immobilised enzymes may be considered to a certain extent in order to make enzyme react under its interior state.Immobilised enzymes, compared with resolvase, has many advantages: 1) easily separate with substrate, product；2) can carry out Reusability in a long time, cost reduces；3) stability of enzyme can be improved；4) can improve (Developments in immobilized-enzyme technology [J] .Powell LW, Biotechnol Genet Eng Rev, 1984,2:409-438.) such as catalytic efficiencies.Up to the present, there is not yet the immobilized research report with regard to 17 β-HSD1 both at home and abroad.
Enzyme immobilizatio method is often divided into absorption method, e, cross-linking method, investment and microencapsulation etc. according to mechanism difference.The present invention, with amido modified silicon ball as carrier, uses Euplotes woodruffi to fix 17 β-HSD1, and the immobilised enzymes stability that obtains improves, and repeatable utilizes 5 times.The present invention, by immobilization 17 β-HSD1, simulated in vivo environment, effective 17 beta-HSD 1 inhibitors of in-vitro screening, simple to operate effectively.
Content of the invention
The technical problem to be solved in the present invention is to provide a kind of method utilizing immobilised enzymes screening 117 beta-HSD 1 inhibitors.Mainly with amido modified silicon ball as carrier, utilize the fixing 17 β-HSD1 extracting from placenta of glutaraldehyde cross-linking.
To achieve these goals, a kind of method utilizing immobilised enzymes screening 117 beta-HSD 1 inhibitors of the present invention, comprises the steps:
1) extraction of 17 β-HSD1
Institute is all carried out in steps on 0～4 DEG C of ice cube.Placenta removes chorion and big blood vessel, with 50mM PBS (pH=7.4 includes 1%KCl), shreds with scissors after cleaning, is homogenized with 50mM PBS (including 0.25M sucrose).Centrifugal 2500 × g30min, discards precipitation, and supernatant centrifuges 60000 × g 60min with precipitation line plastochondria composition.Again discarding precipitation, after adding the ammonium sulfate solids of a certain amount of recrystallization in supernatant, precipitation accumulation, centrifugal rear supernatant discarded, by pellet resuspended in 50mMPBS (including 1mM EDTA and 1mM cysteine hydrochloride), and in-80 DEG C of preservations.
2) preparation of amido modified silicon ball
Taking 200mg silica gel, the hydrochloric acid adding 10～30mL concentration to be 5%～10%, in 90 DEG C of 10～12h that reflux, washing is to neutrality, and 100 DEG C are dried overnight, obtain activated silica gel.Take 50mg activated silica gel, add dry toluene and 3-aminopropyl triethoxysilane (APTES), under nitrogen protection, 90 DEG C of backflow 18～24h.Silica gel toluene and the acetone of upper for bonding APTES are washed successively, is dried overnight in 105 DEG C.
3) preparation of immobilised enzymes
The silicon ball that 100mg amination is modified is placed in the flask of original place, adds 10～20mL concentration to be 1%～2.5% glutaraldehyde solution, stirs 5～12h, and washing, to without glutaraldehyde smell, is dried overnight at 50～70 DEG C, is stored in refrigerator stand-by.Taking the crosslinked silicon ball of 50mg, adding PBS and the 17 β-HSD1 extracts that concentration is 4.8mg/mL, stir 8～12h at 4～25 DEG C, washing, to without resolvase, is dried overnight at 25～35 DEG C, and is stored in 0～4 DEG C.
4) mensuration of activity of the immobilized enzyme
Take described immobilised enzymes temperature and incubate substrates androstenedione 30min～24h, centrifuging and taking supernatant, utilize the content of high effective liquid chromatography for measuring each time point product testosterone, prepare enzymatic curve, calculate enzyme kinetics coefficient, as negative control.
5) screening of 17 beta-HSD 1 inhibitors
Taking described immobilised enzymes, adding compound to be screened, temperature incubates substrates androstenedione 30min～24h, centrifuging and taking supernatant, utilizes the content of high effective liquid chromatography for measuring each time point product testosterone, prepares enzymatic curve, calculate enzyme kinetics coefficient, and compare with negative control.Calculate the inhibiting rate under addition variable concentrations compound and IC50Value, evaluates inhibitory action.
Wherein, step 1) described in, 50mM phosphate buffer PBS (pH=7.4) component is: the content of disodium hydrogen phosphate is 19.332g/L, and the content of sodium dihydrogen phosphate is 1.976g/L, and remaining is water.
Step 2) described in, 10～30mL concentration is the hydrochloric acid of 5%～10%, and preferably 20mL concentration is the hydrochloric acid of 10%.
Step 2) described in, silicon spherolite footpath is 5～50 μm.
Step 2) described in, add toluene to be 3～10mL, APTES is 20～30 μ L, preferably 5mL toluene, 25 μ LAPTES.
Step 3) described in, glutaraldehyde cross-linking reaction system pH is 7.4, owing to glutaraldehyde is shown in that light decomposes, and lucifuge during reaction stirring.
Step 3) described in, add 10～20mL concentration to be 1%～2.5% glutaraldehyde solution, preferably 10mL concentration is the glutaraldehyde solution of 2.5%.
Step 3) described in, add PBS to be 0.5～1mL, 17 β-HSD1 extracts are 50～100 μ L, and preferably PBS is 1mL, and 17 β-HSD1 are 100 μ L.
Step 3) described in, 8～12h is stirred in enzyme immobilization reaction at 4～25 DEG C, stirring stirring 12h at preferably 25 DEG C.
Step 4) and step 5) described in, the temperature condition of incubating of enzyme is: pH=8.0, and temperature is 37～45 DEG C, and androstenedione concentration is 30～40 μM, adds volume to be 100～150 μ L, and NAPDH concentration is 20～25mg/mL, adds volume to be 10～20 μ L.
Step 4) and step 5) described in, high-efficient liquid phase chromatogram determining condition: fixing is octadecylsilane chemically bonded silica mutually, flowing is acetonitrile mutually: 0.1% aqueous formic acid (70: 30), detection wavelength is 240nm, column temperature is 25～35 DEG C, flow velocity is 1.0mL/min, and sampling volume is 20 μ L.
Step 4) and step 5) described in, blank is that the temperature being added without NAPDH incubates system.
Step 5) described in, inhibiting rate computing formula is: I (%)=(A1-A2)/A1 × 100%, A1The peak area of product during for being added without inhibitor, A2 is for adding the peak area of inhibitor afterproduct.With inhibitor concentration as abscissa, inhibiting rate is ordinate, formulates regression curve, calculates IC50 value.
The invention provides a kind of method that external temperature incubates screening 17 beta-HSD 1 inhibitors, abandoned the method carrying out active medicine screening with traditional resolvase, be fixed on the 17 β-HSD1 extracting in placenta on amido modified silicon ball, the tolerance of the enzyme of raising.
Owing to enzyme is fixed in carrier, in pressed powder state, easily separate with substrate, reaction mixture system, the change of the direct detection substrate of high performance liquid chromatography and product assay can be passed through, and, immobilised enzymes can be recovered by centrifugation recycling.
Detailed description of the invention
Embodiment 1: the preparation of immobilised enzymes
1) extraction of 17 β-HSD1
Institute is all carried out in steps on 0～4 DEG C of ice cube.Placenta removes chorion and big blood vessel, with 50mM PBS (pH=7.4 includes 1%KCl), shreds with scissors after cleaning, is homogenized with 50mM PBS (including 0.25M sucrose).Centrifugal 2500 × g 30min, discards precipitation, and supernatant centrifuges 60000 × g 60min with precipitation line plastochondria composition.Again discarding precipitation, after adding the ammonium sulfate solids of a certain amount of recrystallization in supernatant, precipitation accumulation, centrifugal rear supernatant discarded, by pellet resuspended in 50mM PBS (including 1mM EDTA and 1mM cysteine hydrochloride), and in-80 DEG C of preservations.
2) preparation of amido modified silicon ball
Taking the silica gel 200mg that particle diameter is 5 μm, the hydrochloric acid adding 20mL concentration to be 10%, in 90 DEG C of 12h that reflux, washing is to neutrality, and 100 DEG C are dried overnight, obtain activated silica gel.Take 50mg activated silica gel, add 5mL dry toluene and 25 μ L3-aminopropyl triethoxysilane (APTES), under nitrogen protection, 90 DEG C of backflow 24h.Silica gel toluene and the acetone of upper for bonding APTES are washed 3 times successively, is dried overnight in 105 DEG C.
3) enzyme immobilizatio
The silicon ball that 100mg amination is modified, is placed in round-bottomed flask, adds 10mL concentration to be 2.5% glutaraldehyde solution, and under conditions of pH=7.4, lucifuge stirs 12h, and washing, to without glutaraldehyde smell, is dried overnight at 60 DEG C, is stored in refrigerator stand-by.Taking the crosslinked silicon ball of 50mg, adding 1mL PBS and the 17 β-HSD1 extract 100 μ L that concentration is 4.8mg/mL, stir 12h at 25 DEG C, washing, to without resolvase, is dried overnight at 30 DEG C, and is stored in 0～4 DEG C.
Example 2: the zymologic property of immobilised enzymes
1) enzymatic reaction optimum reaction conditions
Take appropriate immobilised enzymes and resolvase, be separately added into the androstenedione of 150 μ L concentration 35 μM, the NAPDH of 20 μ L concentration 25mg/mL, incubate 8h in 37 DEG C of temperature, measure the content of product testosterone.Its optimal reaction pH is 8.0, and optimal reactive temperature is 40 DEG C.After reusing 5 times, immobilised enzymes inactivates.
2) enzyme kinetics research
Taking described immobilised enzymes 20mg, being sequentially added into the NAPDH of 200 μ L concentration 25mg/mL, the androstenedione of 1500 μ L concentration 35 μM, after concussion mixes, average mark dresses up 10 pipes, and under the phosphate buffer of pH=8.0,37 DEG C of temperature incubate 0min, 15min, 30min, 1h, 2h, 4h, 6h, 10h, 12h, 24h, centrifuging and taking supernatant, utilize the content of high effective liquid chromatography for measuring each time point product, prepare enzymatic curve, calculate enzyme kinetics coefficient Vm and Km.
High-efficient liquid phase chromatogram determining condition: fixing is octadecylsilane chemically bonded silica mutually, flowing is acetonitrile mutually: 0.1% aqueous formic acid (70:30), detection wavelength is 240nm, column temperature is 30 DEG C, flow velocity is 1.0mL/min, and sampling volume is 20 μ L, and the detection time is 6min, the retention time of androstenedione is 3.85min, and the retention time of testosterone is 3.15min.
Example 3: the research to immobilised enzymes inhibition for the apiolin
1) apiolin enzymatic suppression curve
Take described immobilised enzymes 20mg, be sequentially added into the NAPDH of 200 μ L concentration 25mg/mL, the androstenedione of 1500 μ L concentration 35 μM, the apiolin of 100 μ L concentration 400nM, after concussion mixes, average mark dresses up 10 pipes, under the phosphate buffer of pH=8.0,37 DEG C of temperature incubate 0min, 15min, 30min, 1h, 2h, 4h, 6h, 10h, 12h, 24h, centrifuging and taking supernatant, utilize the content (deduction is corresponding blank) of high effective liquid chromatography for measuring each time point product, prepare enzymatic curve, calculate enzyme kinetics coefficient VmAnd Km。
2) apiolin IC50The mensuration of value
Take described immobilised enzymes 160mg, it is sequentially added into the NAPDH of 160 μ L concentration 25mg/mL, the androstenedione of 1200 μ L concentration 35 μM, after concussion mixes, average mark dresses up 8 pipes, label 0～7 respectively, No. 0 pipe adds 20 μ LPBS buffer solutions, and it is 5 μM that 1～No. 7 pipe is separately added into 20 μ L concentration, 2 μM, 1 μM, 500nM, 250nM, 100nM, the apiolin of 50nM, after concussion mixes again, incubate 8h in 37 DEG C of temperature, measure the content of product testosterone, calculating the inhibiting rate of variable concentrations apiolin, its computing formula is: I (%)=(A1-A2)/A1× 100%, A1The peak area of product, A during for being added without inhibitor2For adding the peak area of inhibitor afterproduct.With inhibitor concentration as abscissa, inhibiting rate is ordinate, formulates regression curve, calculates IC50Value.
Example 4: the research to immobilised enzymes inhibition for the rheum emodin
1) rheum emodin enzymatic suppression curve
Take described immobilised enzymes 20mg, be sequentially added into the NAPDH of 200 μ L concentration 25mg/mL, the androstenedione of 1500 μ L concentration 35 μM, the rheum emodin of 100 μ L concentration 500 μM, after concussion mixes, average mark dresses up 10 pipes, under the phosphate buffer of pH=8.0,37 DEG C of temperature incubate 0min, 15min, 30min, 1h, 2h, 4h, 6h, 10h, 12h, 24h, centrifuging and taking supernatant, utilize the content (deduction is corresponding blank) of high effective liquid chromatography for measuring each time point product, prepare enzymatic curve, calculate enzyme kinetics coefficient VmAnd Km。
2) rheum emodin IC50The mensuration of value
Take described immobilised enzymes 160mg, it is sequentially added into the NAPDH of 160 μ L concentration 25mg/mL, the androstenedione of 1200 μ L concentration 35 μM, after concussion mixes, average mark dresses up 8 pipes, label 0～7 respectively, No. 0 pipe adds 20 μ LPBS buffer solutions, and it is 5mM that 1～No. 7 pipe is separately added into 20 μ L concentration, 2mM, 1mM, 500 μM, 250 μM, 100 μM, the rheum emodin of 50 μM, after concussion mixes again, incubate 8h in 37 DEG C of temperature, measure the content of product testosterone, calculating the inhibiting rate of variable concentrations rheum emodin, its computing formula is: I (%)=(A1-A2)/A1× 100%, A1The peak area of product, A during for being added without inhibitor2For adding the peak area of inhibitor afterproduct.With inhibitor concentration as abscissa, inhibiting rate is ordinate, formulates regression curve, calculates IC50Value.
The present invention is with by preferred embodiment disclosure as above; but it is not limited to the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, therefore protection scope of the present invention should be with being as the criterion that claims are defined.
Claims (6)
1. the method utilizing immobilised enzymes screening I type 17 beta hydroxysteroid dehydrogenase inhibitors, it is characterised in that comprise the steps:
Step one: the extraction of 17 β-HSD1: institute is all carried out in steps on 0～4 DEG C of ice cube；Placenta removes chorion and big blood vessel, is 7.4 with 50mM pH, shreds with scissors, be homogenized with the PBS of 50mM sucrose containing 0.25M after the PBS cleaning including 1%KCl；Centrifugal 2500 × g 30min, discards precipitation, and supernatant centrifuges 60000 × g 60min with precipitation line plastochondria composition；Again discarding precipitation, after adding the ammonium sulfate solids of a certain amount of recrystallization in supernatant, precipitation accumulation, centrifugal rear supernatant discarded, by pellet resuspended in the PBS of 50mM EDTA containing 1mM and 1mM cysteine hydrochloride, and in-80 DEG C of preservations；
Step 2: the preparation of amido modified silicon ball: take 200mg silica gel, the hydrochloric acid adding 10～30mL concentration to be 5%～10%, in 90 DEG C of 10～12h that reflux, washing is to neutrality, and 100 DEG C are dried overnight, obtain activated silica gel；Take 50mg activated silica gel, add dry toluene and 3-aminopropyl triethoxysilane, under nitrogen protection, 90 DEG C of backflow 18～24h；Silica gel toluene and the acetone of upper for bonding 3-aminopropyl triethoxysilane are washed successively, is dried overnight in 105 DEG C；
Step 3: the preparation of immobilised enzymes: the silicon ball that 100mg amination is modified is placed in round-bottomed flask, adding 10～20mL concentration to be 1%～2.5% glutaraldehyde solution, stirring 5～12h, washing is to without glutaraldehyde smell, it is dried overnight at 50～70 DEG C, be stored in refrigerator stand-by；Taking the crosslinked silicon ball of 50mg, adding PBS and the 17 β-HSD1 extracts that concentration is 4.8mg/mL, stir 8～12h at 4～25 DEG C, washing, to without resolvase, is dried overnight at 25～35 DEG C, and is stored in 0～4 DEG C；
Step 4: the mensuration of activity of the immobilized enzyme: take described immobilised enzymes temperature and incubate substrates androstenedione 30min～24h, centrifuging and taking supernatant, utilize the content of the corresponding blank afterproduct testosterone of high effective liquid chromatography for measuring each time point deduction, prepare enzymatic curve, calculate enzyme kinetics coefficient, as negative control；
Step 5: the screening of 17 beta-HSD 1 inhibitors: take described immobilised enzymes, add compound to be screened, temperature incubates substrates androstenedione 30min～24h, centrifuging and taking supernatant, utilize the content of the corresponding blank afterproduct testosterone of high effective liquid chromatography for measuring each time point deduction, prepare enzymatic curve, calculate enzyme kinetics coefficient, and compare with negative control；Calculate the inhibiting rate under addition variable concentrations compound and IC50Value, evaluates inhibitory action.
2. according to right 1 requires, utilizing the method for immobilised enzymes screening I type 17 beta hydroxysteroid dehydrogenase inhibitors, it is characterised in that in step 2, silicon spherolite footpath is 5～50 μm, adding toluene to be 3～10mL, 3-aminopropyl triethoxysilane is 20～30 μ L.
3., according to right 1 requires, utilize the method for immobilised enzymes screening I type 17 beta hydroxysteroid dehydrogenase inhibitors, it is characterised in that in step 3, glutaraldehyde cross-linking reaction system pH is 7.4, owing to glutaraldehyde is shown in that light decomposes, lucifuge during reaction stirring.
4. according to right 1 requires, utilizing the method for immobilised enzymes screening I type 17 beta hydroxysteroid dehydrogenase inhibitors, it is characterised in that in step 3, adding PBS to be 0.5～1mL, 17 β-HSD1 extracts are 50～100 μ L.
5. according to right 1 requires, utilize the method for immobilised enzymes screening I type 17 beta hydroxysteroid dehydrogenase inhibitors, it is characterized in that, in step 4 and step 5, the condition that enzyme temperature is incubated: pH=8.0, temperature is 37～45 DEG C, androstenedione concentration is 30～40 μM, adding volume to be 100～150 μ L, NAPDH concentration is 20～25mg/mL, adds volume to be 10～20 μ L.
6. according to right 1 requires, utilize the method for immobilised enzymes screening I type 17 beta hydroxysteroid dehydrogenase inhibitors, it is characterized in that, in step 4 and step 5, high-efficient liquid phase chromatogram determining condition: fixing is octadecylsilane chemically bonded silica mutually, flowing is acetonitrile and 0.1% aqueous formic acid mutually, volume ratio is 70: 30, and detection wavelength is 240nm, and column temperature is 25～35 DEG C, flow velocity is 1.0mL/min, and sampling volume is 20 μ L.
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| EP3768838A1 (en) | 2018-03-21 | 2021-01-27 | Regeneron Pharmaceuticals, Inc. | 17ß-HYDROXYSTEROID DEHYDROGENASE TYPE 13 (HSD17B13) IRNA COMPOSITIONS AND METHODS OF USE THEREOF |
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