CN103695497B - Enzymatic preparation of naloxone and pharmaceutical composition thereof - Google Patents
Enzymatic preparation of naloxone and pharmaceutical composition thereof Download PDFInfo
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- CN103695497B CN103695497B CN201310711466.8A CN201310711466A CN103695497B CN 103695497 B CN103695497 B CN 103695497B CN 201310711466 A CN201310711466 A CN 201310711466A CN 103695497 B CN103695497 B CN 103695497B
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- naloxone
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- dihydrocodeinone
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- 229960004127 naloxone Drugs 0.000 title claims abstract description 78
- UZHSEJADLWPNLE-GRGSLBFTSA-N naloxone Chemical compound O=C([C@@H]1O2)CC[C@@]3(O)[C@H]4CC5=CC=C(O)C2=C5[C@@]13CCN4CC=C UZHSEJADLWPNLE-GRGSLBFTSA-N 0.000 title claims abstract 24
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 230000002255 enzymatic effect Effects 0.000 title abstract 2
- 239000008194 pharmaceutical composition Substances 0.000 title description 3
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a method for enzymatic preparation of naloxone. The enzyme is mild in reaction condition and free of harm to a human body. In addition, the invention also provides a naloxone composition prepared by the method and application thereof. The composition contains 14-hydroxy-7,8-dihydro codein ketone impurities, but the medicine performance can not be affected. Furthermore, the invention also provides an identification method of the naloxone composition and enzyme and the like for preparation.
Description
Technical field
The invention belongs to medical technical field, particularly, the present invention relates to naloxone composition prepared by enzyme process, although contain impurity, do not affect its patent medicine performance.
Background technology
Naloxone, chemical name is 17-allyl group-4,5a-epoxy group(ing)-3,14-dihydroxyl morphinan-6-ones, conventionally with the form patent medicine of hydrochloride, be the antagonist of opiate receptor, be mainly used in reversing the narcoticness analgesia and the respiration inhibition that cause due to morphine class material (drugs), and can be used for saving that the materials such as ethanol cause is poisoning etc.
In recent years, to the research of naloxone be mainly conceived to its pharmaceutical preparation and with the coupling of other medicines on, as referring to Chinese patent (application) 03807796.5,200680005969.1,200510080479.5,201180023192.2,200880006847.3 etc.In addition, Chinese patent (application) 200710039118.5 and 201010211706.4 relates to the technology of preparing of naloxone and intermediate thereof, but still adopt chemical synthesising technology, since not jumping out the eighties, utilize Hydrogen bromide or chloroformic acid etc. to carry out synthetic framework, , taking thebaine as raw material, oxidation hydrogenation produces 14-hydroxyl-7, 8-dihydrocodeinone, then pass through complicated process, adding under the condition of protecting group, utilize the reagent such as Hydrogen bromide or chloroformic acid to carry out demethylation, then deprotection base, generate naloxone finally by alkylation, wherein reaction (especially demethylation process) condition is violent, reagent and residual large to harm.
The inventor has finally obtained a kind of demethylase that can identify three-dimensional condensed ring system, can be by 14-hydroxyl-7 under mild conditions, and 8-dihydrocodeinone changes into 14-hydroxyl-7,8-dihydro desmethylmorphine ketone, thus can directly generate naloxone through alkylation.In addition, remove 14-hydroxyl-7 of co-precipitation/crystallization, 8-dihydrocodeinone impurity can bring greater loss, and the inventor studies discovery, if removal of contamination and direct alkylation do not generate naloxone, the naloxone purity obtaining is still greater than 97%, meet medicinal requirements, and 14-hydroxyl-7 wherein, 8-dihydrocodeinone impurity does not form impact to its direct patent medicine, thereby can further reduce bulk drug cost as medicine.
Summary of the invention
The technical problem to be solved in the present invention is the method for preparing naloxone that provides new, and the enzyme that has wherein used prior art not point out makes enzyme reaction mild condition, substantially harmless to human body.In addition, the present invention also provides the naloxone composition and the application thereof that are prepared by the method, although contain impurity in said composition, does not affect its patent medicine performance.Further, the present invention also provides in the authentication method of naloxone composition and preparation enzyme used etc.
Particularly, in first aspect, the invention provides the method that enzyme process is prepared naloxone, it comprises,
(A) optionally thebaine hydrogen oxide is changed into 14-hydroxyl-7,8-dihydrocodeinone;
(B) under the effect of demethylase, by 14-hydroxyl-7,8-paracodin ketogenesis 14-hydroxyl-7,8-dihydro desmethylmorphine ketone purity is high and contain 14-hydroxyl-7, the composition of 8-dihydrocodeinone, the aminoacid sequence of wherein said demethylase:
(1) as shown in SEQ ID No:2, or
(2) be to SEQ ID No:2 disappearance, add and/or replace one or several amino-acid residue and the sequence of the reservation SEQ ID No:2 activity that obtains;
(C) 14-hydroxyl-7 in composition step (B) being obtained, 8-dihydro desmethylmorphine ketoalkylation becomes naloxone, obtains naloxone purity high also containing 14-hydroxyl-7, the composition of 8-dihydrocodeinone; With,
(D) be optionally further purified and/or salt acidification step (C) obtain composition.
This is that the enzyme process of reported first participates in preparing the method for naloxone, wherein use demethylase, its aminoacid sequence: (1) is as shown in SEQ ID No:2, or (2) are to SEQ ID No:2 disappearance, add and/or replace one or several amino-acid residue and the sequence of the reservation SEQ ID No:2 activity that obtains.
In this article, " optionally " has its lexical meaning, selects or do not select.For example, the method for first aspect present invention can select to comprise step (A), prepares naloxone taking thebaine (Thebaine) as raw material; Also can select not comprise step (A), with 14-hydroxyl-7,8-dihydrocodeinone is that raw material is prepared naloxone.Conventionally preferably the former.
And for example, the method for first aspect present invention can select to comprise step (D); Also can select not comprise step (D), produce naloxone purity high also containing 14-hydroxyl-7, the composition of 8-dihydrocodeinone.The preferred the latter of the present invention, preferably the former, because the inventor finds, naloxone purity is high also containing 14-hydroxyl-7, the composition of 8-dihydrocodeinone can be directly as medicine material and patent medicine, even may be useful to patent medicine character, without expensive later stage purge process (remove a small amount of 14-hydroxyl-7 from naloxone, 8-dihydrocodeinone needs expensive chromatographic separation process).So the method for first aspect present invention can be that enzyme process is prepared high 14-hydroxyl-7, the method for the composition of 8-dihydrocodeinone of also containing of naloxone purity.
In this article, as without contrary instruction, in composition, " purity " of composition and " content " can exchange use, all refer to this composition shared proportion in composition, and this area normalization method by HPLC just can calculate easily.Naloxone purity prepared by the method for first aspect present invention is high also containing 14-hydroxyl-7, in the composition of 8-dihydrocodeinone, naloxone purity is high, preferably in the method for first aspect present invention, in the composition obtaining in step (C), the purity of naloxone is greater than 95%, be preferably greater than 96%, more preferably greater than 97%, as be greater than 97.5%.
Because naloxone purity is high, 14-hydroxyl-7 in the composition obtaining in step (C), the content of 8-dihydrocodeinone is just low.Preferably in the method for first aspect present invention, in the composition obtaining in step (C), 14-hydroxyl-7, the content of 8-dihydrocodeinone is less than 5%, is preferably less than 4%, is more preferably less than 3%, as is less than 2.5%.14-hydroxyl-7, the content of 8-dihydrocodeinone cannot be avoided with enzyme process preparation, the statement of " containing 14-hydroxyl-7; 8-dihydrocodeinone " has shown 14-hydroxyl-7 in composition, the content of 8-dihydrocodeinone is not 0, for the purpose of clearer, 14-hydroxyl-7, the content lower limit of 8-dihydrocodeinone can be 0.3%.
Also preferred in the method for first aspect present invention, in step (A), thebaine is become 14-hydroxycodeine ketone by hydrogen peroxide oxidation in formic acid, and then 14-hydroxycodeine ketone is hydrogenated to 14-hydroxyl-7 by hydrogen, 8-dihydrocodeinone.Wherein preferably the catalyzer of hydrogen hydrogenation is Pd/C.In addition, preferably 14-hydroxycodeine ketone and/or 14-hydroxyl-7,8-dihydrocodeinone can precipitate extraction in basic solution, and more preferably wherein the pH of basic solution is 8.0～9.0, especially 8.1～8.5, as 8.2.
Preferably in the method for first aspect present invention, under the effect of demethylase, by 14-hydroxyl-7,8-dihydrocodeinone generates 14-hydroxyl-7 in acidity containing in the solution of halfcystine, 8-dihydro desmethylmorphine ketone purity is high also containing 14-hydroxyl-7, the composition of 8-dihydrocodeinone.Preferably wherein the pH value of acidity is 5.5～6.3, as 6.0.Preferably the temperature of demethylase reaction is 20～33 DEG C in addition, especially 25～30 DEG C, as 28 DEG C; And/or preferably the time of demethylase reaction is 1～10 hour, is preferably 3～8 hours, as 5 hours.For fear of the degrading activity of demethylase of the present invention, improve raw material availability, temperature of reaction can not be higher than 35 DEG C.The composition that also preferred steps (B) obtains in addition can precipitate extraction in basic solution, and more preferably wherein the pH of basic solution is 8.0～9.0, and especially 8.3～8.3, as 8.5.
Like this, in the composition obtaining in step (B), although cannot remove 14-hydroxyl-7,8-dihydrocodeinone impurity, can make its content not form impact to final patent medicine, even may be useful.Preferably in the method for first aspect present invention, in the composition obtaining in step (B), 14-hydroxyl-7, the purity of 8-dihydro desmethylmorphine ketone is greater than 90%, is preferably greater than 92%, more preferably greater than 93%, as is greater than 93.5%.Correspondingly, preferably in the method for first aspect present invention, in the composition obtaining in step (B), 14-hydroxyl-7, the content of 8-dihydrocodeinone is less than 10%, is preferably less than 8%, is more preferably less than 7%, as is less than 6%.So preferably, in the method for first aspect present invention, step (B) does not comprise the further step of purification of composition that step (B) is obtained.The composition that so direct use step (B) obtains can further increase cost advantage.
Preferred in the method for first aspect present invention in addition, in step (C), 14-hydroxyl-7,8-dihydro desmethylmorphine ketone is alkylated into naloxone by bromopropylene.The composition that preferred steps (C) obtains can precipitate extraction in basic solution, and more preferably wherein the pH of basic solution is 8.5～9.5, and especially 8.8～9.3, as 9.0.
Preferred in the method for first aspect present invention in addition, in step (D), purifying is chromatography purification, preferably HPLC purifying; And/or salt acidifying is to add hydrochloric acid neutralization.
In second aspect, the invention provides the composition containing naloxone, it comprises naloxone and contains 14-hydroxyl-7,8-dihydrocodeinone.
Preferably, in the composition of second aspect present invention, naloxone purity is high, and in said composition, the purity of naloxone is greater than 95%, is preferably greater than 96%, more preferably greater than 97%, as is greater than 97.5%.Correspondingly, 14-hydroxyl-7, the content of 8-dihydrocodeinone is low, can be surplus, preferably 14-hydroxyl-7, the content of 8-dihydrocodeinone is less than 5%, is preferably less than 4%, is more preferably less than 3%, as is less than 2.5%.But, in the present invention, 14-hydroxyl-7, the content of 8-dihydrocodeinone is not 0, for the sake of clarity, and 14-hydroxyl-7, the content lower limit of 8-dihydrocodeinone can be 0.3%.
Preferably the composition of second aspect present invention is to be prepared by the method for first aspect present invention.Therefore, each preferred aspect in the method for preferred first aspect present invention.
Also the patent medicine effect of the composition of preferred second aspect present invention and naloxone are without remarkable difference.Wherein, patent medicine effect comprises pharmacodynamics and security effect.In this article, " without significantly difference " has the statistical implication of knowing, as p<0.01.In fact, according to the specific embodiment of the present invention, the patent medicine effect of the composition of second aspect present invention also may have advantage than existing naloxone, can replace existing naloxone patent medicine completely.So preferably the composition of second aspect present invention is pharmaceutical composition.
In the third aspect, the composition that the invention provides second aspect present invention is replacing naloxone (the especially Narlan of equivalent) to prepare the application in medicine, the application of the composition that the invention provides second aspect present invention in the medicine of preparing the disease that naloxone can treat.
The disease that naloxone can be treated comprises, poisoning disease, as acute heroin poisoning, acute alcoholism, Acute Nitrite Poisoning, organophosphate poisoning and/or ACMP; Cental system disease, as cerebral infarction (cerebral apoplexy), acute severe hematencephalon, the swelling of traumatic Diffuse brain, Patients with Severe Brain Injury and/or dizzy; Pediatric disease, as hypoxic ischemic encephalopathy of newborn, asphyxia neonatorum, primary premature breathlessness and/or infantile convulsion continue; And, hepatic disease, schizophrenia and/or severe myocardial ischemia etc.
In fourth aspect, the invention provides the authentication method of the composition of second aspect present invention, it comprises detection naloxone and 14-hydroxyl-7, the existence of 8-dihydrocodeinone.Because prior art adopts chemosynthesis; wherein 14-hydroxyl-7; 8-dihydrocodeinone is added protecting group and 100% conversion when as intermediate product; and utilize fat-soluble qualitative difference to be easy to just can extract and remove 14-hydroxyl-7; 8-dihydrocodeinone; so report does not comprise 14-hydroxyl-7,8-dihydrocodeinone impurity in final Narlan product.Preferred detection can adopt HPLC to detect, and with naloxone and 14-hydroxyl-7, the standard substance of 8-dihydrocodeinone relatively determine whether to exist, thereby whether qualification is the composition of second aspect present invention.
Aspect the 5th, the invention provides demethylase, its aminoacid sequence:
(1) as shown in SEQ ID No:2, or
(2) be to SEQ ID No:2 disappearance, add and/or replace one or several (be preferably no more than 7, more preferably no more than 5, as be no more than 3) amino-acid residue and the sequence of the reservation SEQ ID No:2 activity that obtains.
This enzyme can be with 14-hydroxyl-7, and the so complicated fused ring compound of 8-dihydrocodeinone is substrate, carries out efficiently demethylating reaction, can take off N-methyl and O-methyl.
Aspect the 6th, the invention provides the encoding gene (polynucleotide) of the demethylase of fifth aspect present invention.Those skilled in the art, by recombinant DNA technology, can prepare the misfolded proteins obtaining through disappearance, interpolation and/or substituted amino acid residue.In the specific embodiment of the present invention, demethylase is nucleotide sequence coded by as shown in SEQ ID No:1.This demethylase is to 14-hydroxyl-7,8-dihydrocodeinone and 14-hydroxyl-7, and 8-dihydro desmethylmorphine ketone has certain degrading activity, especially when temperature is during higher than 35 DEG C.So in order to improve raw material availability, the temperature of reaction of this enzyme can not be higher than 35 DEG C.In addition, in order to prevent continuing of degraded, the reaction times can not be long, is conventionally less than 12 hours, and therefore reaction can not completely, can contain a small amount of 14-hydroxyl-7,8-dihydrocodeinone impurity.
Aspect the 7th, the invention provides carrier, it comprises the encoding gene of sixth aspect present invention.Preferred vector is expression vector, if the expression vector of expressing in intestinal bacteria or yeast.
In eight aspect, the invention provides cell, the encoding gene that it comprises sixth aspect present invention, or transform or transfection with the carrier of seventh aspect present invention.By recombinant DNA technology, polynucleotide can directly be imported into, for example, utilize the transfered cell such as microsome, particle gun; Also can indirectly be imported into, for example can be by being structured in transfered cell on plasmid vector.The described polynucleotide that import can be incorporated on the genome of cell and express, and expression also can dissociate.Preferred cell is secreting, expressing cell, as the yeast of secreting, expressing.
Aspect the 9th, the invention provides the method for fermentative production demethylase, it comprises the cell of under fermentation conditions cultivating eighth aspect present invention, isolates the demethylase of fifth aspect present invention from culture.If use secreting, expressing cell, can simplify sepn process, as remove by filter cell and retain supernatant liquor, wherein preferably use 0.22 μ m filter membrane to filter.The supernatant liquor that separation obtains can be directly used in the method for first aspect present invention, also can further purify and/or concentrate.
The present invention has following beneficial effect: in preparation method of the present invention, at least demethylating reaction step is easy, reaction conditions is gentle, reagent and residual substantially harmless; Although the Narlan composition that the present invention obtains contains 14-hydroxyl-7 that more difficult purifying is removed, 8-dihydrocodeinone impurity, does not affect patent medicine effect, even can have some superiority, can substitute existing Narlan completely and use; Owing to having removed impurity separation from, can further reduce costs, make patent medicine advantage more obvious.
For the ease of understanding, below will be described in detail the present invention by specific embodiment.It needs to be noted, these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this specification sheets, many variations of the present invention, change are all apparent concerning one of ordinary skill in the art.In addition, the present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included in and carried out reference herein, and the full text that just looks like them has repeated in this article and clearly recorded the same.
Embodiment
Further illustrate by the following examples content of the present invention.As do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art and commercially available common instrument, reagent, can be referring to references such as the manufacturers instructions of the test handbook such as " organic synthesis " and " molecular cloning experiment guide " and corresponding instrument and reagent.
The expression of embodiment 1 demethylase gene
Design new enzyme gene according to our platform technology, entrust the synthetic codon of Sangon Biotech (Shanghai) Co., Ltd. to express the demethylase gene of optimizing, its nucleotide sequence is as shown in the SEQ ID No:1 of sequence table, and the aminoacid sequence of this genes encoding is as shown in SEQ ID No:2.Then, build the yeast of this enzyme of secreting, expressing according to " molecular cloning experiment guide ", taking synthetic gene as masterplate, with primer 1(SEQ ID No:3, introduce EcoR I restriction enzyme site) and primer 2 (SEQ ID No:4, introduced Not I restriction enzyme site) after pcr amplification with EcoR I and Not I double digestion, and be connected with T4DNA ligase enzyme with pPIC3.5 plasmid through these two endonuclease digestions (can purchased from Invitrogen company), be transformed in bacillus coli DH 5 alpha.Choose positive colony, extract plasmid wherein, after sequence verification is correct, positive plasmid, with after Sal I linearization for enzyme restriction, is transformed into pichia yeast GS115 strain by electrotransformation, choosing positive Yeast transformant containing on the flat board of G418.
Above-mentioned positive Yeast transformant is inoculated into 50ml BMGY liquid nutrient medium, and (formula is: in every 100mM potassium phosphate buffer (pH6.0), comprise, 1% yeast extract, 2% peptone, yeast nitrogenous base 1.34%, vitamin H 0.0004%, glucose 2%, glycerine 1%) in, be cultured to OD600 in 32 DEG C with 180rpm shaking table and reach 4.0.Above-mentioned culture is transferred in 1L BMGY liquid nutrient medium, cultivate 16 hours with 180rpm shaking table in 31 DEG C, add 5ml methyl alcohol, then continuing at 30 DEG C cultivates 10 days with 180rpm shaking table, within every 24 hours during this time, add 5ml methyl alcohol, ventilation is controlled dissolved oxygen (DO) and is greater than 22%, and to control pH be that 5.8(ammoniacal liquor regulates).After cultivation completes, with 0.22 μ m membrane filtration and retain supernatant liquor, this supernatant liquor detects the band that substantially only has obvious 40kDa through SDS-PAGE, shows to ferment to obtain this demethylase.Can will after direct supernatant liquor lyophilize, in 4 DEG C of refrigerators, preserve.
The production of embodiment 2 naloxones
(1) 14-hydroxyl-7, the chemosynthesis of 8-dihydrocodeinone
Substantially be combined to according to prior art hydrogen oxide, concrete improve as follows: take in the anhydrous formic acid that 50g thebaine joins 100mL ice bath, after stirring and dissolving, under condition of ice bath, drip 30%(V/V) superoxol 22mL, then stir 1 hour, be warming up to afterwards 37 DEG C of room temperatures and continue to stir 2.5 hours.Then, adding 60mL acetone and 60mL water to reaction solution, then drip 20%(W/W) sodium hydroxide solution is until pH reaches 8.2, dropwises latter static 0.5 hour, can separate out during this time precipitation, after filtering, be dried to obtain white powder (14-hydroxycodeine ketone) 44.3g.Above-mentioned white powder is dissolved in to 200mL10%(W/W) in acetic acid solution, add Pd/C catalyzer 4g, pass into hydrogen and constantly stir in 20 DEG C of pressure with 4MPa, react thus 3 hours.Then dripping 20%(W/W) sodium hydroxide solution is until pH reaches 8.2, dropwises latter static 0.5 hour, can separate out during this time precipitation, and after filtering, the dry white powder 42.8g of obtaining, is oxidized hydrogenation and has obtained 14-hydroxyl-7,8-dihydrocodeinone.
(2) 14-hydroxyl-7, the enzyme process preparation of 8-dihydro desmethylmorphine ketone
Get 14-hydroxyl-7 of above-mentioned preparation, 8-dihydrocodeinone 10g, add 50mL water, and then drip Glacial acetic acid until pH reaches 6.0 under the condition stirring, then the demethylase lyophilized powder 350mg and the halfcystine 9.6g that add embodiment 1 to prepare, in 28 DEG C (can not higher than 35 DEG C) with 50rpm shaking table oscillatory reaction 5 hours.Then dripping 20%(W/W) sodium hydroxide solution is until pH reaches 8.5, dropwise latter static 0.5 hour, can separate out during this time precipitation, after filtering, be dried to obtain off-white powder 7.6g, through HPLC qualification (area normalization method), wherein 14-hydroxyl-7,8-dihydro desmethylmorphine ketone purity is 93.6%, 14-hydroxyl-7,8-dihydrocodeinone content is 5.7%.Then dripping 20%(V/V) ammoniacal liquor is until pH reaches 8.2, dropwises latter static 0.5 hour, can separate out during this time precipitation
(3) chemosynthesis of naloxone
Substantially synthetic according to prior art alkanisation, concrete improvement is as follows: without purification, the off-white powder 5g that directly gets above-mentioned preparation mixes with 400mL dehydrated alcohol, then adds successively sodium bicarbonate 4.9g and bromopropylene (BrCH
2cH=CH
2) 4.3mL, be heated to 90 DEG C of back flow reaction 5 hours, while being evaporated to 20mL, add 20mL water dissolution.Then dripping 20%(W/W) sodium hydroxide solution is until pH reaches 9, dropwises latter static 0.5 hour, can separate out during this time precipitation, and recrystallization soluble in water after filtration drying, obtains white powder 4.7g, is naloxone composition of the present invention.Through HPLC qualification (area normalization method), in said composition, naloxone purity is 97.7%, 14-hydroxyl-7, and 8-dihydrocodeinone content is 2.0%.After the pure naloxone that HPLC is separated and hydrochloric acid salify, carry out hydrogen nuclear magnetic resonance analysis, 1HNMR (d6-DMSO) result is as follows: δ 9.50 (s1H), 9.39 (s1H), 6.84 (s1H), 6.68 (d1H), 6.60 (d1H), 5.94 (m1H), 5.61,5.61 (dd2H), 5.02 (s1H), 3.94,3.79 (m2H), 3.65 (s1H), 3.39 (t2H), 3.39 (2H), 3.31,3.96,2.67 (m4H), 2.11,1.99 (dd2H), 1.48 (m2H), consistent with bibliographical information.
Drug effect and the safety testing of embodiment 3 naloxone compositions
Naloxone composition prepared by Experimental agents: embodiment 2, without expensive purification, directly uses in hydrochloric acid and rear administration.
Control drug: naloxone hydrochloride injection (Yiqiao (Hunan) Pharmaceutical Co., Ltd.).
Experimental technique: BALB/c mouse (body weight 18-22g, male and female half and half) is divided into following 6 groups at random, every group of 10 animals: 1) blank group (administration distilled water); 2) model group (gastric infusion 50% ethanolic soln 1mL); ; 3) control drug high dose group (drug administration by injection is in naloxone 0.5mg/kg+ gastric infusion 50% ethanolic soln 1mL); 4) control drug low dose group (drug administration by injection is in naloxone 0.1mg/kg days+gastric infusion 50% ethanolic soln 1mL); 5) Experimental agents high dose group (drug administration by injection with naloxone and 14-hydroxyl-7,8-dihydrocodeinone total amount meter 0.5mg/kg+ gastric infusion 50% ethanolic soln 1mL); 6) Experimental agents low dose group (drug administration by injection with naloxone and 14-hydroxyl-7,0.1mg/kg days+gastric infusion of 8-dihydrocodeinone total amount meter 50% ethanolic soln 1mL).Below respectively organize administration every day 1 time, continuous 30 days.At the 30th day, mouse administration, after l2 hour, is weighed mouse, put to death, and take a blood sample and get liver organization.After separation of serum, use gpt detection kit, glutamic-oxal(o)acetic transaminase detection kit not to measure gpt (SGPT) activity and glutamic-oxal(o)acetic transaminase (SGOT) activity; Meanwhile, get rat and get liver, fix, after dehydration, paraffin embedding, film-making (4 μ m are thick), HE dyeing, under opticmicroscope, check and have or not pathology and carry out pathology scoring by pathology professional through 10% formalin solution.
Pharmacodynamic result is as shown in table 1 below, and in model group, the serum glutamic pyruvic transminase of mouse and glutamic-oxal(o)acetic transaminase level all significantly raise; Use naloxone composition of the present invention and commercially available control drug, can significance reduce gpt and the glutamic-oxal(o)acetic transaminase level of mouse, especially in the situation that high dosage uses, the effect of naloxone composition of the present invention is also slightly better than commercially available control drug.Meanwhile, tissue slice microscopy shows, in model group, liver tissue lesions's main manifestations is hepatocellular degeneration, occurs spotty necrosis or focal necrosis in liver, occurs liver internal jugular vein inflammation, the visible a small amount of cell infiltration in liver and portal area and fibroblast proliferation; Use naloxone composition of the present invention and commercially available control drug, can significance reduce liver tissue lesions's degree of mouse, same in the situation that high dosage uses, the effect of naloxone composition of the present invention is also slightly better than commercially available control drug.Expectation is 14-hydroxyl-7 of certain content, and 8-dihydrocodeinone is also favourable to the treatment of liver tissue lesions.
The pharmacodynamic experiment result of table 1 naloxone composition of the present invention
| Group | SGPT（IU） | SGOT（IU） | Pathology scoring |
| 1 | 38±17 | 92±11 | 0 |
| 2 | 186±22 | 143±15 | 0.95±0.25 |
| 3 | 99±16 ** | 81±7 ** | 0.15±0.18 ** |
| 4 | 119±12 ** | 82±25 ** | 0.28±0.20 ** |
| 5 | 83±25 ** | 78±18 ** | 0.08±0.07 ** |
| 6 | 117±9 ** | 85±20 ** | 0.26±0.23 ** |
*represent with respect to model group p<0.01.
In addition BALB/c mouse is carried out to toxicity research, result shows, naloxone composition of the present invention is 112mg/kg to the LD50 of injected in mice, also higher than the LD50(107mg/kg of control drug), expectation and wherein 14-hydroxyl-7 of doses, 8-dihydrocodeinone is relevant.
Claims (36)
1. enzyme process is prepared the method for naloxone, and it comprises,
(A) optionally thebaine hydrogen oxide is changed into 14-hydroxyl-7,8-dihydrocodeinone;
(B) under the effect of demethylase, by 14-hydroxyl-7,8-paracodin ketogenesis 14-hydroxyl-7,8-dihydro desmethylmorphine ketone purity is high and contain 14-hydroxyl-7, the composition of 8-dihydrocodeinone, the aminoacid sequence of wherein said demethylase:
(1) as shown in SEQ ID No:2, or
(2) be to SEQ ID No:2 disappearance, add and/or replace one or several amino-acid residue and the sequence of the reservation SEQ ID No:2 activity that obtains;
(C) 14-hydroxyl-7 in composition step (B) being obtained, 8-dihydro desmethylmorphine ketoalkylation becomes naloxone, obtains naloxone purity high also containing 14-hydroxyl-7, the composition of 8-dihydrocodeinone; With,
(D) be optionally further purified and/or salt acidification step (C) obtain composition.
2. method claimed in claim 1, wherein, does not comprise step (D).
3. method claimed in claim 1, wherein, in the composition obtaining in step (C), the purity of naloxone is greater than 95%.
4. method claimed in claim 3, wherein, in the composition obtaining in step (C), the purity of naloxone is greater than 96%.
5. method claimed in claim 4, wherein, in the composition obtaining in step (C), the purity of naloxone is greater than 97%.
6. method claimed in claim 5, wherein, in the composition obtaining in step (C), the purity of naloxone is greater than 97.5%.
7. method claimed in claim 1, wherein, in the composition obtaining in step (C), 14-hydroxyl-7, the content of 8-dihydrocodeinone is less than 5%.
8. method claimed in claim 7, wherein, in the composition obtaining in step (C), 14-hydroxyl-7, the content of 8-dihydrocodeinone is less than 4%.
9. method claimed in claim 8, wherein, in the composition obtaining in step (C), 14-hydroxyl-7, the content of 8-dihydrocodeinone is less than 3%.
10. method claimed in claim 9, wherein, in the composition obtaining in step (C), 14-hydroxyl-7, the content of 8-dihydrocodeinone is less than 2.5%.
11. methods claimed in claim 1, wherein, in step (C), 14-hydroxyl-7,8-dihydro desmethylmorphine ketone is alkylated into naloxone by bromopropylene.
12. methods claimed in claim 1, wherein, in step (A), thebaine is become 14-hydroxycodeine ketone by hydrogen peroxide oxidation in formic acid, and then 14-hydroxycodeine ketone is hydrogenated to 14-hydroxyl-7 by hydrogen, 8-dihydrocodeinone.
13. methods claimed in claim 1, wherein, in the composition obtaining in step (B), 14-hydroxyl-7, the purity of 8-dihydro desmethylmorphine ketone is greater than 90%.
Method described in 14. claims 13, wherein, in the composition obtaining in step (B), 14-hydroxyl-7, the purity of 8-dihydro desmethylmorphine ketone is greater than 92%.
Method described in 15. claims 14, wherein, in the composition obtaining in step (B), 14-hydroxyl-7, the purity of 8-dihydro desmethylmorphine ketone is greater than 93%.
Method described in 16. claims 15, wherein, in the composition obtaining in step (B), 14-hydroxyl-7, the purity of 8-dihydro desmethylmorphine ketone is greater than 93.5%.
17. methods claimed in claim 1, wherein, in the composition obtaining in step (B), 14-hydroxyl-7, the content of 8-dihydrocodeinone is less than 10%.
Method described in 18. claims 17, wherein, in the composition obtaining in step (B), 14-hydroxyl-7, the content of 8-dihydrocodeinone is less than 8%.
Method described in 19. claims 18, wherein, in the composition obtaining in step (B), 14-hydroxyl-7, the content of 8-dihydrocodeinone is less than 7%.
Method described in 20. claims 19, wherein, in the composition obtaining in step (B), 14-hydroxyl-7, the content of 8-dihydrocodeinone is less than 6%.
21. methods claimed in claim 1, wherein, step (B) does not comprise the further step of purification of composition that step (B) is obtained.
22. compositions containing naloxone, it comprises naloxone and contains 14-hydroxyl-7,8-dihydrocodeinone, wherein the purity of naloxone is greater than 95%, and 14-hydroxyl-7 wherein, and the content of 8-dihydrocodeinone is less than 5%, and 14-hydroxyl-7, the content of 8-dihydrocodeinone is not 0.
Composition described in 23. claims 22, wherein the purity of naloxone is greater than 96%,, and 14-hydroxyl-7 wherein, the content of 8-dihydrocodeinone is less than 4%.
Composition described in 24. claims 23, wherein the purity of naloxone is greater than 97%,, and 14-hydroxyl-7 wherein, the content of 8-dihydrocodeinone is less than 3%.
Composition described in 25. claims 24, wherein the purity of naloxone is greater than 97.5%,, and 14-hydroxyl-7 wherein, the content of 8-dihydrocodeinone is less than 2.5%.
Composition described in 26. claims 22, wherein 14-hydroxyl-7, are limited to 0.3% under the content of 8-dihydrocodeinone.
Composition described in 27. claims 22, it is to be prepared by arbitrary described method of claim 1～21.
Composition described in 28. claims 22, its patent medicine effect and naloxone are without remarkable difference.
The application of the arbitrary described composition of 29. claims 22～28 in the medicine of preparing the disease that naloxone can treat.
Application described in 30. claims 29, the disease that wherein naloxone can be treated is poisoning disease.
Application described in 31. claims 30, wherein poisoning disease is acute heroin poisoning, acute alcoholism, Acute Nitrite Poisoning, organophosphate poisoning or ACMP.
Application described in 32. claims 29, the disease that wherein naloxone can be treated is cental system disease.
Application described in 33. claims 32, wherein cental system disease is cerebral infarction, acute severe hematencephalon, the swelling of traumatic Diffuse brain, Patients with Severe Brain Injury or dizzy.
Application described in 34. claims 29, the disease that wherein naloxone can be treated is pediatric disease.
Application described in 35. claims 34, wherein pediatric disease is that hypoxic ischemic encephalopathy of newborn, asphyxia neonatorum, primary premature breathlessness or infantile convulsion continue.
Application described in 36. claims 29, the disease that wherein naloxone can be treated is hepatic disease, schizophrenia or severe myocardial ischemia.
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| CN101036650A (en) * | 2006-03-16 | 2007-09-19 | 薛京 | Naloxone hydrochloride dropping pills |
| CN101513407A (en) * | 2009-04-03 | 2009-08-26 | 单风平 | Application of naloxone and composition thereof in preparing drug for treating cancer |
| CN101955484A (en) * | 2010-06-26 | 2011-01-26 | 甘肃普安制药有限公司 | Method for synthesizing naloxone or naltrexone |
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| EP1810678A1 (en) * | 2006-01-19 | 2007-07-25 | Holger Lars Hermann | Use of morphine and naloxone for drug substitution |
| CN101437517A (en) * | 2006-04-14 | 2009-05-20 | 夏尔有限责任公司 | Compositions and methods for enhancing analgesic potency of covalently bound compounds, attenuating its adverse side effects, and preventing their abuse |
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| CN101036650A (en) * | 2006-03-16 | 2007-09-19 | 薛京 | Naloxone hydrochloride dropping pills |
| CN101513407A (en) * | 2009-04-03 | 2009-08-26 | 单风平 | Application of naloxone and composition thereof in preparing drug for treating cancer |
| CN101955484A (en) * | 2010-06-26 | 2011-01-26 | 甘肃普安制药有限公司 | Method for synthesizing naloxone or naltrexone |
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