CN101785779B - Separation and preparation of isovaleryl-spiramycin II - Google Patents
Separation and preparation of isovaleryl-spiramycin II Download PDFInfo
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- CN101785779B CN101785779B CN201010119758.9A CN201010119758A CN101785779B CN 101785779 B CN101785779 B CN 101785779B CN 201010119758 A CN201010119758 A CN 201010119758A CN 101785779 B CN101785779 B CN 101785779B
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Abstract
The invention relates to the separation of antibiotic and the application of the antibiotic in resisting infectious diseases, in particular to an isovaleryl-spiramycin II single-component compound. A kelimycin sample is separated by HPLC (high performance liquid chromatography) after crude separation, high-efficiency purification and post-processing, so as to obtain the pure isovaleryl-spiramycin II compound. Biological experiment results show that the antibacterial activity of the isovaleryl-spiramycin II compound is better than that of kelimycin and is much better than that of a control group. The invention lays the foundation for developing the clinical and effective isovaleryl-spiramycin II single-component antibiotic.
Description
Technical field:
The present invention relates to antibiotic separation and the application in anti-infectious disease thereof, particularly isovaleryl spiramycin one pack system.
Background technology:
Rokitamycin is the Novel spiral adm derivative that utilizes technique for gene engineering development, former called after Biotechmycin, and former name is shengjimycin [patent No.: ZL97104440.6].According to " Chinese adopted drug name nomenclature principle " ，Jing Chinese Pharmacopoeia Commission technology, examine and determine, the Universal Chinese character name change of Biotechmycin is rokitamycin, and English name is Calimycin.
Rokitamycin is genetic engineering bacterium tunning; its chemical constitution is to take 4 " isovaleryl spiramycin is main constituent; comprise 4 " isovaleryl Spiramycin I, II, III, secondly also containing having an appointment 6 kind 4, " spiramycin of position hydroxyl acyl group, therefore its chemical name is referred to as 4 " acidylated spiramycins.
The chemical constitution of rokitamycin main constituent is suc as formula shown in (1):
Wherein: R=H, COCH
3, COCH
2cH
3; R '=COCH
2cH (CH
3)
2, COCH
2cH (CH
3)
2, COCH
2cH (CH
3)
2
Rokitamycin is 16 ring macrolide antibiotics, and its mechanism of action is to suppress its protein synthesis by being combined with bacterial ribosome.
In vitro tests result shows, rokitamycin to gram positive bacteria, especially to some fastbacteria (as resistance to beta-lactam gold Portugal bacterium, resistance to erythromycin gold Portugal bacterium etc.) effectively, with similar medicine without obvious cross resistance.It has good antibacterial activity to mycoplasma, chlamydia simultaneously, part gram-negative bacteria is also had to antibacterial activity, and toxoplasma, legionella etc. is had to good antibacterial activity and tissue permeability, also has potential immunoregulation effect.Its antibacterial activity in vivo is obviously better than external [ZL200310122420.9].Clinical research shows, takes rokitamycin tablet 0.2-0.4g 5-7 days every day, the acute bacterial pharyngitis, the acute suppurative tonsillitis that applicable to treatment micrococcus scarlatinae, cause; Bacillary sinusitis, acute bronchitis that sensitive bacterial causes; Light disease pneumonia due to streptococcus pneumoniae, hemophilus influenza and mycoplasma pneumoniae; The non gonococcal urethritis that mycoplasma, chlamydia cause; The infectious disease such as the skin soft-tissue infection that sensitive bacterial causes, periodontitis, otitis media.Its total effective rate is 87.76%, and the untoward reaction rate of rokitamycin is low.
Clinical research proves, rokitamycin is an oral antibiotic safely and effectively.Yet, because rokitamycin itself is multicomponent pharmaceutical, the product obtaining by fermentation, the plurality of impurities producing at sweat, brings certain difficulty to component quantitative assay.The high performance liquid chromatography peak height computing method of setting up at present, can be by several as separated with acetylspiramycin III's with small component, propionylspiramycin II before it with propionylspiramycin III, propionylspiramycin III in isovaleryl spiramycin II and (different) butyryl spiramycin III, (different) butyryl spiramycin II to difficult separated material in rokitamycin sample, degree reach that Chinese Pharmacopoeia stipulates more than 1.5; And the separating degree of acetylspiramycin III and the small component before it is 1.2.The rokitamycin tablet quality control standard of setting up at present, is to adopt high-efficient liquid phase analysis peak height computing method, measures 9 acidylated spiramycin components of rokitamycin, and wherein isovaleryl spiramycin (I+II+III) total content should be not less than 65%; Acidylated spiramycin total content should be not less than 80%.Although according to current tablet manufacturing technique, can obtain controlled, the stay-in-grade rokitamycin of product component ratio, yet, in order to improve extraction and purification process, reduced mass testing process, to improve clinical therapeutic efficacy, be necessary to develop the ejection preparation of the main one-component of isovaleryl spiramycin.Especially for the patient of clinical critical patient or unsuitable oral medication, drug administration by injection takes effect rapidly, more easily accepts.The multicomponent antibiotic producing for fermentation, according to chemical drugs quality control method, be difficult to control its quality, and any tiny variation, the variation of material base all likely caused, the difficulty that increases finished product quality control standard, causes uncertain bad kickback of using medicine.So the multicomponent injection of antibiotic agent producing by fermentation is still rare so far.
Pharmacokinetic result shows, in rokitamycin, the active component of tool activity is mainly isovaleryl Spiramycin I, II, III.After rokitamycin enters in body, very fast metabolism is spiramycin, with the AUC of parent drug isovaleryl Spiramycin I, II, III and active metabolite Spiramycin I, II, III
0-tsummation is calculated, its oral absolute bioavailability average out to 91.6%.Bibliographical information, the oral absolute bioavailability of spiramycin human body is 30-40%[Frydman AM et al J Antimicrob Chemother.1988,22 (suppl B): 93-103].Illustrate that the structure of isovaleryl spiramycin obviously improved the bioavailability of active component spiramycin.Single medication rokitamycin is eliminated slower, T
1/2 βbetween 23-27 hour.
The discovery that the inventor is surprised, the key component isovaleryl spiramycin II of rokitamycin has better anti-infection activity, the invention provides the application of isovaleryl spiramycin II in preparing anti-infectives for this reason.
Summary of the invention:
The object of this invention is to provide a kind of isovaleryl spiramycin II compound and pharmaceutical composition thereof, with and application in anti-infective therapy.The present invention is prepared into isovaleryl spiramycin II compound that a kind of production technology is simplified, quality standard is easily controlled, the one pack system antibiotic that effect of drugs is good.
Isovaleryl spiramycin II compound of the present invention, has following structure:
Wherein: R=COCH
3, R '=COCH
2cH (CH
3)
2
The structure that the invention provides isovaleryl spiramycin II is determined;
The present invention also provides the preparation method of isovaleryl spiramycin II, it comprises the following steps: according to patent [ZL97104440.6], prepare rokitamycin, sample crude separation, efficiently purifying, sample post processing etc., preferably adopt high performance liquid chromatography to carry out separation to rokitamycin sample, obtain isovaleryl spiramycin II pure compounds.
The present invention carries isovaleryl spiramycin II antibacterium, anti-mycoplasma and chlamydia determination of activity is also provided.
The present invention also provides the pharmaceutical composition of preparing as pharmaceutically active substance with isovaleryl spiramycin II of the present invention, and isovaleryl spiramycin II shared percentage by weight in pharmaceutical composition can be 0.01-99.99%, and all the other are medicine acceptable carrier.Pharmaceutical composition of the present invention, exists with unit dosage form, and described unit dosage form refers to the unit of preparation, as every of tablet, and every of capsule, every bottle of oral liquid, every of injection etc.
Pharmaceutical composition of the present invention can be any pharmaceutically useful dosage form, and these dosage forms comprise: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch.Preparation of the present invention, injection type preferably, as: liquid drugs injection, powder pin, the dosage forms such as transfusion.
Pharmaceutical composition of the present invention, the preparation of its oral administration can contain conventional excipient, such as binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, coloring agent, flavoring agent and wetting agent, can carry out coating to tablet if desired.
Applicable filler comprises cellulose, mannitol, lactose and other similar filler.Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivatives, for example sodium starch glycollate.Suitable lubricant comprises, for example magnesium stearate.The acceptable wetting agent of suitable medicine comprises sodium lauryl sulphate.
Can fill by mixing, the method that tabletting etc. are conventional is prepared solid oral composition.Repeatedly mix and can make active substance be distributed in those compositionss of a large amount of filleies of whole use.
The form of oral liquid can be for example aqueous or oily suspensions, solution, Emulsion, syrup or elixir, or can be a kind of available water before use or the composite dry products of other suitable carrier.This liquid preparation can contain conventional additive, such as suspending agent, for example sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl-cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenation edible fat, emulsifying agent, for example lecithin, anhydro sorbitol monooleate or arabic gum; Non-aqueous carrier (they can comprise edible oil), for example almond oil, fractionated coconut oil, such as oily ester, propylene glycol or the ethanol of the ester of glycerol; Antiseptic, for example para hydroxybenzene methyl ester or propyl p-hydroxybenzoate or sorbic acid, and if need, can contain conventional flavouring agent or coloring agent.
For injection, the liquid unit dosage forms of preparation contains active substance of the present invention and sterile carrier.According to carrier and concentration, this compound can be suspended or dissolve.The preparation of solution is normally by active substance being dissolved in a kind of carrier, and filter-sterilized before being packed into a kind of suitable bottle or ampoule, then seals.Adjuvant for example a kind of local anesthetic, antiseptic and buffer agent also can be dissolved in this carrier.In order to improve its stability, can be after packing bottle into, this compositions is freezing, and under vacuum, water is removed.
Pharmaceutical composition of the present invention, when being prepared into medicament, optionally add applicable pharmaceutically acceptable carrier, described pharmaceutically acceptable carrier is selected from: mannitol, sorbitol, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin C, EDETATE SODIUM, Ethylenediaminetetraacetic Acid Calcium Salt, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and derivant thereof, alginate, gelatin, polyvinylpyrrolidone, glycerol, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surfactant, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc.
Pharmaceutical composition of the present invention is determined dosage form and usage and dosage according to patient's situation in use.
Advantage of the present invention and good effect are, for pharmacy corporation provides the easily isovaleryl spiramycin II one-component goods of control of production technology simplification, quality standard; For clinical critical patient or patient that should not oral administration provide, take effect rapidly, be easy to the probability of the drug formulation accepted.
Accompanying drawing explanation:
The HPLC collection of illustrative plates of Fig. 1 isovaleryl spiramycin II
The high resolution mass spectrum figure of Fig. 2 isovaleryl spiramycin II
The hydrogen nuclear magnetic resonance spectrogram of Fig. 3 isovaleryl spiramycin II
The nuclear magnetic resonance, NMR of Fig. 4 isovaleryl spiramycin II
13c spectrogram
Fig. 5 compound and the accumulation antibacterial percentage rate of contrast medicine to 7 strain erythromycin-resistant streptococcus pneumoniae
Wherein:
Fig. 6 compound and the accumulation antibacterial percentage rate of contrast medicine to 6 strain erythromycin-resistant micrococcus scarlatinaes
Wherein:
Embodiment:
Listed embodiment is in order to help those skilled in the art to understand better the present invention below, but does not limit the present invention in any way.
The separation preparation of < embodiment 1>, isovaleryl spiramycin II
Rokitamycin raw material is according to " a kind of method of the utilizing technique for gene engineering MT mycin " patent (patent No.: ZL97104440.6) prepare.Adopt preparative high performance liquid chromatography to carry out separation to rokitamycin sample, obtain isovaleryl spiramycin II pure compounds.Specifically comprise sample crude separation, efficiently purifying, sample post-treating and other steps.
1), crude separation: first from rokitamycin sample, isovaleryl spiramycin II component is carried out to crude separation, adopt silica gel column chromatography method, using ethyl acetate and methanol as eluant, adopt respectively three times of column volume ethyl acetate: methanol (3: 1) and three to five times of column volume ethyl acetate: methanol (1: 1) carries out eluting, collect ethyl acetate: methanol (1: 1) part, collection liquid is concentrated into dry, obtain isovaleryl Spiramycin I, II, III component, for being further purified separation.
2), efficiently purifying: adopt preparative high performance liquid chromatography to carry out purification to crude separation sample, using ODS as chromatograph packing material, with acetonitrile and Ammonium Acetate buffer, carry out gradient elution, pass through ultraviolet detection, record separated uv atlas, and isovaleryl spiramycin II target peak is collected.
Concrete chromatographic condition is as follows: instrument: technical grade preparative hplc.Critical piece comprises binary gradient pump, UV-detector and chromatographic work station; Chromatographic column: ODS preparative hplc post (70i.d. * 380mm, 10 μ); Mobile phase: acetonitrile (A) 25%, 100mM NH
4ac aqueous solution 75% (B); Gradient condition: adopt linear gradient.Flow velocity: 260mL/min; Sample size: 10mL; Sample introduction concentration: 0.5g/mL; Detect wavelength: 231nm;
Collection mode: ultraviolet triggers collects; According to the retention time of isovaleryl spiramycin II (RT 43.34), collect sample.Isovaleryl spiramycin II high pressure liquid chromatography is shown in (Fig. 1).
3), sample post processing: the isovaleryl spiramycin II sample of collection, adopt respectively rotary evaporation to remove acetonitrile, then, with the ethyl acetate extraction of 1 times of amount, with rotary evaporation, remove ethyl acetate in extract, obtain paste sample.With the heavy molten gained sample of petroleum ether, then remove petroleum ether with rotary evaporation, obtain isovaleryl spiramycin II white powder solid.
The evaluation of < embodiment 2> isovaleryl spiramycin II
By the molecular weight 968 of the separated compound of high resolution mass spectrum (BrukerAPEXII, HR-SI-MS) confirmation.Isovaleryl spiramycin II high resolution mass spectrum figure is shown in (Fig. 2), through nuclear magnetic resonance, NMR
1h and
13c composes [Bruker AM500, solvent C DCl
3, interior mark TMS (tetramethylsilane)] etc. furanone, the structure of separated compound is isovaleryl spiramycin II.Isovaleryl spiramycin II nuclear magnetic resonance, NMR
1h and
13c spectrum is shown in (Fig. 3,4), nuclear magnetic resonance, NMR
1h and
13c spectrum data are in Table shown in (1,2).
The nuclear magnetic resonance, NMR of table 1 isovaleryl spiramycin II
1h spectrum data (500MHz, CDCl
3)
| Proton | δ ppm(M，J Hz) | 1H- 1H COSY |
| 2ax | 2.73(dd，J＝11.0，13.2) | 2eq，3 |
| 2eq | 2.26(bd，J＝13.2) | 2ax，3 |
| 3 | 5.13(brd，J＝11.0) | 2eq，2ax，4 |
| 4 | 3.23(brd，J＝9.1) | 5，3 |
| 5 | 3.87(brd，J＝9.1) | 4 |
| 6 | 2.15(m) | 7eq，17b |
| 7ax | 0.97(m) | 7eq，8 |
| 7eq | 1.45(m) | 7ax，6 |
| 8 | 1.93(m) | 7ax，19 |
| 9 | 3.93(dd，J＝9.7，4.0) | 10 |
| 10 | 5.62(dd，J＝9.7，15.2) | 9，11 |
| 11 | 6.55(dd，J＝15.2，10.5) | 10，12 |
| 12 | 6.06(dd，J＝10.5，15.0) | 11，13 |
| 13 | 5.72(ddd，J＝15.0，11.2，3.6) | 12，14ax，14eq |
| 14ax | 2.12(m) | 13，14eq，15 |
| 14eq | 2.47(m) | 13，14ax |
| 15 | 5.06(m) | 14ax，16 |
| 16 | 1.25(d，J＝5.5) | 15 |
| 17a | 2.32(dd，J＝3.0，18.3) | 17b |
| 17b | 2.82(bdd，J＝11.1，18.3) | 6，17a |
| 18 | 9.65(s) | |
| 19 | 0.98(d，J＝6.6) | 8 |
| 21 | 2.27(s) | |
| 22 | 3.52(s) | |
| 1′ | 4.42(d，J＝7.5) | 2′ |
| 2′ | 3.50(dd，J＝7.5，10.4) | 1′，3′ |
| 3′ | 2.46(dd，J＝6.7，10.4) | 2′，4′ |
| 4′ | 3.26(m) | 3′ |
| 5′ | 3.28(m) | 6′ |
| 6′ | 1.19(d，J＝6.6) | 5′ |
| 7′.8′ | 2.50(s) | |
| 1″ | 5.06(d，J＝3.5) | 2″ |
| 2″ax | 1.83(dd，J＝4.0，14.2) | 1″，2″ |
| 2″eq | 2.00(brd，J＝14.2) | 2″ |
| 4″ | 4.61(d，J＝10.2) | 5″ |
| 5″ | 4.44(dq，J＝10.2，6.2) | 4″，6″ |
| 6″ | 1.13(d，J＝6.2) | 5″ |
| 7″ | 1.11(s) | |
| 9″ | 2.29(d，J＝7.0) | 10″ |
| 10″ | 2.14(m) | 9″，11″，12″ |
| 11″，12″ | 0.97(d，J＝6.6) | 10″ |
| Proton | δ ppm(M，J Hz) | 1H-
1 |
| 1′″ | 4.39(bd，J＝9.3) | 2′″ax，2′″ |
| 2′″ax | 1.48(m) | 1′″，2′″eq，3′″ |
| 2′″eq | 1.84(m) | 1′″，2′″ax，3′″ax |
| 3′″ax | 1.43(m) | 2′″eq，3′″eq，4′″ |
| 3′″eq | 1.85(m) | 2′″ax，3′″ax，4′″ |
| 4′″ | 2.25(m) | 3′″ax，3′″eq，5′″ |
| 5′″ | 3.40(dq，J＝6.2，9.3) | 4′″，6′″ |
| 6′″ | 1.20(d，J＝6.2) | 5′″ |
| 7′″，8′″ | 2.21(s) |
The nuclear magnetic resonance, NMR of table 2 isovaleryl spiramycin II
13c spectrum data (125MHz, CDCl
3)
The Determination of Antibacterial Activity of < embodiment 3> isovaleryl spiramycin II
1) compound sample:
Erythromycin: lot number: 0706392, tire: 91.6%, Nat'l Pharmaceutical & Biological Products Control Institute;
Azithromycin: lot number: 0421-9603, tires: 99.3%, Nat'l Pharmaceutical & Biological Products Control Institute;
Clarithromycin: lot number: 0482-9901, tires: 90.4%, Nat'l Pharmaceutical & Biological Products Control Institute.
2) test strain:
Reference culture: streptococcus pneumoniae ATCC49619;
Clinical separated gram positive bacteria;
Erythromycin-resistant streptococcus pneumoniae Streptococcus pneumoniae (7 strain);
Erythromycin-resistant micrococcus scarlatinae Streptococcus pyogenes (6 strain).
Every strain antibacterial before test all through flat board turn live minute pure, with new fresh thalli for test.Each experiment all uses reference culture as sensitive experiment Quality Control bacterium; With not containing the plate of antibacterials as test strain growth control.
3) culture medium and incubation conditions:
M-H (Mueller-Hinton) culture medium (g/L): acid hydrolysis casein 17.5, beef soak bone meal 2, starch 1.5, agar 12.Streptococcus pneumoniae is upper at blood meida (adding 5% defiber Sanguis caprae seu ovis to make in M-H culture medium), 35 ℃ of 5% CO
2environment (CO
2incubator) in, hatch 20-24h.Micrococcus scarlatinae is upper at blood meida (adding 5% defiber Sanguis caprae seu ovis to make in M-H culture medium), hatches 20-24h for 35 ℃.
4) minimum inhibitory concentration (MIC) is measured:
Employing standard plate doubling dilution.Multiple spot inoculation instrument inoculation for tested bacteria suspension, every some inoculum concentration is 10
4cFU.Measure the minimum inhibitory concentration of each antibacterials to various pathogenic bacterium.
Result:
In measured erythromycin-resistant streptococcus, isovaleryl spiramycin II one pack system compound of the present invention is to streptococcic antibacterial action MIC
50(0.5mg/L) and MIC
50(2mg/L) be all better than rokitamycin, meanwhile, be obviously better than contrast medicine erythromycin, azithromycin, clarithromycin (table 3).For the high Antimicrobial Streptococcus Pneumoniae of 7 strain erythromycin (erythromycin MIC 64-> 256mg/L), azithromycin and clarithromycin show as height drug resistance too, and the MIC value of the compounds of this invention is at 1-32mg/L, lower than contrast medicine 32-512 doubly (table 4, Fig. 5).For the low drug resistance micrococcus scarlatinae of 6 strain erythromycin (erythromycin MIC=1-8mg/L), the MIC of the compounds of this invention
50value, at 0.5mg/L, is better than rokitamycin, be more better than contrasting medicine erythromycin, azithromycin, clamycin 2-32 times (table 3,5, Fig. 6).
Table 3. compound and contrast medicine MIC result
Table 4. compound and contrast medicine are to 7 strain erythromycin-resistant streptococcus pneumoniae MIC (mg/L)
Table 5. compound and contrast medicine are to 6 strain erythromycin-resistant micrococcus scarlatinae MIC (mg/L)
The anti-mycoplasma pneumoniae of B and Chlamydia pneumoniae are active
1. compound
The mould II of compound 2 isovaleryl spiral: lot number: 0811214, tire: 93.7%, Tonglian Group Co., Ltd., Shenyang provides
Erythromycin: lot number: 0706392, tire: 91.6%, Nat'l Pharmaceutical & Biological Products Control Institute
Azithromycin: lot number: 0421-9603, tires: 99.3%, Nat'l Pharmaceutical & Biological Products Control Institute
2. mycoplasma pneumoniae strain
Mycoplasma pneumoniae strain (ATCC-FH).
3. mycoplasma pneumoniae culture medium
1. mycoplasma agar culture medium: mycoplasma agar basal medium (Oxoid company, CM0401) 2.84g, add ultra-pure water 80ml, 121 ℃ of high pressure 15min sterilizing, be placed in the water-bath of 50 ℃, add 1 bottle of mycoplasma pneumoniae additive (Oxoid, SR0059C) (20ml), prepare agar culture dish.
2. mycoplasma PPLO broth bouillon: mycoplasma PPLO Mycoplasma Broth Base culture medium (BD company) 2.1g, deionization pure water 70ml, 121 ℃, 15min autoclaving.Be cooled to the phenol red 0.5ml that adds after room temperature lyophilizing mycoplasma pneumoniae to cultivate additive (Oxoid additive) 1 bottle (dissolving of 20ml sterile deionized water), aseptic 50% glucose solution 2.0ml and 0.4%.
4. mycoplasma pneumoniae is cultivated
Mycoplasma pneumoniae FH strain is inoculated in the broth bouillon containing 20ml PPLO, and 72h results scrape with bead, 12000rpm, and 10min centrifugation, abandons supernatant, and precipitation is outstanding again with the fresh PPLO broth bouillon of 8ml, subpackage 8 frozen-80 of pipe ℃ refrigerators.
The titration of mycoplasma pneumoniae FH inoculum
Coat mycoplasma pneumoniae agar plate above-mentioned preparation after the cultivation of mycoplasma pneumoniae FH inoculum is used PPLO broth bouillon to carry out diluting for 1: 10,1: 100 and 1: 1000, and 37 ℃ of 5%CO2 cultivate 7 days, under * 40 power microscopes, count.Calculate mycoplasma pneumoniae titre.
5. the mensuration of mycoplasma pneumoniae MIC
Compound is dissolved in dehydrated alcohol with 16mg/ml, uses mycoplasma PPLO broth bouillon with 10 doubling dilutions.Mycoplasma pneumoniae inoculum adds in the medication tube of above-mentioned serial dilution, and the final inoculum density of mycoplasma pneumoniae FH is 8.25 * 10
5cFU/ml, the variation of color is cultivated and observed every day to postvaccinal culture tube (comprising control tube) in 37 ℃.Test is set up:
(1) positive control: the mycoplasma pneumoniae FH inoculum (1.65 * 10 after dilution
6cFU/ml) 0.5ml+PPLO broth bouillon 0.5ml;
(2) negative control: PPLO broth bouillon 1ml;
(3) dehydrated alcohol is killed and is disturbed contrast: the mycoplasma pneumoniae FH inoculum (1.65 * 10 after dilution
6cFU/ml) 0.5ml, PPLO broth bouillon 0.5ml, ultimate density 0.05% dehydrated alcohol.
When obvious color change (by red flavescence color) occurs positive control pipe, and the antibiotic concentration that color change pipe now do not occur is the minimum antibacterial dense MIC of being concentration.
7. Chlamydia pneumoniae strain
Chlamydia pneumoniae strain: CWL-029 (ATCC VR1310).
8. Chlamydia pneumoniae is cultivated
(1) cell culture fluid: 10% hyclone (HyClone company) Dulbecco ' s MEM (Sigma company) culture fluid.
(2) Chlamydia pneumoniae culture fluid: 10% hyclone Dulbecco ' s MEM culture fluid, containing the cycloheximide (Sigma company) of 2 μ g/ml.
(3) BGMK (green monkey kidney cell, Diagnostic HYBRIDS company) is planted in 96 porocyte culture plates, and 37 ℃, 5%CO2 cultivates becomes cell monolayer for 48 hours.
9. the mensuration of Chlamydia pneumoniae MIC
Compound is dissolved in dehydrated alcohol with 16mg/ml, first uses chlamydia culture fluid with 10 doubling dilutions, by concentration, is 3.3 * 10
7the dilution of cfu (inclusion body formation unit)/1ml Chlamydia pneumoniae inoculum 1: 200, ultimate density is: 1.65 * 10
5cfu/1ml, sucks the cell culture fluid in 96 well culture plates, then by 0.1ml/ hole, inoculates.The complete centrifugal 96 porocyte culture plates of strain inoculation, the J-6MC centrifuge of use Beckman-Coulter company, centrifugal force * 1500g, 35 ℃ of centrifuging temperatures, centrifugation time 60min.
Set up contrast
(1) positive control: BGMK cell, the Chlamydia pneumoniae CWL-029 strain of dilution.
(2) negative control: BGMK cell, Chlamydia pneumoniae culture fluid.
(3) medicine contrast: BGMK cell, the compound of highly diluted concentration (8 μ g/ml), ultimate density 0.05% dehydrated alcohol.
After centrifugal, suck Chlamydia pneumoniae inoculum, add respectively the compound 0.1ml/ hole of serial dilution.37 ℃, 5%CO2 cultivates 72min.Cultivate completely, draw antibiotic medicine solution, PBS (0.01M, pH 7.4) washs 2 times, and 100% dehydrated alcohol is 15min fixedly.
Indirect IF staining is identified: monoclonal antibody to Chlamydia pneumoniae, 50 μ l/ holes, incubation 30min in 37 ℃ of wet boxes, then wash plate machine washing plate 4 times, add again the anti-Mus fluorescent antibody of rabbit (Sigma company), 50 μ l/ holes, same method and condition incubation and wash plate.Add mounting glycerol, 100 μ l/ holes are observed under OlympusX71 inverted fluorescence microscope
The definition of MIC: the minimum antibiotic diluted concentration in the complete suppressed hole of Chlamydia pneumoniae inclusion body growth (inclusion body of fluorescence staining is not found in full hole) in 96 hole bread boards.
10. result:
Isovaleryl spiramycin II activity in measured anti-mycoplasma pneumoniae activity is obviously better than rokitamycin, and is obviously better than contrasting medicine.Activity to Chlamydia pneumoniae, isovaleryl spiramycin II and rokitamycin are suitable, are better than erythromycin.
Table 6 compound is to mycoplasma pneumoniae/chlamydia active (μ g/ml)
The preparation of < embodiment tetra-> isovaleryl spiramycin II pharmaceutical preparatioies
The present invention can be using isovaleryl spiramycin II one pack system as active component, is prepared into useful clinically various dosage form with receivable carrier pharmaceutically.As injection: be dissolved in 1-5ml water after respectively isovaleryl spiramycin II (abbreviation raw material) 100-500mg being mixed homogeneously with solvent adipic acid 18-90mg or citric acid 26-130mg or maleic acid 14-70mg etc. mole, can obtain faint yellow clear and bright solution, pH is between 4.6-5.6.Add mannitol 30-150mg as lyophilizing proppant, after cryogenic quick freezing 9h, lyophilization, obtains faint yellow loose block again, before using, with 2-10ml sterilized water, dissolves.
Claims (1)
1. the separation method of isovaleryl spiramycin II, is characterized in that, step is as follows:
1), crude separation: first from rokitamycin sample, isovaleryl spiramycin II component is carried out to crude separation, adopt silica gel column chromatography method, using ethyl acetate and methanol as eluant, adopt respectively three times of column volume ethyl acetate: methanol=3: 1 and three to five times of column volume ethyl acetate: methanol=1: 1 carries out eluting, collect ethyl acetate: methanol=1: 1 part, collection liquid is concentrated into dry, obtains isovaleryl Spiramycin I, II, III component, for being further purified separation;
2), efficiently purifying: adopt preparative high performance liquid chromatography to carry out purification to crude separation sample, using ODS as chromatograph packing material, with acetonitrile and Ammonium Acetate buffer, carry out gradient elution, pass through ultraviolet detection, record separated uv atlas, and isovaleryl spiramycin II target peak is collected;
Concrete chromatographic condition is as follows: instrument: technical grade preparative hplc, and critical piece comprises binary gradient pump, UV-detector and chromatographic work station; Chromatographic column: ODS preparative hplc post, model 70i.d. * 380mm, 10 μ; Mobile phase A: 25% acetonitrile, Mobile phase B: 100mM NH
4ac aqueous solution 75%; Gradient condition: adopt linear gradient, flow velocity: 260mL/min; Sample size: 10mL; Sample introduction concentration: 0.5g/mL; Detect wavelength: 231nm;
Collection mode: ultraviolet triggers collects; According to the retention time of isovaleryl spiramycin II, RT43.34, collects sample, and isovaleryl spiramycin II high pressure liquid chromatography is shown in Fig. 1;
3), sample post processing: the isovaleryl spiramycin II sample of collection, adopt respectively rotary evaporation to remove acetonitrile, then with the extraction of 1 times of amount ethyl acetate, with rotary evaporation, remove ethyl acetate in extract, obtain paste sample, with the heavy molten gained sample of petroleum ether, then remove petroleum ether with rotary evaporation, obtain isovaleryl spiramycin II white powder solid.
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| PCT/CN2011/071603 WO2011110084A1 (en) | 2010-03-09 | 2011-03-08 | Method for preparing isovalerylspiramycin i, ii or iii and pharmaceutical composition containing compound thereof |
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| CN102311471B (en) * | 2010-05-25 | 2013-10-16 | 沈阳同联集团有限公司 | Levorotary isovaleryl spiramycin II as well as preparation, preparation method and application thereof |
| KR20130041829A (en) | 2010-05-25 | 2013-04-25 | 쉔양 통리엔 그룹 컴패니, 리미티드 | Levoisovalerylspiramycin i, ii or iii, preparations, preparation methods and uses thereof |
| ES2912734T3 (en) | 2017-07-04 | 2022-05-27 | Shenyang Fuyang Pharmaceutical Tech Co Ltd | Use of isovaleryl spiramycin I or III in the preparation of drug to treat and/or prevent tumor and drug |
| RU2771046C2 (en) * | 2018-01-19 | 2022-04-25 | Шенянг Фуянг Фармасьютекал Технолоджи Ко., Лтд. | Use of carrimycin or its active ingredients |
| KR20200112897A (en) * | 2018-01-19 | 2020-10-05 | 쉔양 푸양 파마슈티컬 테크놀로지 컴퍼니 리미티드 | mTOR inhibitor, drug composition and application thereof |
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| KR20220008895A (en) * | 2019-05-16 | 2022-01-21 | 쉔양 푸양 파마슈티컬 테크놀로지 컴퍼니 리미티드 | Drugs, combination products and their applications for preventing, alleviating and/or treating fibrosis |
| CN112239483B (en) * | 2019-07-18 | 2023-10-27 | 沈阳福洋医药科技有限公司 | Compound and pharmaceutical composition |
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