CN107759669A - A kind of aerosporin crystallization and preparation method thereof - Google Patents
A kind of aerosporin crystallization and preparation method thereof Download PDFInfo
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- CN107759669A CN107759669A CN201610694064.5A CN201610694064A CN107759669A CN 107759669 A CN107759669 A CN 107759669A CN 201610694064 A CN201610694064 A CN 201610694064A CN 107759669 A CN107759669 A CN 107759669A
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- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
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Abstract
The invention provides a kind of aerosporin crystallization and preparation method thereof, the preparation method comprises the following steps：1) polymyxin B zymotic fluid is adsorbed with resin, adds aqueous sulfuric acid and/or the acidic ethanol aqueous solution is desorbed, obtain stripping liquid；2) pH for the stripping liquid for obtaining step 1) is adjusted to 5.0~7.0, has then been concentrated into solid precipitation, has been obtained aerosporin saturated solution；3) under agitation, the aerosporin saturated solution in the aerosporin saturated solution that organic solvent instillation step 2) is obtained or obtained step 2) is instilled in organic solvent, to separate out crystal, filtering, by filtration cakes torrefaction, aerosporin crystallization is obtained.The solvent cost used in the preparation method of the present invention is cheap and is readily available, and is adapted to large-scale industrial production, and obtained aerosporin crystalline product has higher purity and clarity, hygroscopicity low.
Description
Technical field
The invention belongs to pharmaceutical technology field, more particularly to a kind of aerosporin crystallization and preparation method thereof.
Background technology
Polymyxin B is caused by aerobacillus polymyxa Donker (bacillus polymyxa), by several amino acids and fat
A kind of alkaline ring type polypeptide class antibiotic of acid composition.Polymyxin B product is multicomponent mixture, including Polymyxin B1,
B2, B3, B1-1 (European Pharmacopoeia requires quality sum >=80.0% of this 4 kinds of components), clinically commonly use its sulfate.Sulfuric acid is more
Colistin B is white or off-white powder, moist with drawing.Aerosporin has potent to gramnegative bacterium
Killing action, particularly there is the extracorporeal sensitivity of height to NDM-1 bacteriums (superbacteria), thus receive much concern.
For the refined of aerosporin finished product, because it is not easy to crystallize, so using spray dry (patent application more
CN201210519331.7) or desivac (patent application CN201510775580.6) is carried out.Crystallized at present on polymyxin B
Patent include as disclosed in patent application CN201210379231.9 Polymyxin B1 two hydrates crystallization, it uses third
Ketone and ether obtain as mixed solvent through precipitation, but ether highly volatile, are oxidized easily in atmosphere and trigger blast, no
It is adapted to industrialized production.In addition, the product of in the market is aerosporin mixture, relevant aerosporin 1 is single
The preparation of body, the patent application that the applicant submits have been authorized (patent No. ZL201110390624.5), its final production
Product are also to be prepared using spray dry.The aerosporin and sulfuric acid commercially prepared using spray dry is more viscous
Rhzomorph B products have strong hygroscopicity, find after testing, and clarity is not high, and certain muddiness is presented in solution, and product quality needs to be carried
It is high.Mother liquor is carried out using conventional crystallization methods to crystallize part that can be removed impurity, but the precipitate of aerosporin is very
It is viscous, easily bond agglomerating, it is difficult to carry out crystallization operation.Therefore, it is necessary to develop new aerosporin crystallization and its prepare
Method.
The content of the invention
For aerosporin product clarity present in prior art and purity is high, crystallization precipitate easily
The problem of agglomerating is bonded, the invention provides a kind of aerosporin crystallization and preparation method thereof.
The preparation method of aerosporin crystallization provided by the invention comprises the following steps：
1) polymyxin B zymotic fluid is adsorbed with resin, adds aqueous sulfuric acid and/or the acidic ethanol aqueous solution enters
Row desorption, obtains stripping liquid；
2) pH for the stripping liquid for obtaining step 1) is adjusted to 5.0~7.0, has then been concentrated into solid precipitation, has been obtained sulphur
Sour polymyxin B saturated solution；
3) while stirring, by the obtained aerosporin saturated solution of organic solvent instillation step 2) or
The aerosporin saturated solution that step 2) is obtained is instilled in organic solvent, to separate out crystal, filtering, and by filtration cakes torrefaction,
Obtain aerosporin crystallization.
Preferably, in the step 1) of the preparation method of the present invention, the concentration of the aqueous sulfuric acid is 0.2mol/L.
One specific embodiment of preparation in accordance with the present invention, in step 1), desorption process is with per hour
The speed of the 0.2mol/L of 0.5 times of column volume aqueous sulfuric acid is desorbed, fraction collection stripping liquid.Stripping liquid potency is higher than
Start to collect during 500 μ g/ml, potency stops collecting when being less than 500 μ g/ml.
Preferably, in the step 1) of the preparation method of the present invention, polymyxin B zymotic fluid is being adsorbed with resin
Before, the polymyxin B zymotic fluid is also by following pretreatment：
A) acidification；
B) plus diatomite filters.
Preferably, it is described that polymyxin B zymotic fluid is inhaled with resin in the step 1) of the preparation method of the present invention
It is attached including：
(i) ion-exchange treatment；
(ii) macroporous absorbent resin is handled.
One specific embodiment of preparation in accordance with the present invention, the pretreatment are included the polymyxin B
The pH of zymotic fluid is adjusted to 1.8, is then added diatomite, is filtered after stirring, then is washed till activity in filtrate with water top and is less than
0.2g/L；Preferably, the acidification is to be adjusted the pH value of polymyxin B zymotic fluid to 1.8 by adding oxalic acid, then
Diatomite is added, with plate-frame filtering after stirring, then activity in filtrate is washed till with water top and is less than 0.2g/L.
One specific embodiment of preparation in accordance with the present invention, the ion-exchange treatment includes will be acidified
It is 6.7 that the polymyxin B fermentating liquid filtrate of processing, which is adjusted to pH value, is then adsorbed, adsorbed with weak-acid ion exchange resin
Quan Hou, by the water washing of 2~3 volumes based on column volume (volume of the weak-acid ion exchange resin i.e. in pillar) to outflow
Liquid is colourless, is then desorbed with aqueous sulfuric acid.Wherein preferably, the weak-acid ion exchange resin is LXD-135 faintly acids
Ion exchange resin.More specifically, the ion-exchange treatment includes filtering the polymyxin B zymotic fluid of acidified pretreatment
Liquid is adjusted to pH value as 6.7 using 2M sodium hydroxides, is then adsorbed with weak-acid ion exchange resin, after absorption completely, with 2~3
The water washing of times column volume is colourless to efflux, is then desorbed with 0.2M aqueous sulfuric acids；Preferably, the weak acid ion is handed over
It is LXD-135 weak-acid ion exchange resins to change resin.
One specific embodiment of preparation in accordance with the present invention, the macroporous resin adsorption processing include passing through
It is 6.5 that pH value is arrived in the polymyxin B zymotic fluid regulation of ion-exchange treatment, after macroporous absorbent resin absorption completely, with
The water top of 2 volumes based on column volume is washed, and is then desorbed with the acidic ethanol aqueous solution；Preferably, at the macroporous resin adsorption
Reason includes the polymyxin B zymotic fluid Jing Guo ion-exchange treatment arriving pH value as 6.5 using the regulation of 2M sodium hydroxides, with big
Macroporous adsorbent resin absorption completely after, top is carried out by the water of 2 volumes based on column volume and washed, then using pH value as 3 40% (v/v)
Ethanol water desorbs.
Preferably, preparation method of the invention is additionally included in step 1), and obtained stripping liquid is concentrated, through activity
Carbon decoloring simultaneously filters.
Preferably, in the step 2) of the preparation method of the present invention, with 0.5M-10M sodium hydroxide solutions by stripping liquid
PH value is adjusted to 5.0~7.0.
Preferably, in the step 3) of the preparation method of the present invention, the organic solvent is selected from C1-C4Alcohol, C3-C4's
One or more in ketone or ethyl acetate, butyl acetate；It is highly preferred that the C1~C4Alcohol be selected from methanol, ethanol, isopropyl
One or more in alcohol, normal propyl alcohol or butanol；It is highly preferred that the C3~C4Ketone be selected from acetone or 2- butanone.
Preferably, in the step 3) of the preparation method of the present invention, the process for separating out crystal is entered at a temperature of 0~40 DEG C
OK.
One specific embodiment of preparation in accordance with the present invention, the preparation method bag of aerosporin crystallization
Include following steps：
1) acidification plus diatomite filtering, ion-exchange treatment, macroporous absorption tree are carried out to polymyxin B zymotic fluid
After fat processing, aqueous sulfuric acid desorption is added, obtains stripping liquid；Obtained stripping liquid is concentrated and with mistake after activated carbon decolorizing
Filter, obtains filtrate；
2) pH value of filtrate is adjusted to 5.0~7.0 with sodium hydrate aqueous solution, has been concentrated into solid precipitation, has obtained sulphur
Sour polymyxin B saturated solution；
3) organic solvent is added dropwise to analysis in the saturated solution of the aerosporin obtained while stirring to step 2)
Go out crystal, process temperature is added dropwise in the range of 0~10 DEG C；Continue that 0.5~5 based on aerosporin saturated solution is added dropwise
The organic solvent of times volume simultaneously stirs 0~8 hour, filtering, abandons filtrate, filter cake is dried in vacuo 3~20 hours, obtains crystallinity
Aerosporin.
Present invention also offers the crystallinity aerosporin prepared according to above-mentioned preparation method, i.e. the present invention also carries
A kind of aerosporin has been supplied to crystallize, the wherein crystallization is radiated using Cu-Ka, spread out with the X-ray powder that 2 θ angles represent
It is as shown in Figure 3 to penetrate collection of illustrative plates.As can be seen from Figure 3, aerosporin prepared according to the methods of the invention has crystal formation.
Further, present invention also offers a kind of pharmaceutical composition, it is more viscous that described pharmaceutical composition includes the sulfuric acid
Rhzomorph B is crystallized, and pharmaceutically acceptable carrier.Optionally, described pharmaceutical composition also includes one or more sulfuric acids
Antibacterial Constituents beyond polymyxin B.Preferably, the formulation of described pharmaceutical composition is tablet, capsule or granule；
It is highly preferred that the tablet is selected from fast-release tablet, chewable tablets, dispersible tablet, effervescent tablet, sustained release tablets, controlled release tablet or enteric coatel tablets, the glue
Wafer is selected from hard shell capsules, soft capsule, spansule, controlled release capsule or capsulae enterosolubilis, and the granule is selected from mix suspension grain, effervesce
Particle, enteric coated particles, slow-releasing granules or controlled release granule.
Present invention also offers aerosporin crystallization to prepare antibacterials, especially thin in anti-Gram-negative
Purposes in the medicine of bacterium.Present invention also offers aerosporin crystallization to prepare overriding resistance bacterium, and especially anti-leather is blue
Purposes in family name's feminine gender drug-resistant bacteria, such as the medicine of the super drug-resistant bacteria of Gram-negative.
In other words, present invention also offers for antibacterial, the aerosporin knot of especially anti-gramnegative bacterium
It is brilliant.Present invention also offers super for overriding resistance bacterium, especially anti-Gram-negative drug-resistant bacteria, such as Gram-negative
The aerosporin crystallization of drug-resistant bacteria.
Present invention also offers one kind prevention and/disease caused by treatment bacterium infection, especially gramnegative bacterium
Or the method for illness, this method include giving subject or patient in need prevention and/or therapeutically effective amount comprising described
The pharmaceutical preparation of aerosporin crystallization.
Present invention also offers one kind prevention and/treatment drug-resistant bacteria to infect, especially anti-Gram-negative drug-resistant bacteria,
Such as the method for disease or illness caused by the super drug-resistant bacteria of Gram-negative, this method include giving subject in need
Or the pharmaceutical preparation for including aerosporin crystallization of patient's prevention and/or therapeutically effective amount.
Aerosporin crystalline product is prepared in the present invention, test result indicates that crystal formation, clarity is presented in the product
More preferable with purity, hygroscopicity is low.In addition, preparation method provided by the invention is easily operated, used solvents used for crystallization
Cost is cheap and is readily available, and is adapted to large-scale industrial production, and the clinical practice for extension aerosporin crystallization is established
Basis.
Brief description of the drawings
Figure 1A shows crystallization of the aerosporin in n-butanol-ethanol water；
Figure 1B shows dried aerosporin crystallized sample；
Fig. 2A is the microphoto under aerosporin 40 times of object lens of crystallization；
Fig. 2 B are the microphotos under aerosporin 100 times of object lens of crystallization；
Fig. 3 is X-ray powder diffraction (XRD) spectrogram of aerosporin crystallized sample；
Fig. 4 A are the HPLC spectrograms and chromatogram peak data of aerosporin spray drying sample；
Fig. 4 B are the HPLC spectrograms and chromatogram peak data of aerosporin crystallized sample；
Fig. 5 shows the clarity contrast situation of aerosporin crystallization (left side) and spray drying sample (right side).
Embodiment
In order to which the present invention is better described, technical scheme is readily appreciated, enters one in conjunction with drawings and examples
Step illustrates the present invention, it will be appreciated that specific embodiment of the invention is for illustration purposes only, rather than limitation of the present invention.
In this application, used polymyxin B zymotic fluid is prepared via a method which：Using aerobacillus polymyxa Donker,
Using flour and cotton seed meal as primary raw material, by the pure-blood ferment culture of 40-50 hours, polymyxin B zymotic fluid is obtained.Contrast
The sample that sample is aerosporin material sample and in the market is bought.
Embodiment 1
Polymyxin B zymotic fluid 5L, potency 1.8g/L are taken, it is 1.8 to add oxalic acid solid and adjust pH.After stirring 30 minutes,
250g diatomite is added, continues stirring 30 minutes, then with plate-frame filtering, is washed till potency with water top and is less than 0.2g/L, stopped
Filter, obtains filtrate 7.1L, contains 8.5g polymyxin Bs in filtrate.Filtrate adjusts pH to 6.7 with 2M sodium hydroxides, is then loaded into
In chromatographic column containing 60ml LXD-135 weak-acid ion exchange resins, post absorption is crossed.After absorption completely, using by LXD-
The water washing of 2-3 times of volume of the stereometer of 135 weak-acid ion exchange resins is colourless to efflux, then with 0.2M sulfuric acid water
Solution stripping, obtain stripping liquid 800ml (containing aerosporin 7.4g).Stripping liquid adjusts pH with 2M sodium hydroxide solutions
To 6.5, the chromatographic column for being then loaded into the LXT-081 macroporous absorbent resins containing 80ml is adsorbed.Absorption completely after, with by
The water top of 2 volumes of column volume meter is washed, and is then desorbed with 40% (v/v) the acidic ethanol aqueous solution (pH 3), is obtained stripping liquid
1600ml (sulfur acid polymyxin B 6.74g).
At a temperature of 50 DEG C, after being concentrated into 1/3 liquor capacity with Rotary Evaporators, add based on the volume of concentrate
1% (w/v) activated carbon 5g, stir 30 minutes, filter, filtrate 6.1g containing polymyxin B at 60 DEG C.Filtrate is with 2M hydrogen-oxygens
Change sodium and adjust pH to 5.0-7.0, then proceed to be concentrated into 70ml or so, open stirring, slowly dropwise addition absolute ethyl alcohol to solution has mixed
Stop that absolute ethyl alcohol is added dropwise during turbid phenomenon, be then cooled to 0-5 DEG C, stir to solid and continue to be added dropwise after separating out by the volume of concentrate
The absolute ethyl alcohol of 1-2 times of volume of meter, until the absolute ethyl alcohol for 3 times of amounts being added dropwise altogether based on the volume of concentrate.Then, in 0-5
Continue stirring 6 hours under conditions of DEG C, filter out solid, the solid filtered out is dried in vacuo 4 hours at a temperature of 60 DEG C, obtained
To 5.4g white solids, crystallization yield 88.5%, purity 91.38%.The X ray powder of gained aerosporin crystal
Last diffraction (XRD) spectrogram is as shown in figure 3, clarity detection (two B of annex Ⅸ of Chinese Pharmacopoeia version in 2010) result is less than No. 1
(shown on the left of Fig. 5), aerosporin clarity on the market are more than No. 2 (shown on the right side of Fig. 5).Pass through cylinder-plate method
Aerosporin prepared by the method for the measure present invention, obtained potency is 8300U/g.Gained aerosporin crystallizes
The HPLC spectrograms of sample and spray drying sample are respectively as shown in Fig. 4 B and Fig. 4 A.
Embodiment 2
Polymyxin B zymotic fluid 5L is taken, potency 1.8g/L, stripping liquid is then prepared according to the technique of embodiment 1.This reality
Apply stripping liquid that example obtains it is concentrated after volume be 50ml (sulfur acid polymyxin B 5.3g) concentrate.Stirring is opened, to dense
Ethyl acetate to solution is slowly added dropwise in contracting liquid and when having research of chaotic phenomenon stops that ethyl acetate is added dropwise.Then 0-5 DEG C is cooled to, stirring
Continue that residual acetic acid ethyl ester is added dropwise after separating out to solid, until the ethyl acetate for 3 times of amounts being added dropwise altogether based on the volume of concentrate.So
Continue stirring 7 hours under the conditions of 0-5 DEG C afterwards, filter out solid.It is small that the solid filtered out is dried in vacuo at a temperature of 60 DEG C to 3
When, 4.82g white solids are obtained, crystallization yield 91%, purity 89.58%, clarity testing result is No. 1, passes through cylinder-plate method
The potency of measure is 8355U/g.
Embodiment 3
Polymyxin B zymotic fluid 7L is taken, potency 1.8g/L, stripping liquid is then prepared according to the technique of embodiment 1.This reality
Apply example stripping liquid it is concentrated after obtain volume be 85ml concentrate (sulfur acid polymyxin B 6.7g).Stirring is opened, to dense
Slowly slow be added dropwise when acetone to solution becomes turbid stops that acetone is added dropwise in contracting liquid.Then 0-5 DEG C is cooled to, is stirred to solid
Body after separating out continues that remaining acetone is added dropwise, until the acetone for 3.5 times of amounts being added dropwise altogether based on the volume of concentrate.Then at 0-5 DEG C
Under the conditions of continue stirring 6 hours, filter out solid, the solid filtered out be dried in vacuo 4 hours at a temperature of 60 DEG C, obtained
5.83g white solids, crystallization yield 87%, purity 90.98%, clarity 1, the potency determined through cylinder-plate method is 8100U/g.
Under similarity condition, the product that the crystallized sample of the present embodiment and market are bought is subjected to minor biological cycling respectively and HPLC is examined
Survey.As a result show：The biological value of the crystallized sample of the present embodiment is higher by 8.8% than the biological value of market purchase product.Phase
To the product of in the market, it is relatively difficult that biological value, which improves 8.8%,.For quality product, sulfuric acid is more in the present invention
The amplitude that the biological value of Colistin B crystalline product improves is very big.From the HPLC of market purchase product (spray dry preparation)
As a result the chromatographic purity of middle principal component (B1, B2, B3, B1-1) is 89.99% (Fig. 4 A), the HPLC of the crystallized sample of the present embodiment
As a result the chromatographic purity of middle active ingredient (B1, B2, B3, B1-1) is 92.62% (Fig. 4 B), far above in European Pharmacopoeia standard
The requirement of 4 kinds of component sum >=80.0%.Relative to the spray drying technique generally used at present, use the method for the present invention can be with bright
The aobvious chromatographic purity and content for improving crystalline product, so as to improve product quality.
Embodiment 4
Polymyxin B zymotic fluid 4L is taken, potency 1.5g/L, stripping liquid is then prepared according to the technique of embodiment 1.It incite somebody to action this
The stripping liquid of embodiment is concentrated into about 25ml saturated solution (sampling detection, sulfur acid polymyxin B 2.60g), then will be full
250ml (10 times of volumes of aerosporin saturated solution) is slowly dropped into solution to be in the isopropanol under stirring,
The temperature of isopropanol is controlled in the range of 5-10 DEG C all the time during dropwise addition.The aerosporin separated out during dropwise addition
Crystallization gradually increase, in homogeneously dispersed state, adhesion.Continue stirring 1 hour, filtering, by temperature of the solid filtered out at 50 DEG C
The lower vacuum drying of degree 10 hours, obtains aerosporin crystal powder 2.40g, crystallization yield 92.3%.
Embodiment 5
Polymyxin B zymotic fluid 10L is taken, potency 2.0g/L, stripping liquid is then prepared according to the technique of embodiment 1.Will
The stripping liquid of the present embodiment is concentrated into about 140ml saturated solution (sampling detection, sulfur acid polymyxin B 13.50g), then
Saturated solution is slowly dropped into the n-butanol that 700ml (5 times of volumes of aerosporin saturated solution) is under stirring
In, the temperature of butanol solution system is controlled in the range of 25-30 DEG C all the time under dropwise addition process.The sulphur separated out during dropwise addition
Sour polymyxin B gradually increases, and crystallizes dispersed, adhesion, and graininess is good.The aerosporin aqueous solution drips
Afterwards, continue to stir 30min, filtering, the solid filtered out is dried in vacuo 20 hours at a temperature of 40 DEG C, obtains the more Acarasiales of sulfuric acid
Plain B crystal powders 12.40g, crystallization yield 91.9%.
Embodiment 6
Polymyxin B zymotic fluid 7L is taken, potency 1.5g/L, stripping liquid is then prepared according to the technique of embodiment 1.Will solution
Imbibition is concentrated into about 50ml saturated solution (sampling detection, sulfur acid polymyxin B 5.00g), then that saturated solution is slow
Instill ethanol (200ml)+n-butanol that 400ml (8 times of volumes of aerosporin saturated solution) is under stirring
(200ml) in the mixed solvent.Temperature is controlled in the range of 0-5 DEG C all the time during dropwise addition.The sulfuric acid separated out during dropwise addition
Polymyxin B gradually increases, and is uniformly dispersed, and has good graininess (Fig. 1).After the aerosporin aqueous solution drips,
Continue stirring 2 hours, filtering, the solid filtered out is dried in vacuo 8 hours at a temperature of 60 DEG C, obtains aerosporin
Crystal powder 4.52g, crystallization yield 90.4%.Observed by 40 times and 100 times object lens of microscope, aerosporin crystallization is non-
Chang Mingxian, single crystal is uniformly (Fig. 2A and Fig. 2 B).
Embodiment 7
Polymyxin B zymotic fluid 15L is taken, potency 1.5g/L, stripping liquid is then prepared according to the technique of embodiment 1.Will
Stripping liquid is concentrated into about 100ml saturated solution (sampling detection, sulfur acid polymyxin B 10.00g), then by saturated solution
It is slowly dropped into ethanol (500ml) that 1000ml (10 times of volumes of aerosporin saturated solution) is under stirring+different
In propyl alcohol (500ml).Temperature is controlled in the range of 5-10 DEG C all the time during dropwise addition, separate out continue to drip after crystal it is molten
Agent, obtained crystal are uniformly dispersed, and have graininess, can realize that solid-liquid is layered (30min) after standing quickly.Continue to stir
30min, filtering, the solid filtered out is dried in vacuo 10 hours at a temperature of 55 DEG C, obtains aerosporin crystal powder
9.23g, crystallization yield 92.3%.
Although the embodiment of the present invention is described and illustrated in detail, it should be recognized that this hair
It is bright not limited by the embodiment.Without departing from the spirit and scope of the present invention, the present invention is made respectively
Kind improvement, modification and transformation are within the scope of the present invention.
Claims (10)
1. a kind of preparation method of aerosporin crystallization, it is characterised in that the preparation method comprises the following steps：
1) polymyxin B zymotic fluid is adsorbed with resin, adds aqueous sulfuric acid and/or the acidic ethanol aqueous solution is solved
Inhale, obtain stripping liquid；
2) pH for the stripping liquid for obtaining step 1) is adjusted to 5.0~7.0, has then been concentrated into solid precipitation, it is more to obtain sulfuric acid
Colistin B saturated solution；
3) under agitation, by the obtained aerosporin saturated solution of organic solvent instillation step 2) or by step 2)
Obtained aerosporin saturated solution is instilled in organic solvent, to separate out crystal, filtering, by filtration cakes torrefaction, obtains sulfuric acid
Polymyxin B crystallizes.
2. preparation method according to claim 1, it is characterised in that in step 1), the concentration of the aqueous sulfuric acid
For 0.2mol/L.
3. preparation method according to claim 1 or 2, it is characterised in that the preparation method is additionally included in step 1),
Before being adsorbed with resin to polymyxin B zymotic fluid, the polymyxin B zymotic fluid is also by pretreatment, the pre- place
Reason comprises the following steps：
A) acidification；
B) plus diatomite filters；
Preferably, it is described absorption is carried out to polymyxin B zymotic fluid with resin to include：
(i) ion-exchange treatment；
(ii) macroporous absorbent resin is handled；
It is highly preferred that the pretreatment includes adjusting the pH of the polymyxin B zymotic fluid to 1.8, then add in step b)
Enter diatomite, filtered after stirring, then activity in filtrate is washed till with water top and is less than 0.2g/L；More preferably, the pH regulations are logical
Cross the progress of addition oxalic acid；
More preferably, the ion-exchange treatment includes adjusting the pH of the polymyxin B fermentating liquid filtrate of acidified processing
For 6.7, then adsorbed with weak-acid ion exchange resin, after absorption completely, by the water washing of 2~3 volumes based on column volume
It is colourless to efflux, then desorbed with 0.2mol/L aqueous sulfuric acid；Preferably, the weak-acid ion exchange resin is
LXD-135 weak-acid ion exchange resins；The pH regulations are carried out using 2M sodium hydroxide solutions；
Again preferably, the macroporous resin adsorption processing is included the pH of the polymyxin B zymotic fluid Jing Guo ion-exchange treatment
6.5 are adjusted to, after macroporous absorbent resin absorption completely, is washed by the water top of 2 volumes based on column volume, then with acidic ethanol
The aqueous solution desorbs；Preferably, pH regulations are carried out using 2M sodium hydroxide solutions；It is further preferred that the acidic ethanol is water-soluble
Liquid is the 40 volume ratio % ethanol waters that pH value is 3；
It is further preferred that the preparation method is additionally included in step 1), obtained stripping liquid is concentrated, through activated carbon decolorizing
After filter.
4. preparation method according to any one of claim 1 to 3, it is characterised in that in step 2), with 0.5M~
10M sodium hydroxide solutions adjust the pH of stripping liquid to 5.0~7.0.
5. preparation method according to any one of claim 1 to 4, it is characterised in that described organic molten in step 3)
Agent is selected from C1~C4Alcohol, C3~C4Ketone or ethyl acetate or butyl acetate in one or more；Preferably, the C1~C4
One or more of the alcohol in methanol, ethanol, isopropanol, normal propyl alcohol or butanol；More preferably, the C3~C4Ketone choosing
One or more from acetone or 2- butanone.
6. preparation method according to any one of claim 1 to 5, it is characterised in that in step 3), separate out crystal
Process is carried out at a temperature of 0~40 DEG C；
Preferably, the preparation method is additionally included in step 3), continues stirring after being added dropwise to complete 0~8 hour；
It is highly preferred that in step 3), the drying is to be dried in vacuo 3~20 hours at a temperature of 40~60 DEG C.
7. a kind of aerosporin crystallization, it is characterised in that the aerosporin crystallization is radiated using Cu-Ka, with 2
The X-ray powder diffraction pattern that θ angles represent is as shown in Figure 3.
8. a kind of antibacterial combination, it is characterised in that described pharmaceutical composition is included according to any in claim 1 to 6
Aerosporin crystallization or aerosporin knot according to claim 7 prepared by the preparation method described in
Crystalline substance, and pharmaceutically acceptable carrier；Preferably, the formulation of described pharmaceutical composition is tablet, capsule or granule；
It is highly preferred that the tablet is selected from fast-release tablet, chewable tablets, dispersible tablet, effervescent tablet, sustained release tablets, controlled release tablet or enteric coatel tablets, the glue
Wafer is selected from hard shell capsules, soft capsule, spansule, controlled release capsule or capsulae enterosolubilis, and the granule is selected from mix suspension grain, effervesce
Particle, enteric coated particles, slow-releasing granules or controlled release granule.
9. pharmaceutical composition according to claim 8, it is characterised in that described pharmaceutical composition also includes one or more
Antibacterial Constituents beyond sulfuric acid polymyxin B.
10. aerosporin prepared by the preparation method as any one of claim 1 to 6 crystallizes or according to right
It is required that the aerosporin described in 7 crystallizes the application in antibacterials are prepared；Preferably it is used for anti-gram-negative in preparation
Application in the medicine of property bacterium；Sulfuric acid prepared by preparation method more preferably according to any one of claim 1 to 6
Polymyxin B crystallizes or aerosporin according to claim 7 crystallization is in the medicine for preparing overriding resistance bacterium
Using the drug-resistant bacteria is preferably Gram-negative drug-resistant bacteria.
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| CN113121646A (en) * | 2021-03-02 | 2021-07-16 | 河北圣雪大成制药有限责任公司 | Method for preparing polymyxin methanesulfonic sodium by crystallization process |
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