CN1864755A - Preparation method of endotoxin absorbent for blood perfusion - Google Patents
Preparation method of endotoxin absorbent for blood perfusion Download PDFInfo
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- CN1864755A CN1864755A CN 200510046452 CN200510046452A CN1864755A CN 1864755 A CN1864755 A CN 1864755A CN 200510046452 CN200510046452 CN 200510046452 CN 200510046452 A CN200510046452 A CN 200510046452A CN 1864755 A CN1864755 A CN 1864755A
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- polymyxin
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Abstract
The invention relates to an endotoxin adsorbent, which in detail relates to a method of preparing endotoxin adsorbent for blood perfusion. The method employs spherical agarose gel with good biocompatibility as base material, employs epichlorohydrin, hexamethylene diamine, 1, 1'-carbonyldiimidazole as activating agent, and bonds polymyxin B for endotoxin removal. The invention is characterized in that it overcomes the toxicity of cyanogen bromide and instability of aglycone, improves the safty and reliability of operation, reducesnon-specific adsorption through bonding polymyxin B with 1, 1'-carbonyldiimidazole, and improves biocompatibility of adsorbent and suits for treating endotoxemia. The invention is also characterized by simple experimental procedure and high clearance for endotoxin.
Description
Technical field
The present invention relates to endotoxin absorbent, specifically a kind of preparation method that is used for the endotoxin absorbent of hemoperfusion.
Background technology
Endotoxin is a lipoid polysaccharose substance that discharges in gram negative bacilli when growth or produced by the cell wall cracking when dead, and denier (nanogram level) endotoxin enters human body and will cause hyperpyrexia, diarrhoea, and vasodilation, even faint or dead.Endotoxemia is one of clinical modal deadly disease, and mortality rate can reach 40%-90%, does not still have effective Therapeutic Method clinically.How accomplishing to remove timely and effectively the intravital endotoxin of patient, is the key of treatment endotoxemia.In recent years, developed multiple anti-endotoxic molecular biosciences preparation short of money abroad, but its complex manufacturing, the medicine source is few, cost is high, and does not still have so far and a kind ofly can pass through III phase clinical experiment [Yao Yongming etc., China's critical illness emergency medicine, 11:456 (1999)].
Haematogenic immunity absorption therapy is emerging in recent years a kind of clinical treatment technology, the difficult and complicated illness that does not have obvious curative effects for some medicines, carry out external immunoadsorption blood purification as autoimmune systemic disease, organ transplantation rejection, hepatitis gravis, malignant tumor, the nephrotic syndrome, jaundice etc., often can obtain good effect.Haematogenic immunity adsorbing therapy technology just is being widely used in various clinical fields such as urinary system, hepatopathy, blood, heart failure.Abroad begun the method for blood purification is used for the treatment of endotoxemia, the research worker of Japan is coated with polymyxin B and is used for the endotoxic removal of blood plasma [Tani T on the inwall that invests hollow-fibre membrane, et al., Artif.Organs 22 (12): 1038-1044 (1998)].The research worker of University of Vienna is utilized self-designed MDS system, polymine is bonded in has carried out zoopery on the cellulose, and the endotoxin removal effect is [K.Von Appen, et al., Artif.Organs, 20 (5): 420425 (1996)] better.Domesticly utilize the endotoxic zoopery of activated carbon adsorption report, but DeGrain [Wang Jinda. Chinese critical emergency medicine, 10:578 (1998)]; Nankai University is a carrier with the agarose, through epoxidation, and amination, bonding affinity ligand behind the glutaraldehyde cross-linking has prepared a series of endotoxic adsorbents of blood plasma [Yuan Zhi etc., Chinese invention patent, publication number: CN1493368A, 2004] that are used for removing.
Summary of the invention
The invention provides a kind of preparation method that is used for the endotoxin absorbent of hemoperfusion.The present invention is that the Sepharose CL-4B agarose gel with good biocompatibility is a substrate, through epoxidation, and amination, 1, after the activation of 1 '-carbonyl dimidazoles, the bonding polymyxin B is used for endotoxic removal.This method has overcome the toxicity of cyanogen bromide activation and the unstability of aglucon, has improved the stability of affinity ligand and the safety and the reliability of operation; Adopt 1,1 '-carbonyl dimidazoles activation has bonding affinity ligand polymyxin B behind the amino agarose gel, unreacted active imidazole group complete hydrolysis in aqueous solution is a hydroxyl, reduced the non-specific adsorption of adsorbent, improve the biocompatibility of adsorbent, more be applicable to the blood purification treatment endotoxemia; 1,1 '-carbonyl dimidazoles activation method has been avoided sealing, the reduction reaction of glutaraldehyde bonding method, has simplified experimental procedure.
Technical scheme of the present invention is:
A kind of preparation method that is used for the endotoxin absorbent of hemoperfusion is a substrate with Sepharose CL-4B agarose gel, through (epoxychloropropane) epoxidation, (hexamethylene diamine) amination, through 1, after the activation of 1 '-carbonyl dimidazoles, the bonding polymyxin B is used for endotoxic removal again.
Specific operation process is as follows,
1) preparation of epoxy substrate (epoxychloropropane activated agarose gel)
Measure an amount of Sepharose CL-4B agarose gel, the abundant drip washing of water is as substrate; Substrate is in 1-2mol/L NaOH solution, and 30-50 ℃ is descended and epichlorohydrin reaction 1-4 hour, and epoxychloropropane is fully excessive; The abundant drip washing of water after reaction is finished, filter is done, and obtains epoxy substrate;
2) preparation of amino substrate (hexamethylene diamine carries out ring-opening reaction to the agarose gel that has epoxide group)
Above-mentioned epoxy substrate is mixed with hexamethylene diamine solution, and at pH 9-12, reaction temperature is 50-80 ℃, reacts 1-4 hour, and hexamethylene diamine is fully excessive; The abundant drip washing of water after reaction is finished, filter is done, and obtains amino substrate.
3) preparation of active imidazoles substrate (1, the activation of 1 '-carbonyl dimidazoles has the agarose gel of active amino)
Or/and acetonitrile repeatedly washs above-mentioned amino substrate, add 1 with exsiccant dioxane, acetone, 1 '-carbonyl dimidazoles, reaction is 1-4 hour under the room temperature; 1,1 '-carbonyl dimidazoles is excessive; After reaction is finished, with dioxane, acetone or/and the acetonitrile flush away is unreacted 1,1 '-carbonyl dimidazoles;
4) 1, activatory agarose gel of 1 '-carbonyl dimidazoles and polymyxin B prepared in reaction endogenous toxin adsorbent, reaction temperature is 10-30 ℃, response time 8-24 hour, pH 7-12, polymyxin B is excessive;
Use 0.2mol/L NaOH, 20% ethanol (v/v), 1.5mol/L NaOH flushing after reaction is finished respectively, be washed till neutrality with apirogen water at last; Obtain endotoxin absorbent.Put into and preserve liquid, 4 ℃ of preservations; The present invention adopts and contains 0.02-0.10% (w/w) NaN
3The phosphate buffer of 0.1mol/L pH 7.4 do preservation liquid.
The endotoxin absorbent that described hemoperfusion is used is used for the endotoxin of clinical removing blood samples of patients, treatment endotoxemia disease.
The invention has the beneficial effects as follows:
1, the present invention is by adopting epoxychloropropane, hexamethylene diamine, 1, and behind 1 '-carbonyl dimidazoles activation SepharoseCL-4B agarose gel, the bonding polymyxin B provides a kind of preparation method of the new endotoxin absorbent that is used for hemoperfusion.
2, adsorbent of the present invention is that the agarose gel with good biocompatibility is a substrate, through epoxidation, and amination, 1, after the activation of 1 '-carbonyl dimidazoles, the bonding affinity ligand is used for endotoxic removal.The present invention has overcome the toxicity and the unstability of cyanogen bromide method, has increased the safety of preparation.
3, owing to unreacted active imidazole group, complete hydrolysis is a hydroxyl in aqueous solution, has improved the biocompatibility of adsorbent, has reduced the non-specific adsorption of adsorbent, is more suitable for the blood purification treatment endotoxemia.
4, the present invention adopts 1, and behind 1 '-carbonyl dimidazoles activation Sepharose CL-4B agarose gel, the bonding polymyxin B is compared with the glutaraldehyde bonding method, has avoided sealing, reduction reaction, has simplified experimental procedure.
The specific embodiment
With specific embodiment the present invention is described below, but they do not impose any restrictions to the present invention.
Embodiment 1
1) preparation of epoxy substrate
Measure Sepharose CL-4B agarose gel 50mL with graduated cylinder, pour sand core funnel into, the abundant drip washing of water is drained.Change in the conical flask, add 1mol/L NaOH 50mL, epoxychloropropane 25mL seals back in shaking table 40 ℃, and 200r/min reacted 2 hours.After reaction is finished, the abundant drip washing of water, filter is done standby;
2) preparation of amino substrate
Above-mentioned epoxy substrate is changed in the conical flask, add the 0.1mol/L NaHCO of 13g/L hexamethylene diamine
3Buffer 250mL.Shaking table 50-55 ℃, 150r/min reacted 2 hours.After reaction is finished, pour the sand core funnel sucking filtration into, the abundant drip washing of water, filter is done standby;
3) affinity ligand is immobilized
The dioxane of getting certain volume adds an amount of sodium metal in round-bottomed flask, after reflux 6-12 hour, steam dioxane and use.
Repeatedly wash above-mentioned amino substrate with the 250mL dioxane that steams, and be transferred in the conical flask.Take by weighing 5-10g 1,1 '-carbonyl dimidazoles is dissolved in the 100-150mL dioxane, mixes with above-mentioned amino substrate.Under the room temperature, shaking table 120r/min reacted 2 hours.After reaction is finished, pour the sand core funnel sucking filtration into, wash several times with dioxane, flush away is unreacted 1, and 1 '-carbonyl dimidazoles is transferred in the conical flask;
Take by weighing the 1.0-2.0g polymyxin B and be dissolved in 250mL 0.1mol/L NaHCO
3Solution, the pH to 7-10 of 4mol/LNaOH or 4mol/L hydrochloric acid conditioning solution.With above-mentioned 1, the activatory agarose gel of 1 '-carbonyl dimidazoles mixes, and under the 120r/min, reacts 24 hours in the room temperature shaking table.After reaction is finished, pour the sand core funnel sucking filtration into, wash with 0.2mol/L NaOH, 20% ethanol (v/v), the 1.5mol/LNaOH of 3-5 times of volume respectively again, be washed till neutrality with apirogen water at last, drain, put into and preserve liquid, 4 ℃ of preservations.
Embodiment 2 adsorbents are used for the endotoxic removal of aqueous solution
Get the adsorbent of 50mg bonding polymyxin B, add 1mL 16Eu/mL endotoxin solution, 25 ℃ of vibrations are after 2 hours, and the centrifuging and taking supernatant adopts azo development process to measure endotoxic concentration.Experimental result shows endotoxic clearance greater than 90%.
The bonded amount of 3 two kinds of endotoxin absorbent polymyxin Bs of embodiment reaches to endotoxic removal result in the aqueous solution relatively
Adsorbent 1:Sepharose CL-4B agarose gel after epoxychloropropane activation, Direct Bonding polymyxin B, the endotoxin absorbent that obtains.
Adsorbent 2:Sepharose CL-4B agarose gel is through epoxychloropropane, hexamethylene diamine, 1, and 1 '-carbonyl dimidazoles activates back bonding polymyxin B, the endotoxin absorbent that obtains.
The bonded amount of adsorbent 1,2 polymyxin Bs reaches endotoxic removal effect in the aqueous solution is seen the following form.The assay method of polymyxin B is seen document [D.Petsch et al, J.Chromatogr.B 693:79-91 (1997)].Endotoxin adsorption experiment condition: get each 50mg of clean adsorbent, add the endotoxin solution of 1mL 16Eu/mL, 25 ℃ of following joltings 2 hours are measured endotoxic concentration in the supernatant with azo development process.
| Polymyxin B bonded amount (mg/g) | Clearance (%) | |
| Adsorbent 1 | 0.31 | 80.80 |
| Adsorbent 2 | 6.12 | 91.13 |
Claims (3)
1. preparation method that is used for the endotoxin absorbent of hemoperfusion is characterized in that: with the agarose gel substrate, and with epoxychloropropane, the hexamethylene diamine reaction, again through 1, after the activation of 1 '-carbonyl dimidazoles, the bonding polymyxin B obtains endotoxin absorbent;
2. according to the described preparation method that is used for the endotoxin absorbent of hemoperfusion of claim 1, it is characterized in that:
1) epoxychloropropane activated agarose gel carries out in 1-2mol/LNaOH, and reaction temperature is 30-50 ℃, and the response time is 1-4 hour, and epoxychloropropane is fully excessive;
2) hexamethylene diamine carries out ring-opening reaction to the agarose gel that has epoxide group, and reaction temperature is 50-80 ℃, and the response time is 1-4 hour, and hexamethylene diamine is fully excessive;
3) 1, when 1 '-carbonyl dimidazoles activation has the agarose gel of active amino, at exsiccant dioxane or/and acetone or/and carry out in the acetonitrile, the response time is 1-4 hour, 1,1 '-carbonyl dimidazoles is excessive;
4) 1, activatory agarose gel of 1 '-carbonyl dimidazoles and polymyxin B prepared in reaction endogenous toxin adsorbent, reaction temperature is 10-30 ℃, response time 8-24 hour, pH 7-12, polymyxin B is excessive; Obtain endotoxin absorbent.
3. according to the described preparation method that is used for the endotoxin absorbent of hemoperfusion of claim 2, it is characterized in that: described step 3) 1,1 '-carbonyl dimidazoles at room temperature reacts when activating the agarose gel that has active amino.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102000550A (en) * | 2010-10-20 | 2011-04-06 | 广州康盛生物科技有限公司 | Synthetic method used for preparing adsorbent for clearing pathogenic antibody by oxidizing periodate |
| CN106334541A (en) * | 2016-10-09 | 2017-01-18 | 南开大学 | Adsorbent for removing inflammatory factors in blood of patient with inflammatory reaction on whole body and preparation method thereof |
| WO2017071601A1 (en) * | 2015-10-29 | 2017-05-04 | 重庆郑博生物科技有限公司 | Adsorbing material for multiple pathogenic factors of sepsis as well as preparation method and application thereof |
| JP2019508074A (en) * | 2015-10-02 | 2019-03-28 | エシコン・インコーポレイテッドＥｔｈｉｃｏｎ， Ｉｎｃ． | Method and system for treating medical devices and fluids |
| CN113351185A (en) * | 2021-06-17 | 2021-09-07 | 华南理工大学 | Microsphere adsorbent for removing toxin through blood perfusion and preparation method thereof |
-
2005
- 2005-05-18 CN CN 200510046452 patent/CN1864755A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102000550A (en) * | 2010-10-20 | 2011-04-06 | 广州康盛生物科技有限公司 | Synthetic method used for preparing adsorbent for clearing pathogenic antibody by oxidizing periodate |
| CN102000550B (en) * | 2010-10-20 | 2012-08-29 | 广州康盛生物科技有限公司 | Synthetic method used for preparing adsorbent for clearing pathogenic antibody by oxidizing periodate |
| JP2019508074A (en) * | 2015-10-02 | 2019-03-28 | エシコン・インコーポレイテッドＥｔｈｉｃｏｎ， Ｉｎｃ． | Method and system for treating medical devices and fluids |
| WO2017071601A1 (en) * | 2015-10-29 | 2017-05-04 | 重庆郑博生物科技有限公司 | Adsorbing material for multiple pathogenic factors of sepsis as well as preparation method and application thereof |
| US10898632B2 (en) | 2015-10-29 | 2021-01-26 | Chongqing Zhengbo Biotechnology Co. Ltd. | Adsorbing material for multiple pathogenic factors of sepsis as well as preparation method and application thereof |
| CN106334541A (en) * | 2016-10-09 | 2017-01-18 | 南开大学 | Adsorbent for removing inflammatory factors in blood of patient with inflammatory reaction on whole body and preparation method thereof |
| CN106334541B (en) * | 2016-10-09 | 2018-07-27 | 南开大学 | A kind of adsorbent and preparation method thereof removing inflammatory factor in systemic inflammatory response blood samples of patients |
| CN113351185A (en) * | 2021-06-17 | 2021-09-07 | 华南理工大学 | Microsphere adsorbent for removing toxin through blood perfusion and preparation method thereof |
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Application publication date: 20061122 |