CN109608526B - Application of the SNase in preparation treatment inflammatory bowel medicine - Google Patents
Application of the SNase in preparation treatment inflammatory bowel medicine Download PDFInfo
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- CN109608526B CN109608526B CN201910073368.3A CN201910073368A CN109608526B CN 109608526 B CN109608526 B CN 109608526B CN 201910073368 A CN201910073368 A CN 201910073368A CN 109608526 B CN109608526 B CN 109608526B
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
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- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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Abstract
The present invention relates to application of the staphylococcal nuclease (staphylococcal nuclease, SNase) in preparation prevention or treatment inflammatory bowel medicine.The object of the present invention is to provide SNase in the active new application of inflammatory enteropathy.SNase can be relieved the colitis of C57BL/6J mouse as the result is shown, can preferably control the weight of C57BL/6J mouse, colon lengths, fecal occult blood and inflammatory infiltration etc., and significantly improve the survival rate of C57BL/6J mouse.It follows that SNase can be relieved C57BL/6J mouse intestinal inflammation, it can be used for preventing and treating the exploitation of inflammatory bowel medicine.
Description
Technical field
The present invention relates to a kind of Nuclease SN ase with inflammatory enteropathy effect, and spreading out containing the nuclease
Biological and similar pharmaceutical preparation, the present invention relates to pharmacy and medicine related fieldss.
Background technique
Inflammatory bowel disease (inflammatory bowel disease, IBD) is the unknown chronic gut disease of one group of cause of disease
Disease, including inflammatory bowel disease (Ulcerative Colitis, UC) and Crohn disease (Crohn ' s disease, CD).In west
Country is common, and it is the common disease in digestion field that Europe and north america illness rate, which are up to 120-200/100000,.With life
The trend risen year by year is presented in China for the variation and improvement of living standard of mode, IBD disease incidence, oneself digests through becoming China
The Etiological of the common disease and chronic diarrhea of system, hematochezia.UC and CD site of pathological change has differences, and UC is a kind of chronic non-
Special colitis, lesion mainly involve colonic mucosa and submucosa, and range can drive in the wrong direction proximally mostly since lower distal colon
Development, or even involve total colectomy and terminal ileum, it is distributed in continuity；Clinical manifestation is diarrhea, abdominal pain and mucus purulence blood
Just.CD is a kind of chronic granulomatous inflammation, and lesion can be involved each position of gastrointestinal tract, is with terminal ileum and its neighbouring colon
It is main, it is in transmural inflammation, mostly segmental, investigation on asymmetric distribution；Clinical manifestation is abdominal pain, diarrhea, fistula, anus lesion
Deng.
IBD is typically considered a kind of based on heredity, multifactor to participate in jointly, organ specific autoimmune
Disease.Currently, the conventional medicament of the treatment listing for IBD, such as aminosalicylic acids mainly include salicylazosulfapyridine, but
It is that it cannot eradicate disease, and there is higher recurrence rates, and glucocorticoids adverse reaction is more, improper use is easily drawn
Hair shock, current common biological agent have anti-TNF monoclonal antibody such as Infliximab, adalimumab and cell factor to inhibit
Agent, but the expensive price of biological agent also have a degree of adverse reaction, apply the treatment of biological agent not at present in native land
It is more, and most of administration modes are non-oral administration, the compliance of patient is poor.Therefore illustrate IBD be a complexity, it is multifactor
Cause autoimmune disease, only immunosupress can not play good result, therefore find novel targets, study new treatment method
It is imperative.
Present invention firstly discovers that SNase oral preparation has the function of alleviating inflammatory bowel disease intestinal inflammatory, and pass through
The fixed SNase of calcium alginate can take orally, and overcoming nuclease the problems such as cannot take orally.
Summary of the invention
Goal of the invention:
The object of the present invention is to provide SNase new application technical solutions:
Application of the SNase in preparation prevention or treatment inflammatory bowel medicine.
The SNase is obtained by prokaryotic expression.
SNase reference literature the method used in the present embodiment prepares (mighty torrent, Lang Junchao, He Dongmei, Liu Kun
Cutting edge of a knife or a sword, the preparation of Wu Jie staphylococcal nuclease and property research [J] China Medicine University journal, 2017, (the 2nd phase).
Injection, sustained release agent, subdermal implants, tablet, powder is prepared in the SNase and pharmacy usual excipients
Agent, granule, capsule or oral solution.
The excipient includes calcium alginate etc..
The utility model has the advantages that
1, the present invention has found that it has alleviation inflammatory bowel disease colitis using staphylococcal nuclease (SNase) for the first time
Effect.And it can be taken orally, the present invention relates to its new applications.
2, the prior art indicate that many albumen such as TNF monoclonal antibody etc can be used for the prevention and treatment of inflammatory bowel disease, work
Journey bacterium has broken its yoke for being difficult to produce in enormous quantities for the expression of this albuminoid, and the efficiency of inducing expression is higher, SNase
With biggish pH tolerance range, enzyme activity can be stabilized and kept between pH4-10, while also being had and not lost at 135 DEG C
Characteristic living, therefore can be to avoid engineering bacteria the purification process the problem of, such as egg is merged during isolating and purifying
White degradation, control manipulation condition is stringent, and purification efficiency is excessively low.The invention has dexterously evaded protein degradation, has saved life
Produce cost, for produce on a large scale with for inflammatory bowel disease prevention and treatment and treatment be of great significance.
3, conventional medicament is administered generally by hypodermic mode, but is subcutaneously injected for sufferer excessively
Pain, so being difficult to be received, in addition, there is also serious safety issues for this administration mode.And involved in this patent
SNase can be administered orally, and overcome above-mentioned various limitations, be easy to as sufferer receiving.Calcium-alginate-immobilized SNase is simultaneously
Suspension is prepared for being administered orally by ultrasonic disperse.Sodium alginate is the day being present in the brown algaes such as kelp, sargassum
Right polysaccharide, white in appearance or pale yellow powder, it is odorless, tasteless, it is practically insoluble in the organic solvents such as ethyl alcohol, ether, alginic acid
Calcium is resistant to acid, will not be degraded under the acidic environment of gastric acid, and reachable colon lesions position discharges the nuclease, protects
SNase is from the degradation of gastric acid, while bioavailability is improved largely, the day that biological compatibility is good, biodegradable
Right high molecular material has preferable film forming and mouldability, is widely used in pharmaceutical carrier field.
The SNase is the non-specific ribalgilase of one kind of staphylococcus aureus secretion, the SNase of mature form
It is made of 149 amino acid residues (see Seq NO.1), wherein homologous sequence SNc contains 135 amino acid residues, catalytic site
Containing 67 amino acid residues, can non-specifically degradation of dna and RNA, and release 3 '-phosphoric acid mononucleotides and dinucleotide
Acid, it has excision enzyme and endonuclease activity simultaneously, in addition compared with other DNase also have molecular weight is small, thermal stability is good,
The advantages that pH field of activity wide (pH4-10).
Detailed description of the invention
The influence control group mice weight that Fig. 1 SNase oral preparation changes C57BL/6J mouse weight is in experimental period
Remain growth slightly in the process, the 8th day weight of modeling group mouse falls to 15.37g, have dropped 22.3% than before, say
Bright modeling success, SNase administration group mouse weight before modeling also than being declined, and fall is lower than modeling group, although not having
There is significant difference, but has shown and alleviated weight sharply downward trend.
Influence of Fig. 2 SNase oral preparation to C57BL/6J mouse Colon length
Influence of Fig. 3 SNase oral preparation to C57BL/6J mouse spleen weight
Influence of Fig. 4 SNase oral preparation to C57BL/6J mouse Colon supernatant MPO level
Specific embodiment
With reference to the document (mighty torrent, Lang Junchao, He Dongmei, Liu Kunfeng, the preparation and property of Wu Jie staphylococcal nuclease
Research [J] China Medicine University journal, 2017, (the 2nd phase)) prepare solid solution involved in SNase. following examples
For quality percent by volume.
The preparation of embodiment 1:SNase oral preparation
Round-bottomed flask, 40 DEG C of water-bath magnetic agitations will be added after the mixing of 57ml span 85 and 22.8ml dehydrated alcohol
Then 10min adds 0.25% sodium alginate soln (drugloading rate 5mg SNase) 8ml dropwise, continue after stirring 10min, decompression is taken out
It goes moisture to clarifying, pours into the calcium chloride ethanol solution (0.1%) that 1ml is added, continue pumping stirring 30min curing nano grain extremely
Bubble-free.Mixed liquor is centrifuged 30min in 8000rpm, collects precipitating, it is secondary with petroleum ether, precipitating is collected, distilled water is used
Precipitating is resuspended, with ultrasonic cell disruptor ultrasonic disperse, 400W, 50 beats/min of dispersions obtain SNase nanoparticle suspension.
Embodiment 2:SNase oral preparation inflammatory enteropathy pharmacodynamic evaluation.
21 8 week old female C57BL/6J mouse (are purchased from Jiangning county's Qinglongshan animal reproduction factory, licensing
Number: 3 groups SCXK (Soviet Union) 2017-0001) are randomly divided into, is blank group and modeling group and calcium-alginate-immobilized SNase respectively
Group (i.e. embodiment 1 prepares nanoparticle suspension), every group 7, adaptable fed three days, naive mice freely drank life
Salt water is managed, modeling group will be made 3%DSS (sodium dextran sulfate) with physiological saline solvent, so that mouse free water, embodiment
1 obtains SNase nanoparticle suspension SNase (2.5mg/ml) group starts gastric infusion, every intragastric administration on mice on the day of modeling
200ul, modeling 7 days, daily stomach-filling was primary, and cervical dislocation puts to death mouse within the 8th day, carried out pharmacodynamics scoring.
(1) influence that SNase oral preparation changes C57BL/6J mouse weight
From experiment, weighing body weight is carried out to each group experiment mice using electronic balance daily.It is small after modeling
Mouse weight loss, fecal occult blood etc. can be used as the successful mark of modeling, as a result as shown in Figure 1.
(2) influence of the SNase oral preparation to C57BL/6J mouse Colon length
At the end of experiment, mouse is put to death using cervical dislocation and mouse is dissected, takes its colonic tissue, measurement knot
Intestines length, and take pictures to it, experimental result is as shown in Figure 2.
(3) influence of the SNase oral preparation to C57BL/6J mouse spleen weight
The spleen immune organ important as mouse, after inflammatory bowel disease modeling success, spleen meeting enlargement, before observation administration
Whether can alleviate spleen enlargement situation afterwards, at the end of experiment, mouse be put to death using cervical dislocation and mouse is dissected,
Its spleen, the adipose tissue around separating spleen are taken, and is weighed to it, experimental result is as shown in Figure 3.
(4) influence of the SNase oral preparation to C57BL/6J stool in mice occult blood and lethality
On the day of testing modeling, Mice Mice fecal occult blood situation is observed, and count the lethality of each experimental mice, tested
The results are shown in Table 1.
Influence of the table 1SNase oral preparation to C57BL/6J stool in mice occult blood and lethality
Note :+soft stool ++ loose stools +++ diarrhea ++++bleeding
(5) influence of the SNase oral preparation to C57BL/6J mouse Colon supernatant MPO level
Myeloperoxidase (MPO) activity is to evaluate one of the index of neutrophil infiltration.3,3 ', 5,5 '-tetramethyls
Benzidine (TMB) is converted into blue under the catalysis of peroxidase, and is converted into final yellow, color under the action of an acid
The depth and the concentration of MPO be positively correlated, absorbance is surveyed at 450nm.After mouse 3.0%DSS modeling success, using cervical vertebra
Dislocation method is put to death mouse and is dissected, and colonic tissue is separated, and the adipose tissue around colonic tissue is separated completely, and excrement
Just remove, and weigh to it, its colonic tissue is shredded with surgical instrument, by 1:10 (g/ml) be added physiological saline or
PBS buffer solution is fully ground using electric grinder, and lapping liquid is collected, and in 12000r/min, is centrifuged 15min, is taken supernatant standby
With.10% colon of 10uL is added in 96 orifice plates and is homogenized supernatant 100uL TMB solution, 37 DEG C of reaction 5min.Every hole adds
The sulfuric acid of the 2mol/L of 50 μ L terminates reaction, measures absorbance at 450nm wavelength.Experimental result is as shown in Figure 4.
Conclusion: it follows that calcium-alginate-immobilized SNase can be relieved C57/BL6J mouse weight it is violent under
Drop, improvement colon shortens, red and swollen, improves mouse splenomegaly, colonic tissue MPO level is reduced, so that it is small to alleviate inflammatory bowel disease
The occurrence and development of the intestinal inflammatory of mouse can improve the survival ability of C57/BL6J mouse significantly, can be used for inflammatory bowel disease
The exploitation of drug.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although passing through ginseng
Issuing as before bright certain preferred embodiments, invention has been described, it should be appreciated by those of ordinary skill in the art that
Various changes can be made to it in the form and details, without departing from this hair defined by the appended claims
Bright spirit and scope.
Sequence table
<110>China Medicine University
<120>application of the SNase in preparation treatment inflammatory bowel medicine
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 171
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
Met Asn Ala Ser Gln Thr Asp Asn Gly Val Asn Arg Ser Gly Ser Glu
1 5 10 15
Asp Pro Thr Val Tyr Ser Ala Thr Ser Thr Lys Lys Leu His Lys Glu
20 25 30
Pro Ala Thr Leu Ile Lys Ala Ile Asp Gly Asp Thr Val Lys Leu Met
35 40 45
Tyr Lys Gly Gln Pro Met Thr Phe Arg Leu Leu Leu Val Asp Thr Pro
50 55 60
Glu Thr Lys His Pro Lys Lys Gly Val Glu Lys Tyr Gly Pro Glu Ala
65 70 75 80
Ser Ala Phe Thr Lys Lys Met Val Glu Asn Ala Lys Lys Ile Glu Val
85 90 95
Glu Phe Asp Lys Gly Gln Arg Thr Asp Lys Tyr Gly Arg Gly Leu Ala
100 105 110
Tyr Ile Tyr Ala Asp Gly Lys Met Val Asn Glu Ala Leu Val Arg Gln
115 120 125
Gly Leu Ala Lys Val Ala Tyr Val Tyr Lys Pro Asn Asn Thr His Glu
130 135 140
Gln His Leu Arg Lys Ser Glu Ala Gln Ala Lys Lys Glu Lys Leu Asn
145 150 155 160
Ile Trp Ser Glu Asp Asn Ala Asp Ser Gly Gln
165 170
Claims (3)
- Application of the 1.SNase in preparation prevention or treatment inflammatory bowel medicine, it is characterised in that: the ammonia of the SNase Base acid sequence is SEQ NO.1.
- 2. application according to claim 1, it is characterised in that: the SNase is prepared with pharmacy usual excipients Injection, subdermal implants, tablet, pulvis, granule, capsule or oral solution.
- 3. application according to claim 2, it is characterised in that: the oral solution is nanosuspension, the nanometer Suspension is formed by following steps system: after the mixing of span 85 and dehydrated alcohol, 40 DEG C of stirring in water bath 10min, then dropwise Add the sodium alginate soln containing SNase, after stirring 10min, remove water to clarification, pour into and calcium chloride ethanol solution is added, continues Mixed liquor is centrifuged to bubble-free, collects precipitating, with petroleum ether, collect precipitating, with distilled water to precipitating weight by pumping stirring Outstanding, ultrasonic disperse obtains nanoparticle suspension.
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