CN102504018A - Melittin pamoate capable of being continuously released and preparation agent thereof - Google Patents
Melittin pamoate capable of being continuously released and preparation agent thereof Download PDFInfo
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- CN102504018A CN102504018A CN2011103327280A CN201110332728A CN102504018A CN 102504018 A CN102504018 A CN 102504018A CN 2011103327280 A CN2011103327280 A CN 2011103327280A CN 201110332728 A CN201110332728 A CN 201110332728A CN 102504018 A CN102504018 A CN 102504018A
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- mellitin
- palmoxiric acid
- palmoxiric
- acid salt
- injection
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Abstract
The invention relates to the technical field of medicine and discloses a melittin pamoate capable of being continuously released and a preparation agent and a preparation method of the same. The melittin pamoate is used for treatment of rheumatic arthritis and scapulohumeral periarthritis, is capable of enabling effective constituent to be continuously released for one to four weeks, overcomes the shortcoming that solution type free melittin is short in plasma half-life and poor in stability and requires frequency injection, and improves adaptability of patients for pharmacy.
Description
Technical field
The present invention relates to medical technical field, exactly provide a kind of mellitin palmoxiric acid salt and preparation and preparation method of sustainable release.
Background technology
Mellitin (melittin) is the main composition that plays pharmacological action and BA in the bee venom, accounts for 50% of bee venom dry weight.Its primary structure is made up of 26 amino-acid residues: NH
2-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly
-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-CONH
2, molecular formula C
131H
229O
31, molecular weight 2840 Da.Wherein the 1st to the 20th amino acid is hydrophobic grouping, and the 21st to the 26th amino acid is hydrophilic radical; 2 amino acid of N-terminal and 4 equal positively chargeds of amino acid of C-terminal, to make mellitin be the strong basicity peptide in the existence of three Methionins and two arginine residues in the molecule, its iso-electric point is 12.06.
The bee venom Toplink stimulates pitutary-adrenal system that cortin is discharged and increases and the generation anti-inflammatory action; It is one of the strongest material of the human up to now anti-inflammatory activity of being known; Its anti-inflammatory activity is 100 times of HYDROCORTISONE INJECTIONS; And can suppress the growth and breeding of more than 20 kind of Gram-negative and positive bacteria, especially resist penicillium mould is had chemical sproof streptococcus aureus.Mellitin has significant analgesic activity simultaneously; Its restraining effect to prostaglandin synthetase is 70 times of indoles magnesium suffering; Analgesia intensity is 40% of morphine; Can improve pain threshold, prolong the time length of analgesic activity, and not have the immunosuppressive action of bigcatkin willow acids gastral stimulation and steroidal class.In addition, mellitin can have direct killing effect to kinds of tumors, can stop the hyperplasia of tumour cell at the mellitin of external 1 μ mol concentration, and not suppress Normocellular growth and cloning efficiency.Compare with macromole toxalbumin such as ricin, diphtheria toxalbumin, the mellitin immunogenicity is low, is difficult for producing anaphylaxis.
Because mellitin has significant anti-inflammatory, analgesia and antitumor action; At present quite active about the basis and the clinical study of mellitin; But clinical application is still very limited; Major cause is that this type of medicine internal metabolism has the characteristic that the tangible transformation period is short, clearance rate is high, and will reach the Plasma Concentration and the result of treatment that need during clinical application needs heavy dose medication repeatedly.Be generally intraarticular injection once a day like the current clinical administration scheme of present Beetoxin injection in treatment of arthritis, continuous 20～30 days courses of treatment, need use several courses of treatment repeatedly.Body injury that long-term frequent drug administration by injection is caused and spirit, economic pressures often make the patient be difficult to bear, and very easily cause treating failure midway owing to poor compliance.
CN03110871.7 discloses a kind of bee venom biodegradable microsphere injection and preparation method thereof, adopts emulsion-liquid drying method directly the solution-type mellitin to be wrapped up in the polylactic acid-based biodegradable polymer, around the sustainable release of medicine.This is the method for traditional coated water-soluble medicine, and organic solvent that relates among the preparation technology and high shear can produce very big influence to the biological activity of the free polypeptide of solution-type.
Summary of the invention
The salt type mellitin that the purpose of this invention is to provide a kind of sustainable release; It can overcome defectives such as short, the organic solvent that needs to relate among frequent injection and the existing microballoon preparation technology of common free type mellitin transformation period and high shear, 2～4 weeks of sustained release drug after subcutaneous or intramuscular injection.
Mellitin palmoxiric acid salt provided by the invention has following general structure:
In the formula, n=0.5～6.Preferred n=1～3.
The method of the above-mentioned mellitin palmoxiric acid of preparation provided by the invention salt comprises the steps:
(1) palmoxiric acid is suspended in the water for injection, adds sodium hydroxide solution to palmoxiric acid under stirring and dissolve fully, process the palmoxiric acid sodium salt.Said material molar ratio is: 1 part of palmoxiric acid, 2 parts in sodium hydroxide.
(2) mellitin is dissolved in the water for injection, stirs and to add the palmoxirate sodium salts solution down, separate out to crystallization.Said material molar ratio is: 1 part of mellitin, 0.5～6 part of palmoxiric acid sodium salt.
(3), dry with the water for injection washing with the crystalline deposit filtering separation.
Another object of the present invention is to provide the preparation of mellitin palmoxiric acid salt.Mellitin palmoxiric acid salt of the present invention can be processed injection suspensoid, oil solution or injectable sterile powder with acceptable accessories.Wherein, preferred injectable sterile powder.
The free mellitin of solution-type is short plasma half-life, and physico-chemical property is unstable, and (pH7.4) is extremely unstable under the physiological pH condition, and being used for treatments such as rheumatic arthritis, scapulohumeral periarthritis needs frequently inject every day.And have beyond thought slow release effect behind mellitin of the present invention and the palmoxiric acid salify, according to sustainable 1～4 week of release effective constituent of treatment needs.
Description of drawings
Fig. 1 is the accumulation stripping curve of embodiment 3,4,5,6 prepared mellitin palmoxiric acid salt.
Fig. 2 is the average Plasma Concentration-time curve of embodiment 8 (the aseptic freeze-dried powder of injection mellitin palmoxiric acid salt) and reference examples (solution-type mellitin).
Embodiment
The separation and purification of embodiment 1 mellitin
Take by weighing dry bee venom 10g, place centrifuge tube, and adding formic acid-ammonium formiate (0.1M, pH4.5) damping fluid fully dissolves it, with the centrifugal 5min of 4000r/min, draws supernatant, through the 0.45um filtering with microporous membrane; The centrifuge tube bottom residues with damping fluid washing, centrifugal, get supernatant, filtration, twice filtrating is merged, subsequent use.With Sephadex G-50 post on the above-mentioned pretreatment sample, carry out wash-out with formic acid-ammonium formiate damping fluid as elutriant, flow velocity 10mL/h detects wavelength 280nm; After collection main peak elutriant also concentrated, last Sephadex G-50 post carried out the secondary chromatography with the same terms, collects main peak elutriant and lyophilize.It is 96.4% that the efficient gel permeation chromatography is measured separation mellitin purity.
The preparation of embodiment 2 palmoxirate sodium salts solutions (0.1M)
The 3.88g palmoxiric acid is suspended in the 20ml water for injection, adds 20ml 1.0M sodium hydroxide solution to palmoxiric acid under stirring and dissolve fully, add water for injection and be diluted to 100ml.
The preparation of embodiment 3 mellitin palmoxiric acid salt (1:0.5 mol/mol)
The 1.42g mellitin is dissolved in the 20ml water for injection, stirs and to add 10ml palmoxiric acid disodium salt solution (0.1M) down, separate out to crystallization.Deposition is leached, dry with the water for injection washing.Product contains 2:1 peptide palmoxiric acid salt (mol/mol).
The preparation of embodiment 4 mellitin palmoxiric acid salt (1:1 mol/mol)
The 2.84g mellitin is dissolved in the 40ml water for injection, stirs and to add 10ml palmoxiric acid disodium salt solution (0.1M) down, separate out to crystallization.Deposition is leached, dry with the water for injection washing.Product contains 1:1 peptide palmoxiric acid salt (mol/mol).
The preparation of embodiment 5 mellitin palmoxiric acid salt (1:3 mol/mol)
The 2.84g mellitin is dissolved in the 40ml water for injection, stirs and to add 30ml palmoxiric acid disodium salt solution (0.1M) down, separate out to crystallization.Deposition is leached, dry with the water for injection washing.Product contains 1:3 peptide palmoxiric acid salt (mol/mol).
The preparation of embodiment 6 mellitin palmoxiric acid salt (1:6 mol/mol)
The 2.84g mellitin is dissolved in the 40ml water for injection, stirs and to add 60ml palmoxiric acid disodium salt solution (0.1M) down, separate out to crystallization.Deposition is leached, dry with water washing.Product contains 1:6 peptide palmoxiric acid salt (mol/mol).
The release in vitro behavior of embodiment 7 mellitin palmoxiric acid salt
The mellitin palmoxiric acid salt that embodiment 3～6 is obtained is packed in the releasing tube, is release medium with 0.1M phosphate buffer 1 0ml, and (37 ℃, 100r/min), timing sampling is measured the amount of mellitin in the release medium in vibration in the constant temperature horizontal oscillator tube.The result is as shown in Figure 1.Each prescription all can be kept the time of releasing in 1～3 week, and along with the increase of palmoxiric acid salt molar ratio, the release rate of mellitin slows down gradually, and as the 1:3 that surpasses of molar ratio, rate of release does not have noticeable change.
The cumulative release percentage of table 1 embodiment 3,4,5,6 prepared mellitin palmoxiric acid salt
The preparation of the aseptic freeze-dried powder of embodiment 8 injection mellitin palmoxiric acid salt
Prescription (100 dosage):
Mellitin palmoxiric acid salt (making) 1.0 g by embodiment 4
N.F,USP MANNITOL 4.0 g
Water for injection 100ml
With mellitin palmoxiric acid salt, N.F,USP MANNITOL and water for injection thorough mixing and homogenize, be sub-packed in the cillin bottle, lyophilize obtains pressed powder.
Pharmacokinetics in the embodiment 9 single dose bodies
For further specifying the advantage of mellitin palmoxiric acid salt of the present invention, the aseptic freeze-dried powder of mellitin palmoxiric acid salt that embodiment 8 makes is rebuild with the 1ml sterile water for injection; Embodiment 1 being separated the mellitin lyophilized powder that obtains oozes mannitol solution (4%) with 1ml etc. and rebuilds as reference examples.Two preparations are injected (2.0 mg/kg) respectively at the subcutaneous single dose of tame rabbit neck part.Get rabbit auricular vein blood 0.5mL in the time point that configures, room temperature leaves standstill 30 min, and centrifugal 5 min of 5000 r/min get upper serum, and in-20 ℃ of preservations, ELISA measures preceding 4 ℃ of multiple melting.Adopt the double-antibody sandwich elisa method to measure mellitin level in the serum, the result is as shown in Figure 2.The aseptic freeze-dried powder of embodiment 8 injection mellitin palmoxiric acid salt after the rabbit injected, its transformation period t
1/2Compare with mellitin solution with average retention time MRT in the body, prolonged 51 times and 40 times respectively, have tangible slow releasing function.
The pharmacokinetic parameters of table 2 embodiment 8 (the aseptic freeze-dried powder of injection mellitin palmoxiric acid salt) and reference examples (solution-type mellitin).
Claims (8)
1. mellitin palmoxiric acid salt, it is characterized in that: general structure is following:
In the formula, n=0.5～6.
2. mellitin palmoxiric acid salt according to claim 1 is characterized in that: n=1～3.
3. the preparation method of mellitin palmoxiric acid salt according to claim 1 is characterized in that may further comprise the steps:
(1) palmoxiric acid is suspended in the water for injection, adds sodium hydroxide solution to palmoxiric acid under stirring and dissolve fully, process the palmoxiric acid sodium salt, said material molar ratio is: 1 part of palmoxiric acid, 2 parts in sodium hydroxide;
(2) mellitin is dissolved in the water for injection, stirs adding palmoxirate sodium salts solution down, separate out to crystallization, said material molar ratio is: 1 part of mellitin, 0.5～6 part of palmoxiric acid sodium salt;
(3), dry with the water for injection washing with the crystalline deposit filtering separation.
4. preparation method according to claim 3 is characterized in that: the ratio of mellitin and palmoxiric acid sodium salt is 1:1～1:3 in the step (2).
5. a compsn that contains the said mellitin palmoxiric acid of claim 1 salt is characterized in that comprising mellitin palmoxiric acid salt and acceptable accessories.
6. mellitin palmoxiric acid salt Injectable sterile lyophilized powder is characterized in that prescription is:
Mellitin palmoxiric acid salt 1.0 g
N.F,USP MANNITOL 4.0 g
Water for injection 100ml.
7. mellitin palmoxiric acid salt Injectable sterile lyophilized powder according to claim 6, its preparation method is: with mellitin palmoxiric acid salt, N.F,USP MANNITOL and water for injection thorough mixing and homogenize, be sub-packed in the cillin bottle, lyophilize obtains pressed powder.
8. the application of mellitin palmoxiric acid salt according to claim 1 in preparation anti-inflammatory, analgesia and antitumor drug.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011103327280A CN102504018A (en) | 2011-10-28 | 2011-10-28 | Melittin pamoate capable of being continuously released and preparation agent thereof |
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| CN2011103327280A CN102504018A (en) | 2011-10-28 | 2011-10-28 | Melittin pamoate capable of being continuously released and preparation agent thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104258373A (en) * | 2014-09-17 | 2015-01-07 | 吉林大学 | Application of antineoplastic polypeptide TT-1 in preparing antineoplastic medicines |
| CN104784677A (en) * | 2014-05-13 | 2015-07-22 | 东北师范大学 | Application of melittin in preparation of drug used for inhibiting invasion metastasis of breast cancer cells |
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| CN1294519A (en) * | 1999-02-18 | 2001-05-09 | 英法马有限公司 | Pharmaceutical Compsns. contg. compounds with activity for enhancement of absorption of active ingredients |
| CN1494919A (en) * | 1993-12-09 | 2004-05-12 | �����ɷ� | Embonate medicine composition containing bombesin-like product |
| CN102199150A (en) * | 2011-04-01 | 2011-09-28 | 中国药科大学 | Optical active oxazolidinone derivatives as well as preparation methods and application to preparing medicaments |
| CN102199163A (en) * | 2011-04-01 | 2011-09-28 | 中国药科大学 | 2-hydroxy tetrahydrothiophene derivative, preparation method thereof and application thereof in pharmacy |
| US20120245202A1 (en) * | 2010-09-17 | 2012-09-27 | Rock Creek Pharmaceuticals, Inc. | Methods and products for treating inflammation |
-
2011
- 2011-10-28 CN CN2011103327280A patent/CN102504018A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1494919A (en) * | 1993-12-09 | 2004-05-12 | �����ɷ� | Embonate medicine composition containing bombesin-like product |
| CN1294519A (en) * | 1999-02-18 | 2001-05-09 | 英法马有限公司 | Pharmaceutical Compsns. contg. compounds with activity for enhancement of absorption of active ingredients |
| US20120245202A1 (en) * | 2010-09-17 | 2012-09-27 | Rock Creek Pharmaceuticals, Inc. | Methods and products for treating inflammation |
| CN102199150A (en) * | 2011-04-01 | 2011-09-28 | 中国药科大学 | Optical active oxazolidinone derivatives as well as preparation methods and application to preparing medicaments |
| CN102199163A (en) * | 2011-04-01 | 2011-09-28 | 中国药科大学 | 2-hydroxy tetrahydrothiophene derivative, preparation method thereof and application thereof in pharmacy |
Non-Patent Citations (1)
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| 郭艳红 等: "蜂毒肽的研究概述", 《蜜蜂杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104784677A (en) * | 2014-05-13 | 2015-07-22 | 东北师范大学 | Application of melittin in preparation of drug used for inhibiting invasion metastasis of breast cancer cells |
| CN104258373A (en) * | 2014-09-17 | 2015-01-07 | 吉林大学 | Application of antineoplastic polypeptide TT-1 in preparing antineoplastic medicines |
| CN104258373B (en) * | 2014-09-17 | 2015-06-10 | 吉林大学 | Application of antineoplastic polypeptide TT-1 in preparing antineoplastic medicines |
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Application publication date: 20120620 |