CN105342999A - Exenatide oral nanoparticle - Google Patents
Exenatide oral nanoparticle Download PDFInfo
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- CN105342999A CN105342999A CN201410406341.9A CN201410406341A CN105342999A CN 105342999 A CN105342999 A CN 105342999A CN 201410406341 A CN201410406341 A CN 201410406341A CN 105342999 A CN105342999 A CN 105342999A
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Abstract
The invention provides an exenatide oral nanoparticle and a preparation method thereof. The exenatide oral nanoparticle contains exenatide and CSK-chitosan. The exenatide oral nanoparticle can substantially improve the oral administration absorption effect, improves the bioavailability, and realizes fast blood sugar lowering effect and stable blood sugar.
Description
Technical field
The present invention relates to field of pharmaceutical preparations, be specifically related to containing Exenatide oral administration nanometer grain and preparation method thereof.
Background technology
The general metabolism disease of diabetes to be a kind of with chronic hyperglycemia be feature, shows as defect of insulin secretion or obstacle appears in biological insulin effect.According to statistics, in current global range, about have 1.4 hundred million diabeticss, and due to aged tendency of population, the factors such as incidence of obesity increase, estimate will reach 300,000,000 at global diabetics in 2025.Insulin-dependent (IDDM, I type) and non-insulin-depending type (NIDDM, II type) diabetes can be divided into according to Pathologic Characteristics.Type i diabetes, it is characterized in that insulin secreting ability reduces or thoroughly loses, clinical treatment needs subcutaneous insulin injections every day; Type ii diabetes, is characterized in that human body effectively cannot utilize insulin, has 90% to be type ii diabetes in diabetics.Current type ii diabetes is clinically based on Drug therapy, and a part of patient needs insulin and oral antidiabetic drug conbined usage.
Glucagon-like-peptide-1 (GLP-1) analog Exenatide (exenatide) be develop in type ii diabetes treatment field in recent years with one of two Hormone Deficiency newly-developed hypoglycemic agent that is targeting.Exenatide is secretin's analog, it is glucagon-like-peptide-1 (GLP-1) receptor stimulating agent, can the effect of class GLP-1 be played with GLP-1 receptors bind thus reduce blood glucose, there is promotion insulin secretion, increase insulin sensitivity and improve the effect of islet cell function, having the advantage that traditional treatment diabetes medicament is incomparable.
(hundred secrete and reach Exenatide injection
) went on the market in 2005 in the U.S., within 2007, in Europe listing, within 2009, obtain Chinese SFDA and ratify listing, as the adjuvant therapy medicaments of metformin medicine.
half-life short (t
1/2about 2.4h), can not Sustainable Control blood glucose, cause drug effect to be subject to certain limitation, patient's blood concentration fluctuation is large, is unfavorable for Disease epizootic.AstraZeneca drugmaker exists
basis is researched and developed further Exenatide long-acting slow-release microsphere
obtain European EMEA in 2011 and ratify listing, within 2012, obtain U.S. FDA approval listing.
Exenatide is polypeptide drug, and current clinical administration is drug administration by injection.Long term injections administration brings very large misery to patient, patient's poor compliance, and oral route is administering mode most widely used, convenient at present, but because protein and peptide drugs oral administration biaavailability is low, most protein polypeptide drug is little or through gastrointestinal absorption, can not limit its clinical practice, and its reason mainly contains: (1) molecular weight is large, fat-soluble difference, is difficult to pass through membranes barriers; (2) there is a large amount of peptidohydrolase and proteolytic enzyme degradable peptide and protein in gastrointestinal tract; (3) easily eliminated by liver after absorbing; (4) there is chemistry and conformation instability problem.
Therefore, in order to expand the clinical practice of Exenatide formulation, bed is badly in need of the convenient Exenatide oral formulations of a kind of administration of exploitation.
Summary of the invention
The invention provides a kind of Exenatide oral administration nanometer grain, it is characterized in that containing Exenatide and CSK-chitosan.The weight ratio of Exenatide and CSK-chitosan is 0.5:10 ~ 3:10, preferred 1:10.
Described CSK-chitosan is the copolymer of CSK polypeptide and chitosan, and described CSK refers to CSK polypeptide, and its aminoacid sequence is CSKSSDYQC; CSK and chitosan weight ratio are 2:1 ~ 1:4, preferred 1:1.
Also containing ion crosslinking agent in Exenatide oral administration nanometer grain provided by the invention, described ion crosslinking agent is sodium tripolyphosphate (TPP), tetrasodium pyrophosphate, sodium tetrapolyphosphate, eight polyphosphate sodiums and hexapolyphosphate sodium, be preferably TPP, in nanoparticle, the weight ratio of peptide modified chitosan and ion crosslinking agent is 10:0.6 ~ 10:4.5.Preferred 10:0.9 ~ 10:1.
Described Exenatide oral administration nanometer grain adopts following method to prepare: be dissolved in low-concentration acetic acid by chitosan peptide modified for CSK; add Exenatide aqueous solution under magnetic stirring, dropwise add ionomer agent solution with method and continue to stir until form nanoparticle suspension.
Described Exenatide oral administration nanometer grain exists with oral dosage form, preferred oral liquid.Described oral formulations can adopt pharmacy conventional method to prepare, and dosage is oral administration 1.25-2.5 μ g/kg every day.
Accompanying drawing explanation
The Drug-time curve of Fig. 1: the embodiment 3-6 nanoparticles oral administration prepared
Change of blood sugar situation after Fig. 2: the embodiment 3-6 nanoparticles oral administration prepared
Detailed description of the invention
Further illustrate the present invention with test example by the following examples, but do not limit the present invention in any way.
The synthesis of embodiment 1CSK polypeptide
1. resin swelling
100 resins are put into reaction tube, adds 1500ml dimethyl formamide (DMF), swelling 30min.
2. deprotection
Removing DMF, adds 1500ml20% Piperidine/DMF solution, removes after 5min, then add 20% Piperidine/DMF solution 1500ml, 15min.
3. detect
Take out piperidine solution, get tens grainy resins, wash three times, add 1,2,3-indantrione monohydrate with ethanol, potassium cyanide (KCN), each one of phenol solution, 105C-110C heats 5min.
4. wash
Respectively wash twice with 1000mlDMF, methanol, DMF respectively.
5. condensation
The protected aminoacid of 10mmol aminoterminal is added by required polypeptid acid sequence; 30mmol coupling agent 2-(7-azo BTA)-tetramethylurea hexafluorophosphoric acid ester (HBTU); all with DMF dissolving less as far as possible; add reaction tube, add the methyl morpholine (NMM) that more than 100mmol measures at once. reaction 30min.
6. wash
With 1000mlDMF washing once, 1000ml methanol wash twice, 1000mlDMF washes twice.
7. repeat 2-6 operation, when the 5th step adds the protected aminoacid of aminoterminal, add required aminoacid successively.
8. wash for the last time
Wash twice with the ratio DMF of 1000ml, 1000ml methanol wash twice, 1000mlDMF washes twice, and 1000ml dichloromethane (DCM) washes twice.
9. cracking
Preparation lysate is containing trifluoroacetic acid (TFA) 94.5%; Water 2.5%; 1，2-ethandithiol (EDT) 2.5%; Tri isopropyl silane (TIS) 1%, reaction 120min.
10. dry up washing to be dried up by lysate nitrogen, wash six times with ether, then room temperature volatilizes and get final product as far as possible.
The preparation of embodiment 2CSK-chitosan
1, by polypeptide 100mg good for purification, dissolve with PBS, it is for subsequent use that ice bath soaks 15min.
2. in the polypeptide PBS solution in 1, added 1-ethyl-3-(dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) of 200mg and 200mgN-N-Hydroxysuccinimide (NHS) low-temp reaction 1h.
3. in reactant liquor, add chitosan 100mg, reaction 4h.
4. configuring PH is 7.4 phosphate buffer 2000ml.
5. reactant liquor bag filter is installed and tighten, put into buffer and to dialyse 48h.
6. the product lyophilizing of will dialyse, can obtain CSK-chitosan (1:1).
Other weight ratio CSK-chitosans adopt above-mentioned same procedure, required reagent and consumption as shown in table 1:
Table 1 prepares Different Weight than reagent and consumption needed for CSK-chitosan
Embodiment 3
10ml1mg/ml chitosan solution (being dissolved in 0.25% acetic acid), under magnetic stirring, 0.5ml2mg/mlExenatide solution is added under gravity with 5ml syringe, 2.5mg/mlTPP aqueous solution 1.8ml is added under gravity with 5ml syringe, centrifugal 30min under magnetic stirrer 50min, 3500rpm at 500 rpm.Add potassium sorbate preservative 0.1mg to the nanoparticle solution prepared, natrium malicum correctives 1mg, obtains nanoparticles oral liquid formulation.
Embodiment 4
10ml1mg/mlCSK-chitosan (1:1) solution (being dissolved in 0.25% acetic acid), under magnetic stirring, 0.5ml2mg/mlExenatide solution is added under gravity with 5ml syringe, 0.5mg/mlTPP aqueous solution 2.0ml is added under gravity with 5ml syringe, centrifugal 30min under magnetic stirrer 50min, 3500rpm at 500 rpm.Add potassium sorbate preservative 0.5mg to the nanoparticle solution prepared, natrium malicum correctives 5mg, obtains nanoparticles oral liquid formulation.
Embodiment 5
10ml1mg/mlCSK-chitosan (2:1) solution (being dissolved in 0.25% acetic acid), under magnetic stirring, 0.5ml2mg/mlExenatide solution is added under gravity with 5ml syringe, 0.5mg/mlTPP aqueous solution 1.8ml is added under gravity with 5ml syringe, centrifugal 30min under magnetic stirrer 50min, 3500rpm at 500 rpm.Add potassium sorbate preservative 0.5mg to the nanoparticle solution prepared, natrium malicum correctives 5mg, obtains nanoparticles oral liquid formulation.
Embodiment 6
10ml1mg/mlCSK-chitosan (1:4) solution (being dissolved in 0.25% acetic acid), under magnetic stirring, 0.5ml2mg/mlExenatide solution is added under gravity with 5ml syringe, 0.5mg/mlTPP aqueous solution 2.0ml is added under gravity with 5ml syringe, centrifugal 30min under magnetic stirrer 50min, 3500rpm at 500 rpm.Add potassium sorbate preservative 0.5mg to the nanoparticle solution prepared, natrium malicum correctives 5mg, obtains nanoparticles oral liquid formulation.
Embodiment 7
10ml1mg/mlCSK-chitosan (2:1) solution (being dissolved in 0.25% acetic acid), under magnetic stirring, 1.5ml2mg/mlExenatide solution is added under gravity with 5ml syringe, 0.6mg/ml hexapolyphosphate sodium aqueous solution 1.0ml is added under gravity with 5ml syringe, centrifugal 30min under magnetic stirrer 50min, 3500rpm at 500 rpm.Add potassium sorbate preservative 0.5mg to the nanoparticle solution prepared, natrium malicum correctives 10mg, obtains nanoparticles oral liquid formulation.
Embodiment 8
10ml1mg/mlCSK-chitosan (1:4) solution (being dissolved in 0.25% acetic acid), under magnetic stirring, 0.25ml2mg/mlExenatide solution is added under gravity with 5ml syringe, 1.0mg/mlTPP aqueous solution 4.5ml is added under gravity with 5ml syringe, centrifugal 30min under magnetic stirrer 50min, 3500rpm at 500 rpm.Add potassium sorbate preservative 0.1mg to the nanoparticle solution prepared, natrium malicum correctives 10mg, obtains nanoparticles oral liquid formulation.
Embodiment 9
10ml1mg/mlCSK-chitosan (1:1) solution (being dissolved in 0.25% acetic acid), under magnetic stirring, 0.5ml2mg/mlExenatide solution is added under gravity with 5ml syringe, 0.625mg/ml sodium tetrapolyphosphate saline solution 1.5ml is added under gravity with 5ml syringe, centrifugal 30min under magnetic stirrer 50min, 3500rpm at 500 rpm.Add potassium sorbate preservative 0.5mg to the nanoparticle solution prepared, natrium malicum correctives 5mg, obtains nanoparticles oral liquid formulation.
Embodiment 10
10ml1mg/mlCSK-chitosan (1:2) solution (being dissolved in 0.25% acetic acid), under magnetic stirring, 0.5ml2mg/mlExenatide solution is added under gravity with 5ml syringe, 0.625mg/mlTPP aqueous solution 1.5ml is added under gravity with 5ml syringe, centrifugal 30min under magnetic stirrer 50min, 3500rpm at 500 rpm.Add potassium sorbate preservative 0.5mg to the nanoparticle solution prepared, natrium malicum correctives 5mg, obtains nanoparticles oral liquid formulation.
Bioavailability study test in test example 1 body
Trial drug:
Exenatide-chitosan nano: prepare according to embodiment 3
Exenatide-CSK-chitosan nano: prepare according to embodiment 4-6
Exenatide solution: take 1mg Exenatide, adds in 1000ml deionized water, mixes for subsequent use.
Experimental animal: SD rat 30, often organizes 6, male and female half and half, body weight 200-250g.
Test apparatus: high performance liquid chromatograph (AgilentTechnololies), microplate reader SPECTRAMAXM5
Test method: experimental animal is divided into 6 groups at random, wherein 1-5 group dosage is 50.0 μ g/kg, gastric infusion (i.g.) Exenatide-chitosan nano, Exenatide-CSK-chitosan nano and Exenatide solution respectively, 6th group of dosage, for being 5.0 μ g/kg, subcutaneous injection (s.c.) Exenatide solution, distinguishes 0h upon administration, 0.5h, 1h, 2h, 3h, 4h, blood about 0.5ml is got after 6h, 8h, 10h and 12h, 5000rpm, centrifugal 10 minutes, get supernatant, adopt euzymelinked immunosorbent assay (ELISA) to measure Exenatide content in serum.Result of the test is in table 2, table 3 and Fig. 1.
Result shows: relative to oral Exenatide solution, and Exenatide nanoparticle prepared by employing chitosan, CSK-chitosan can significantly improve oral absorption effect; And the Exenatide nanoparticle better effects if adopting CSK-chitosan to obtain, can improve bioavailability about 1.5 times relative to chitosan, when CSK and chitosan weight ratio are 1:1, bioavailability reaches maximum.
The concentration (ng/ml) of Exenatide in table 2 different time points rat body
The pharmacokinetic parameter of table 3 different modes of administration
Test example 2 reduces blood sugar test
Trial drug: with test example 1
Experimental animal: db/db mice 30, often organizes 6, male and female half and half, body weight 40-45g.
Test apparatus: high performance liquid chromatograph (AgilentTechnololies), the integrated system for detecting blood sugar of Luo Kang (German Roche Diagnostics GmbH)
Test method: animal is divided into 6 groups at random, wherein 1-5 group dosage is 30.0 μ g/kg, respectively gastric infusion (i.g.) Exenatide-chitosan nano, Exenatide-CSK-chitosan nano and Exenatide solution; 6th group of dosage is 5.0 μ g/kg, subcutaneous injection (s.c.) Exenatide solution.0h, 1h, 2h, 4h, 6h, 8h, 10h and 12h, at tail venous blood sampling, measure blood glucose value upon administration respectively.Result of the test is in table 4 and Fig. 2.
Result shows: compared with oral Exenatide solution, CSK-chitosan Exenatide nanoparticle has obvious blood sugar decreasing effect, the hypoglycemic effect of CSK-chitosan (1:1) is best, in administration after 1 hour, blood glucose can be reduced to original 62%, and compared with subcutaneous injection Exenatide solution, hypoglycemic effect is close, and in 8 subsequently hour, blood glucose can maintain lower level.Compared with chitosan Exenatide nanoparticle, the onset of CSK-chitosan Exenatide nanoparticle is faster.
After the administration of table 4 mice, blood glucose reduces ratio (%)
Claims (8)
1. an Exenatide oral administration nanometer grain, is characterized in that containing Exenatide and CSK-chitosan.
2. nanoparticle according to claim 1, is characterized in that the weight ratio of Exenatide and CSK-chitosan is 0.5:10 ~ 3:10, preferred 1:10.
3., according to the arbitrary described nanoparticle of claim 1 or 2, it is characterized in that CSK and chitosan weight ratio are 2:1 ~ 1:4, preferred 1:1.
4., according to the arbitrary described nanoparticle of claim 1-3, it is characterized in that also containing ion crosslinking agent.
5. nanoparticle according to claim 4, is characterized in that described ion crosslinking agent be described ion crosslinking agent is sodium tripolyphosphate, tetrasodium pyrophosphate, sodium tetrapolyphosphate, eight polyphosphate sodiums and hexapolyphosphate sodium, is preferably sodium tripolyphosphate.
6. nanoparticle according to claim 5, is characterized in that the weight content of ion crosslinking agent is 10:0.6 ~ 10:4.5, preferred 10:0.9 ~ 10:1.
7. nanoparticle according to claim 1; it is characterized in that adopting following method to prepare; chitosan peptide modified for CSK is dissolved in low-concentration acetic acid; add Exenatide aqueous solution under magnetic stirring, dropwise add ionomer agent solution with method and continue to stir until form nanoparticle suspension.
8. nanoparticle according to claim 1, is characterized in that existing with oral dosage form, preferred oral liquid.
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| CN201410406341.9A CN105342999A (en) | 2014-08-18 | 2014-08-18 | Exenatide oral nanoparticle |
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|---|---|---|---|
| CN201410406341.9A CN105342999A (en) | 2014-08-18 | 2014-08-18 | Exenatide oral nanoparticle |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107773552A (en) * | 2016-08-26 | 2018-03-09 | 中国科学院上海药物研究所 | PTHrP analog nanoparticles |
| CN109675020A (en) * | 2019-01-11 | 2019-04-26 | 浙江大学 | A kind of oral GLP-1 polypeptide nanometer formulation and its preparation method and application |
| CN111053891A (en) * | 2019-11-15 | 2020-04-24 | 浙江工业大学 | Polypeptide nanoparticles for treating diabetes, polypeptide nanoparticle microneedles and preparation methods thereof |
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| CN101607080A (en) * | 2009-07-22 | 2009-12-23 | 南京凯瑞尔纳米生物技术有限公司 | Oral drugs of treatment diabetes and preparation method thereof |
| WO2013171570A1 (en) * | 2012-05-16 | 2013-11-21 | Glaxo Group Limited | Polypeptide loaded poca nanoparticles for oral administration |
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2014
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101607080A (en) * | 2009-07-22 | 2009-12-23 | 南京凯瑞尔纳米生物技术有限公司 | Oral drugs of treatment diabetes and preparation method thereof |
| WO2013171570A1 (en) * | 2012-05-16 | 2013-11-21 | Glaxo Group Limited | Polypeptide loaded poca nanoparticles for oral administration |
Non-Patent Citations (2)
| Title |
|---|
| MENGSHU WANG ET AL.: "Permeability of Exendin-4-Loaded Chitosan Nanoparticles across MDCK Cell Monolayers and Rat Small Intestine", 《BIOLOGICAL AND PHARMACEUTICAL BULLETIN》 * |
| YUN JIN: "Goblet celltargeting nanoparticles for oral insulin delivery and the influence of mucus on insulin transport", 《BIOMATERIALS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107773552A (en) * | 2016-08-26 | 2018-03-09 | 中国科学院上海药物研究所 | PTHrP analog nanoparticles |
| CN107773552B (en) * | 2016-08-26 | 2021-04-20 | 中国科学院上海药物研究所 | PTHrP analogue nanoparticle |
| CN109675020A (en) * | 2019-01-11 | 2019-04-26 | 浙江大学 | A kind of oral GLP-1 polypeptide nanometer formulation and its preparation method and application |
| CN111053891A (en) * | 2019-11-15 | 2020-04-24 | 浙江工业大学 | Polypeptide nanoparticles for treating diabetes, polypeptide nanoparticle microneedles and preparation methods thereof |
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Application publication date: 20160224 |