CN103965287A - Deuterohemin-beta-Ala-His-Lys(DhHP-3), and preparation method and application thereof - Google Patents
Deuterohemin-beta-Ala-His-Lys(DhHP-3), and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of chemical medicaments and medicinal preparations, and particularly relates to Deuterohemin-beta-Ala-His-Lys (DhHP-3), derivatives and new medical purposes of the DhHP-3 and the derivatives, in particular to the application of the DhHP-3 and the derivatives in preparation of type 2 diabetes-resistant medicaments. The preferred DhHP-3 and the derivatives thereof have the activity of reducing blood sugar, cholesterol and triglyceride, serve as the medicaments for treating and preventing the type 2 diabetes, have the advantages of high cell membrane permeability, high plasma stability, good blood reducing effect, low side effects and the like, and have a good application prospect.
Description
Technical field
The invention belongs to the technical field of a kind of chemicals and pharmaceutical preparation, particularly a kind of secondary heme tripeptides in the preparation of anti-diabetes B medicine and preparation method thereof that is applied in.
Background technology
Diabetes B (Type2Diabetes Mellitus, T2DM) is a kind of metabolic syndrome, and main manifestations is hyperglycemia, insulin resistant and islet beta cell function obstacle etc., compare with type 1 diabetes, its sickness rate is high, also comparatively common, accounts in diabetic subject 90%.Along with the development of diabetes B, patient often can produce the complication of various ways, and these complication are often to cause dead direct factor, the serious harm mankind's life and health.
In recent years, ferriporphyrin-short peptide compound is more concerned because having good biologic activity, and such ferriporphyrin short peptide compound is all that the carboxyl on protoheme or secondary heme is connected with amido linkage with the amino on peptide chain.Chinese patent CN02144902.3 discloses ferriporphyrin and derivative-short peptide compound and synthetic method thereof; Chinese patent CN200610131682.5 has reported the purposes of ferriporphyrin short peptide compound in medicament for resisting coronary heart disease preparation.
Further result of study shows, protoheme-short peptide compound or secondary heme-short peptide compound that this protoheme or secondary heme are connected with small peptide and form, due to the amino-acid residue composition of small peptide and the difference of order, its sterie configuration, water-soluble, peroxidase activity, permeability of cell membrane and biologic activity are also different, even widely different.
Chinese patent CN200910066991.2 has reported the purposes of a kind of secondary heme six peptides (DhHP-6) compound in anti-cerebral ischemia drugs preparation, a little less than water-soluble low, the permeability of cell membrane of the shortcoming of this compound, cause its bioavailability low, and the poor stability in blood plasma; Chinese patent CN201210448286.0 has reported the application of ferriporphyrin-short peptide compound in anti-diabetes B medicine preparation, although these short peptide compounds show certain hypoglycemic activity, yet, due to the amino-acid residue composition of small peptide and the difference of order, still do not overcome above-mentioned water-soluble low, shortcoming that permeability of cell membrane is weak, and a little less than hypoglycemic activity.
In order to overcome the deficiency of existing ferriporphyrin-short peptide compound, the invention provides a kind of secondary heme tripeptide compound and derivative thereof that is preferably connected with tripeptides with secondary heme and forms, with and in the application for the treatment of aspect diabetes B.
Summary of the invention
The technical problem to be solved in the present invention, to overcome the deficiencies in the prior art, a kind of specific secondary heme tripeptides Deuterohemin-β-Ala-His-Lys (DhHP-3) and derivative thereof are provided, and the application in preparation prevention and treatment diabetes B medicine.
The technical scheme of secondary heme tripeptides of the present invention is as follows:
A secondary heme tripeptides, is that the carboxyl of secondary heme (representing with Dh) is connected with amido linkage with the amino on alanine residue in three peptide molecules, it is characterized in that, and described tripeptides, on-link mode (OLM) is followed successively by β-Ala residue, His residue, Lys residue; Concrete structure formula (being designated as structural formula I) is:
or/and
That is, in the present invention, the secondary heme tripeptides of indication is the mixture of A and two kinds of structural compounds of B in structural formula I, also comprises that A's and two kinds of structural compounds of B in structural formula I is any; Result of study demonstration, in structural formula I, two kinds of compound biologic activity of A and B are identical, there is no difference.
The molecular formula of secondary heme tripeptides of the present invention is expressed as Dh-β-Ala-His-Lys; By three amino-acid residue order changes in peptide chain or replace combination with optional 1～3 seed amino acid residue in existing 20 seed amino acids respectively, can obtain the secondary heme tripeptide analog thing that secondary heme tripeptides multiple and of the present invention is close, as: analogue I Dh-β-Ala-Lys-His; Analogue II Dh-His-β-Ala-Lys; Analogue III Dh-His-Lys-β-Ala; Analogue IV Dh-Lys-β-Ala-His; Analogue V Dh-Lys – His-β-Ala; Analogue VI Dh-Lys-His-β-Ala; Analogue VIII Dh-Ala-His-Lys; Analogue IX Dh-β-Ala – Thr-Lys; Analogue X Dh-β-Ala-His – Glu etc.
Experimental result shows, secondary heme tripeptide analog thing activity is poor, only have secondary heme and Beta-alanine, Histidine, Methionin is with amido linkage combination, and binding sequence is Dh, β-Ala, His, during Lys, the secondary heme tripeptides forming, good water solubility, more easily enter in cell, bioavailability is high, in blood plasma, stability is better, and show higher pharmacologically active, especially in induction diabetes B animal model, shown good blood sugar reducing function, compare and there is outstanding advantage and feature with secondary heme six peptides and other secondary heme-short peptide compound.
The said secondary heme tripeptides of the present invention is to make by solid phase synthesis process, compare with traditional liquid-phase synthesis process have simple to operate, yield is high, purity advantages of higher; Furthermore, secondary heme tripeptides of the present invention is to make by Fmoc solid phase synthesis process, and the method is compared with Boc solid-phase peptide synthetic method, and reaction conditions is controlled, it is little to pollute, yield is higher, purity is higher.
The present invention further improves bioavailability and the protease inhibitor degradation capability of secondary heme tripeptides, on the basis of secondary heme tripeptides structure, by modification, obtains secondary heme tripeptide derivative of the present invention.
The present invention modifies the polyoxyethylene glycol (PEG, molecular weight is 200～20000 dalton) that secondary heme tripeptides structure is used, and is a kind of nontoxic polymer with wetting ability and biocompatibility; It is furthermore the activated polyethylene glycol derivative of commodity in use; It is further the derivative (representing with m-SC-PEG) that uses mono methoxy polyethylene glycol succinimide ester.
The derivative of said mono methoxy polyethylene glycol succinimide ester is to take polyoxyethylene glycol as main chain, and has the macromolecular compound of the succinimide ester terminal of a methoxyl group end group and an activation, and structural formula (being designated as structural formula II) is as follows:
In structural formula II, 44≤n≤447, k=0～3, molecular weight 2000～20000 dalton.
The present invention be take structural formula A in structural formula I or/and structural formula B is basis, and obtains secondary heme tripeptide derivative after modifying on this basis.Described secondary heme tripeptide derivative, the derivative that is mono methoxy polyethylene glycol succinimide ester is connected with amido linkage by nucleophilic substitution reaction with the epsilon-amino on lysine residue in secondary heme three peptide molecules, obtain the secondary heme tripeptides (representing with m-SC-PEG-DhHP-3) that a kind of mono methoxy polyethylene glycol is modified, structural formula (being designated as structural formula III) is as follows:
In structural formula III, Dh represents secondary heme; 44≤n≤447, k=0～3; Molecular weight polyethylene glycol scope is 2000～20000 dalton.
Research shows, the secondary heme tripeptides (m-SC-PEG-DhHP-3) of process the inventive method PEG Derivatives Modified is when keeping original pharmacologically active, its bioavailability and protease inhibitor degradation capability are improved significantly, simultaneously than not having better stability through the secondary heme tripeptides of modifying.
It is active that secondary heme tripeptides of the present invention and derivative thereof have the blood sugar of reduction, cholesterol and triglyceride level, according to conventional pharmaceutical technology, add tackiness agent, disintegrating agent, weighting agent, lubricant, seasonings, solubilizing agent, solvent, sanitas, vehicle etc. wherein any one or a few, can be prepared into according to ordinary method any conventional dose such as tablet, pill, capsule, electuary, sprays, oral liquid, suspension, cutaneous permeable agent, injection.
The present invention further extends secondary heme tripeptides drug half-life, reduce administration number of times, improve utilization ratio of drug, reach controlled release drug administration, with Poly(D,L-lactide-co-glycolide (PLGA) or polyethylene glycol-polylactic acid-co-glycolic acid (PEG-PLGA) high molecular polymer, secondary heme tripeptides is carried out to embedding, select more conventional emulsion solvent evaporation method to prepare two kinds of polyester medicine-carried sustained-release micro-spheres of secondary heme tripeptides with good biocompatibility and biological degradability.That is,
Described secondary heme tripeptides, can make medicine carrying polyester microsphere; Described medicine carrying polyester microsphere can be by Poly(D,L-lactide-co-glycolide (PLGA) or polyethylene glycol-polylactic acid-co-glycolic acid (PEG-PLGA), secondary heme tripeptides to be carried out to embedding to obtain.
Wherein, PLGA is the degradation material that a kind of biocompatibility is good, is by lactic acid (L-LA) and oxyacetic acid (GA) through polycondensation by a certain percentage, the multipolymer being polymerized by ester bond.PLGA has good biocompatibility and the tolerance to this base polymer human body, is finally completely degraded as hydration carbonic acid gas, without accumulation problems, can not cause significantly Inflammatory response, immune response and cell-cytotoxic reaction in body.As biodegradable medicine carrier, PLGA volume is small simultaneously, easily sees through tissue space, can regulate degradation rate by changing the composition and ratio of molecular weight and interpolymer, and then reach controlled release drug administration, the prolong drug transformation period, reduce administration number of times, improve utilization ratio of drug etc.The structural formula of PLGA (being designated as structural formula V) is as follows:
PEG-PLGA be on PLGA molecular chain, introduce that wetting ability is nontoxic, non-immunogenicity PEG segment, form PEG-PLGA segmented copolymer, can make PEG-PLGA material have amphipathic, can also be by changing the molecular weight of multipolymer and the character that composition and ratio regulates material, and then control degradation speed, improve rate of releasing drug.The made medicament-carrying nano-microsphere with specified particle diameter and distribution of this multipolymer being formed by lactic acid, oxyacetic acid and polyoxyethylene glycol, first pass through in vivo ester linkage hydrolyzing, be degraded to lactic acid and oxyacetic acid, finally be completely degraded into carbonic acid gas and water, metabolic system by human body excretes, and is usually used in preparing the pharmaceutical carrier of the various ways such as gel and microballoon.PEG-PLGA structural formula (being designated as structural formula VI) is as follows:
Experimental result shows, PEG-PLGA medicine carrying microballoons with respect to PLGA medicine carrying microballoons spheroid size more evenly, mean diameter is large, drug loading is high, encapsulation rate is high.
Further say, the invention provides the PEG-PLGA medicine carrying microballoons of the embedding secondary heme tripeptides that a kind of effect obviously improves, and aspect treating diabetes potential application prospect.
As the combination medicine that contains secondary heme tripeptides of the present invention and derivative thereof, it consists of and contains the compounds of this invention for the treatment of significant quantity, said compound can be independent use in the middle of pharmaceutical composition, also can combine N1,N1-Dimethylbiguanide, Exenatide, Regular Insulin or other ofhypoglycemic medicine and be used in conjunction with.
The present invention is preferred secondary heme tripeptides in secondary heme short peptide, amino-acid residue in secondary heme tripeptides is preferred β-Ala residue, His residue, Lys residue, preferred again specific order in tripeptides section, make peptide secondary heme tripeptides of the present invention and derivative thereof not only there is reduction blood sugar, cholesterol and triglyceride level are active, can be as the medicine for the treatment of or prevent anti-diabetes B, and it is better to have compared with the prior art permeability of cell membrane, plasma stability is better, hypoglycemic effect is better, the advantages such as side effect is little, there is good application prospect.Therefore, the application in anti-diabetes B medicine preparation of a kind of secondary heme tripeptides of the present invention and derivative thereof is very necessary.
Accompanying drawing explanation
Fig. 1 is the mass spectrum of the secondary heme tripeptides (DhHP-3) that makes of embodiment 1.
Embodiment
Below in conjunction with embodiment, secondary heme tripeptides and derivative thereof in the present invention are described further, but protection scope of the present invention is not limited to embodiment.
Abbreviation language vocabulary
The solid phase synthesis of embodiment 1 secondary heme tripeptides
1. resin swelling: take 2mmol Rink-NH
2resin, adds 50ml methylene dichloride swelling 2h, and suction filtration is used DMF (dimethyl formamide) washing resin 6 times;
2. deprotection: add 100ml20% piperidines/DMF solution, in room temperature concussion 30min, suction filtration, with DMF washing resin 6 times, detects amino deprotection situation with developer, when resin particle all become blue for deprotection complete;
3. connect amino acid: by Fmoc-β-Ala-OH, PyBOP (phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus), HOBT (I-hydroxybenzotriazole), NMM (N-methylmorpholine) and Rink-NH
2resin adds in reactor for 3:3:3:3:1 in molar ratio successively, add 50ml dimethyl formamide room temperature concussion 1h, suction filtration, uses dimethyl formamide washing resin 6 times, with developer, detect amino acid connection, resin particle all becomes transparent yellow for reacting completely; 2. 3. repeating step, connects follow-up Fmoc-His (Trt)-OH and Fmoc-Lys (Trt)-OH;
4. connect protoheme: 2. repeating step, adds 4 times of excessive heme reactions 3 hours; Suction filtration, uses DMF washing resin 6 times;
5. cutting: add 100ml cutting reagent (95% trifluoroacetic acid, 2.5% tri isopropyl silane and 2.5% ultrapure water), room temperature reaction 3h, suction filtration, steams filtrate rotation;
6. precipitate: gained debris is added in 300ml ether and precipitated, centrifugal, collect, obtain 1.7 grams of secondary heme tripeptides crude products;
7. purifying: the thick peptide obtaining is purified with anti-phase preparative liquid chromatography, moving phase: 0.1% trifluoroacetic acid/10～90% acetonitrile/water gradient elution, receive main peak, steam solvent rotary evaporation, obtained aqueous solution freeze-drying, obtains 1.5 grams of product secondary heme tripeptides sterlings.
The secondary heme tripeptides that embodiment 1 is made is through mass spectrometric detection, and theoretical value is 899.3 (C
45h
53feN
11o
6), practical measurement result is 899.3[M+H
+], consistent with theory, confirm that synthetic what obtain is target product secondary heme tripeptides, mass spectrum is shown in Fig. 1 in Figure of description.
Embodiment 2 secondary heme tripeptides and other secondary heme-short peptide compound attribute comparison
Use content well known in the art, to secondary heme tripeptides of the present invention and in the past molecular weight, water-soluble, the cytolemma perviousness of the secondary heme-short peptide compound of report compare, the results are shown in Table I:
Table I: secondary heme tripeptides and other secondary heme-short peptide compound attribute comparison
Table I is visible, and secondary heme tripeptides of the present invention is compared with the secondary heme-short peptide compound of report in the past, and molecular weight is relatively little, good water solubility, permeability of cell membrane are strong.
Embodiment 3 secondary heme tripeptides and the comparison of secondary heme hexapeptide compounds plasma stability
Use content well known in the art, secondary heme tripeptides and this protoheme hexapeptide compounds measured at plasma stability, the results are shown in Table II:
Table II secondary heme tripeptides and the comparison of secondary heme hexapeptide compounds plasma stability
Table II is visible, and DhHP-3 (secondary heme tripeptides) and the stability of m-SC-PEG-DhHP-3 (secondary heme tripeptide derivative) in blood plasma are better than time DhHP-6 (protoheme six peptides) and D9; And the secondary heme tripeptide derivative protease inhibitor degradation capability obtaining after modified is improved significantly.
The preparation of embodiment 4DhHP-3 derivative (m-SC-PEG-DhHP-3)
Methoxy poly (ethylene glycol) succinimdyl carbonate (structure is shown in structural formula II I, k=0 wherein, the weight-average molecular weight of PEG is 20000, n ≈ 447) (m-SC-PEG20000) modification to secondary heme tripeptides:
Getting 0.1mL concentration is the DhHP-3 aqueous solution of 1mg/ml, with the phosphate buffered saline buffer (PBS) of PH=8, be diluted to 1mL, the m-SC-PEG20000 (m-SC-PEG20000 and DhHP-3 mol ratio are 10:1) that adds respectively 20mg, after dissolving completely, in 25 ℃ of reactions 2 hours, add glycine termination reaction, with RPLC, detect transformation efficiency, recording transformation efficiency is 68.2%.With preparative RPLC, purify.
Purification condition:
Chromatographic column: prepLC, 25mmModule;
Moving phase: A:0.5% trifluoroacetic acid water-soluble (TFA, V/V) liquid, B:90% acetonitrile solution is (containing 0.5% trifluoroacetic acid (TFA, V/V);
Wavelength: 380nm
Gradient: 0-10min, 100%A phase; Elution time 10-30 minute, B phase gradient is from 30%-90%
Under elutriant after collecting is reduced pressure at 40 ℃, rotation is evaporated to without obvious Bubble formation, obtains the DhHP-3 derivative solid that PEG modifies.
The preparation of embodiment 5PLGA
With anhydrous lactitol liquid system, preparing biodegradable polyesters material, is the multipolymer of glycollide and rac-Lactide.The re-crystallizing in ethyl acetate that L-rac-Lactide is crossed with drying and distilling is purified for three times, and the re-crystallizing in ethyl acetate that glycollide is crossed with drying and distilling is purified for five times.Under vacuum state, the moisture of gaslight heating cleaning reaction bottle internal adsorption, inflated with nitrogen is cooling; in triplicate, under nitrogen protection, (4:1) adds rac-Lactide and the glycollide after refining in proportion, with the ratio of 10mL/g, adds the toluene after refining; take stannous octoate as catalyzer; phenylcarbinol is initiator, 120 ℃ of reaction 24h, the ethanol sedimentation of doubly measuring with toluene 5-8; obtain white solid; after collection, with chloroform, dissolve, then purify with ethanol sedimentation, obtain in triplicate product P LGA.PLGA: rac-Lactide: glycollide mol ratio be 4:1; Molecular weight is 22000-24000, meets the requirements.
The preparation of embodiment 6PEG-PLGA
According to embodiment 5 methods, obtain after PLGA, with azeotropic water removing method polymerization PLGA and PEG, post-treating method, as PLGA synthetic method aftertreatment in embodiment 5, makes product P EG-PLGA.PEG-PLGA:PLGA=50000, PEG=5000, meets the requirements.
The preparation of embodiment 7DhHP-3 medicine carrying PLGA polyester microsphere
Adopt compound emulsion method (W/O/W) to prepare the polyester microsphere of embedding.The aqueous solution of medicine is added in the dichloromethane solution of certain density polymkeric substance and emulsifying agent, ultrasonic emulsification forms water in oil colostrum (W/O), then transfer them to immediately in 1%PVA solution, under the effect of high speed shear Mixingemulsificationmachine, carry out emulsion (W/O/W), last normal temperature magnetic agitation 6-8h, makes the methylene dichloride volatilization in solution complete.Centrifugal collection product, and with second distillation water washing 3-4 time, lyophilize, obtains the PLGA polyester microsphere that bag carries secondary heme tripeptides (DhHP-3), after testing, the median size of PLGA medicine carrying microballoons is 5.13 μ m
The preparation of embodiment 8DhHP-3 medicine carrying PEG-PLGA polyester microsphere
Adopt compound emulsion method (W/O/W) to prepare the polyester microsphere of embedding DhHP-3.The aqueous solution of medicine is added in the dichloromethane solution of certain density polymkeric substance and emulsifying agent, ultrasonic emulsification forms water in oil colostrum (W/O), then transfer them to immediately in 1%PVA solution, under the effect of high speed shear Mixingemulsificationmachine, carry out emulsion (W/O/W), last normal temperature magnetic agitation 6-8h, makes the methylene dichloride volatilization in solution complete.Centrifugal collection product, and with second distillation water washing 3-4 time, lyophilize, obtains the PEG-PLGA polyester microsphere that bag carries secondary heme tripeptides (DhHP-3), after testing, the median size of PEG-PLGA medicine carrying microballoons is 17.52 μ m.
Embodiment 9 polyester microsphere drug loading are measured
Drug loading and the encapsulation rate of polyester microsphere are measured by ultraviolet-visible spectrophotometer.
Microballoon drug loading is measured: precision takes dry DhHP-3 microballoon 10mg, add 0.1mol/L NaOH (containing 0.5%SDS) solution 2mL, put into constant temperature oscillation case shaken overnight, after microballoon is degradable, get supernatant, by ultraviolet-visible pectrophotometer, measure its absorbancy, by typical curve, calculate drug loading.
Drug loading (%)=microballoon Chinese traditional medicine quality/microspheres quality * 100
Theoretical dosage * 100 in actual drug amount/microballoon in encapsulation rate (%)=microballoon
The drug loading of two kinds of medicine carrying microballoonss and encapsulation rate are in Table III
Drug loading and the encapsulation rate of the different polyester microspheres of Table III
| Index | PLGA | PEG-PLGA |
| Drug loading (%) | 8.785 | 9.27 |
| Encapsulation rate (%) | 56.6 | 76.7 |
Table III is visible, two kinds of medicine carrying microballoonss have higher drug loading and encapsulation rate, this illustrates that by emulsion solvent evaporation method, secondary heme tripeptides successfully being wrapped is written in two kinds of microballoons, and the drug loading of PEG-PLGA medicine carrying microballoons and encapsulation rate are all higher than PLGA medicine carrying microballoons, illustrate that PEG-PLGA is better than PLGA implementation result.
Embodiment 10DhHP-3 and derivative thereof (m-SC-PEG-DhHP-3) and the comparison of DhHP-6 to the effect of promotion C2C12 cell sugar consumption
By medicine after the administration of reduction enzymatic assays, to glucose content in differentiation C2C12 cell culture supernatant, observe medicine and promote the effect of C2C12 cell consumption sugar.
Experimental technique:
(1) clone: C2C12 is mice skeletal clone;
(2) drug effect is 10 μ M in C2C12 final concentration of cells;
(3) cell is bred to 80%～90% time in culturing bottle, and 0.25% trysinization is centrifugal, adds substratum and blows and beats into gently cell suspension, counting, 10
4individual/hole is laid in 96 orifice plates, within every 2 days, changes liquid, when cell proliferation to 70%, adding inducible factor induction C2C12 cytodifferentiation is myotube cell, changes low sugar culture-medium and spends the night, and again changes low sugar culture-medium morning next day and gives different pharmaceutical (10 μ mol/L) (every group arranges 6 multiple holes) simultaneously, drug effect 24h, draw supernatant nutrient solution, reduction enzymatic assays is glucose concn wherein, adds MTT, supernatant discarded after 4h, add DMSO Rong Xie formazan, 492nm reads each hole light absorption value, analytical results.
Table IV medicine is to the effect of C2C12 cell consumption sugar
Compare * p<0.05, * * p<0.01 with blank.
Because C2C12 is mice skeletal clone, therefore test in vitro the ability of metabolism sugar can direct reaction Mice Body endoskeleton myocyte to the picked-up of glucose and metabolic capacity.By the following result of table 1, can be drawn, DhHP-3 and m-SC-PEG-DhHP-3 can increase the consumption sugar effect to Skeletal Muscle Cell, DhHP-3 and m-SC-PEG-DhHP-3 all can promote the consumption sugar effect of C2C12 when drug level is 10 μ M, and effect is better than DhHP-6 and D9.
Embodiment 11DhHP-3 and derivative thereof (m-SC-PEG-DhHP-3) and the DhHP-6 therapeutic action comparison to rats with type 2 diabetes
(1) foundation of Rat Model of Type 2 Diabetes
Body weight is that the clean level male Wistar rat of 200 ± 20g is purchased from Jilin University's experimentation on animals center.Before experiment starts, rat is normally cultivated 1 week to adapt to laboratory condition.All rats all can ad lib and drinking-water.All experiments are all to carry out according to local experimentation on animals Ethical Demand.
Rat is cultivated at 21-22 ℃, and 55 ± 5% relative humidity, in the culturing room that every 12h light and dark replaces.We select high fat diet associating STZ induction rats with type 2 diabetes in this experiment.After rat condition of compatibility 1 week, randomly drawing 40 rats, to correct normal feed be that high lipid food is fed 4 weeks.Rats by intraperitoneal injection STZ (30mg/kg body weight).STZ is after mono-week in injection, rat fasting 12h, but can freely intake.Tail venous blood sampling is surveyed blood sugar concentration by blood glucose meter.Wherein fasting plasma glucose concentration >=7.8mmol/L is considered as diabetes B modeling success.Diabetes B rat is divided into four groups at random, ten every group, every day drug treatment.Be grouped as follows:
(I) Normal group (NC), normal rat abdominal injection every day 0.9%NaCl;
(II) diabetic model group (DM), diabetes B rat abdominal injection every day 0.9%NaCl;
(III) N1,N1-Dimethylbiguanide group (Metformin), diabetes B rat gastric infusion every day 300mg/kg N1,N1-Dimethylbiguanide;
(IV) DhHP-65.0mg/kg group, diabetes B rat abdominal injection every day 5.0mg/kg DhHP-3;
(V) DhHP-33.0mg/kg group, diabetes B rat abdominal injection every day 3.0mg/kg DhHP-3;
(VI) m-SC-PEG-DhHP-330mg/kg group, diabetes B rat abdominal injection every day 30mg/kgDhHP-3;
Administration every day of all animals, administration 5 weeks.
(2) body weight and blood sugar detection
Body weight and the blood sugar of rat detect once weekly.Rat fasting 12h before detecting, but can freely intake.By wide range balance weighing apparatus body weight.Tail vein blood, detects blood sugar concentration by blood glucose meter.
(3) abdominal injection glucose-tolerant experiment (IPGTT)
While finishing (the 5th week) in administration, after rat fasting 12h, the glucose of abdominal injection 2g/kg body weight.Respectively at 0min, 30min, 60min and 120min, tail venous blood sampling, detects blood sugar concentration by blood glucose meter.
(4) DhHP-3 and and the impact of derivative m-SC-PEG-DhHP-3 on high fat associating STZ induction diabetes B rat blood sugar, and the impact on empty stomach cholesterol and triglyceride level.
Table V rat fasting blood-glucose value
Compare * p<0.05, * * p<0.01 with model group.
Table V is visible, DhHP-3, m-SC-PEG-DhHP-3 and N1,N1-Dimethylbiguanide medicine were respectively organized intraperitoneal injection after 3 weeks, DhHP-3 has compared obvious reduction with m-SC-PEG-DhHP-3 medicine group and N1,N1-Dimethylbiguanide group fasting plasma glucose with model group, and action effect is remarkable, and is all better than DhHP-6 and D9.
DhHP-3 and derivative m-SC-PEG-DhHP-3 thereof and the DhHP-6 effect comparison to diabetes B rat limosis cholesterol and triglyceride level
Table VI is cholesterol and triglyceride level on an empty stomach
Compare * p<0.05, * * p<0.01 with model.
Table VI is visible, DhHP-3, m-SC-PEG-DhHP-3 and N1,N1-Dimethylbiguanide medicine were respectively organized intraperitoneal injection after 3 weeks, respectively to empty stomach cholesterol after medicine group and the treatment of N1,N1-Dimethylbiguanide group, reduce and compared notable difference with model group, each treatment group of Serum Triglyceride level all has obvious reduction, significant difference is obvious, and action effect DhHP-3 and m-SC-PEG-DhHP-3 are all better than DhHP-6 and D9.
As can be seen here, DhHP-3 and m-SC-PEG-DhHP-3 have significant blood sugar reducing function to rats with type 2 diabetes, and diabetes B action effect, higher than DhHP-6 and D9, is illustrated to DhHP-3 and derivative m-SC-PEG-DhHP-3 thereof are the medicines of a potential treatment diabetes B.
The preparation of embodiment 12DhHP-3 injection liquid
Take l00mg DhHP-3, be dissolved in 0.9% sodium chloride solution 1000ml, after mixing, be distributed into the injection liquid that 0.1mg/ml/ props up concentration and seal in medicine bottle, product is made in sterilizing.
The preparation of embodiment 13DhHP-3 analogue lyophilized powder
Take l.0g DhHP-3 derivative, be dissolved in 1000ml water and make the aqueous solution, add 3.83% N.F,USP MANNITOL, 0.9% sodium-chlor, mixes, and is distributed into the injection liquid that 1.0mg/ml/ props up concentration, and lyophilized powder is made in freeze-drying.
Embodiment 14 secondary heme tripeptides and derivative thereof, in anti-diabetes B medicine preparation, are combined the application of other treatment diabetes B medicine, and described other treatment diabetes B medicine can be N1,N1-Dimethylbiguanide, Exenatide, Regular Insulin.
Claims (10)
1. a secondary heme tripeptides, is that the carboxyl of secondary heme is connected with amido linkage with the amino on alanine residue in three peptide molecules, it is characterized in that, and described tripeptides, on-link mode (OLM) is followed successively by β-Ala residue, His residue, Lys residue; Concrete structure formula is:
or/and
2. secondary heme tripeptides according to claim 1, it is characterized in that, take secondary heme tripeptides structure as basis, the derivative of mono methoxy polyethylene glycol succinimide ester is connected with amido linkage by nucleophilic substitution reaction with the epsilon-amino on lysine residue in secondary heme three peptide molecules, obtains the secondary heme tripeptides that a kind of mono methoxy polyethylene glycol is modified; Concrete structure formula is:
In structural formula, Dh represents secondary heme; 44≤n≤447, k=0～3; Molecular weight polyethylene glycol scope is 2000～20000 dalton.
3. secondary heme tripeptides according to claim 1 and 2, is characterized in that, described protoheme tripeptides, makes medicine carrying polyester microsphere; Described medicine carrying polyester microsphere is by Poly(D,L-lactide-co-glycolide or polyethylene glycol-polylactic acid-co-glycolic acid, secondary heme tripeptides or protoheme tripeptide derivative to be carried out to embedding to obtain.
4. a preparation method for the secondary heme tripeptides of claim 1, is Fmoc solid phase synthesis process, and detailed process is:
1. resin swelling: take 2mmol Rink-NH
2resin, adds 50ml methylene dichloride swelling 2h, and suction filtration is used dimethyl formamide washing resin 6 times;
2. deprotection: add 100ml20% piperidines/dimethyl formamide solution, in room temperature concussion 30min, suction filtration, with dimethyl formamide washing resin 6 times, detects amino deprotection situation with developer, when resin particle all become blue for deprotection complete;
3. connect amino acid: by Fmoc-β-Ala-OH, PyBOP, HOBT, NMM and Rink-NH
2resin adds in reactor for 3:3:3:3:1 in molar ratio successively, add 50ml dimethyl formamide room temperature concussion 1h, suction filtration, uses dimethyl formamide washing resin 6 times, with developer, detect amino acid connection, resin particle all becomes transparent yellow for reacting completely; 2. 3. repeating step, connects follow-up Fmoc-His (Trt)-OH and Fmoc-Lys (Trt)-OH;
4. connect protoheme: 2. repeating step, adds 4 times of excessive heme reactions 3 hours; Suction filtration, uses dimethyl formamide washing resin 6 times;
5. cutting: add 100ml cutting reagent; Cutting reagent consists of 95% trifluoroacetic acid, 2.5% tri isopropyl silane and 2.5% water, room temperature reaction 3h, and suction filtration, steams filtrate rotation;
6. precipitate: gained debris is added in 300ml ether and precipitated, centrifugal, collect, obtain 1.7 grams of secondary heme tripeptides crude products;
7. purifying: the thick peptide obtaining is purified with anti-phase preparative liquid chromatography, moving phase: 0.1% trifluoroacetic acid/10～90% acetonitrile/water gradient elution, receive main peak, steam solvent rotary evaporation, obtained aqueous solution freeze-drying, obtains 1.5 grams of product secondary heme tripeptides sterlings.
5. the application of the secondary heme tripeptides of a claim 1 in preparation prevention and treatment diabetes B medicine.
6. the application of secondary heme tripeptides according to claim 5, it is characterized in that, in secondary heme tripeptides, add one or more in tackiness agent, disintegrating agent, weighting agent, lubricant, seasonings, solubilizing agent, solvent, sanitas, vehicle, be prepared into tablet, pill, capsule, electuary, sprays, oral liquid, suspension, cutaneous permeable agent or injection.
7. according to the application of the secondary heme tripeptides described in claim 5 or 6, it is characterized in that secondary heme tripeptides and N1,N1-Dimethylbiguanide, Exenatide, insulin combination medication.
8. an application for the secondary heme tripeptide derivative of claim 2, for the preparation of prevention and treatment diabetes B medicine.
9. the application of secondary heme tripeptide derivative according to claim 8, it is characterized in that, in secondary heme tripeptide derivative, add in tackiness agent, disintegrating agent, weighting agent, lubricant, seasonings, solubilizing agent, solvent, sanitas, vehicle any one or a few, be prepared into tablet, pill, capsule, electuary, sprays, oral liquid, suspension, cutaneous permeable agent or injection.
10. the application of secondary heme tripeptide derivative according to claim 8 or claim 9, is characterized in that secondary heme tripeptide derivative and N1,N1-Dimethylbiguanide, Exenatide, insulin combination medication.
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| CN104478990A (en) * | 2014-11-15 | 2015-04-01 | 吉林大学 | Deuterohemin-beta-Ala-His (DhHP-2) and preparation method and use thereof |
| CN105859840A (en) * | 2015-01-22 | 2016-08-17 | 长春百益制药有限责任公司 | Coupling method of Deuterohaemin molecule with peptide chain, cyclized Deuterohaemin peptide, and preparation and application thereof |
| CN105985441A (en) * | 2015-01-27 | 2016-10-05 | 长春百益制药有限责任公司 | Deuterohaemin hexapeptide derivative, preparation method and application thereof |
| CN107011413A (en) * | 2017-05-31 | 2017-08-04 | 浙江省农业科学院 | Tripeptides CGP with function of blood sugar reduction and application thereof |
| CN107312065A (en) * | 2017-07-05 | 2017-11-03 | 吉林大学 | The application of ferriporphyrin and its derivative short peptide compound and ferriporphyrin and its derivative short peptide compound |
| WO2021129579A1 (en) * | 2019-12-24 | 2021-07-01 | 吉林省澳宇生物科技有限公司 | Heme derivative and preparation method and use thereof |
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