CN105777892A - Pramlintide polyethylene glycol derivative, and preparation method and application thereof - Google Patents
Pramlintide polyethylene glycol derivative, and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of pharmaceutical synthesis, and discloses a pramlintide polyethylene glycol derivative. The pramlintide polyethylene glycol derivative is composed of pramlintide and polyethylene glycol covalently connected with pramlintide, the N end of pramlintide is modified with polyethylene glycol, and the N end of pramlintide is connected to one or two ends of polyethylene glycol. The invention also discloses a method for preparing the pramlintide polyethylene glycol derivative, and an application of the derivative in the preparation of medicinal preparations for treating diabetes. The pramlintide polyethylene glycol derivative improves the in vivo half life of pramlintide, pharmacological experiments prove that the pramlintide polyethylene glycol derivative combined with insulin makes the blood sugar reduction keeping time be obviously longer than that of pramlintide unmodified with PEG.
Description
Technical field
The present invention relates to technical field of medicine synthesis, be specifically related to Pramlintide polyethyleneglycol derivative, its preparation method and application thereof.
Background technology
Pancreas amyloid polypeptide, it it is a kind of polypeptide hormone being made up of 37 amino acid residues, discharge by pancreatic beta cell after the meal, there is different physiological roles, if the food that slows down (including glucose) is in the infiltration rate of small intestinal, by suppressing glucagon to reduce the generation of glycogen, reduce patient's appetite, assist body to regulate blood sugar level etc..But, natural pancreas amyloid polypeptide is also unstable in the solution, and facile hydrolysis has toughness feature big, agglutinophilic, thus is not suitable for treatment.
Pramlintide is a kind of stable pancreas amyloid polypeptide analog through screening, synthesizing.The amino acid sequence differences of Pramlintide and pancreas amyloid polypeptide shows that the former is substituted by proline on the 25th, 28 and 29.The production of Pramlintide is based on solid phase synthesis, for instance the method for the synthesizing pramlintide disclosed in CN200910189086.6.Research confirms, Pramlintide can the absorption of delay glucose, it is suppressed that the secretion of glucagon, reduces glycogen and generates and release, thus have blood glucose fluctuation frequency and fluctuating margin in reduction diabetics body, improves the effect that overall glycemic controls.Can be used as the adjuvant therapy medicaments of 1 type and type 2 diabetes mellitus clinically, be mainly used in alone insulin, and use in conjunction insulin and sulfonylurea drugs and/or metformin still cannot obtain the diabetics of expected effect.
Polyethylene Glycol (polyethyleneglycol, the PEG) modification of protein or polypeptide and PEGization (pegylation), be coupled on protein or peptide molecule with covalent bond by chemical method by the PEG of activation.Since Davis in 1977 adopts PEG to modify bovine serum albumin first, PEG modification technique develops rapidly, and is widely used in the chemical modification of multiple proteins and polypeptide, and PEG modification technique has also moved towards from theory in the practical application of medicine.
PEG modifies and can give proteins and peptides class multiple premium properties, be embodied in circulating half-life extend, immunogenicity reduce or disappear, toxic and side effects reduce and physics, chemistry and biological stability enhancing etc., widened to a great extent proteins and peptides range of application.
At present, the proteins and peptides that existing multiple PEG modifies is for biomedicine field, come into the market by FDA approval, such as the ADA Adenosine deaminase (PEG-ADA) of PEG modification, asparaginase (PEG-L-asparaginase), Interferon Alpha-2b (peginterferonalfa-2b), Intederon Alpha-2a (peginterferonalfa-2a), recombinant human granulocyte colony stimulating factor (pegfilgrastim) etc..
The modification target spot that PEGization is modified mostly is the amino of protein or polypeptide, including the ε-NH of lysine2α-NH with end2.Amino is the nucleophile group that in protein or polypeptide, quantity is maximum, and is frequently exposed to molecular surface, thus is easier to and PEG reagent reacting.Be target spot with amino polyethyleneglycol modified dose, mainly has PEG-succinimidyl succinate (PEG-SS), PEG-succinimdyl carbonate (PEG-SC), PEG-tresylate (PEGtresylate) and PEG-aldehyde (PEGaldehyde) etc..
The past research data that analyzing proteins class medicine PEG modifies, it has been found that, to the same object that is modified, the kind of PEG decorating site, the quantity of covalently bound PEG and PEG, all it is likely to the activity to modified outcome and produces large effect.Further, being modified between thing and PEG, PEG molecular weight is more big, it is possible to the activity decrease causing modified outcome is more many, is modified thing molecular weight more little, and the probability that its active site is shielded is more big.
At present, it is concentrated mainly on for the research of Pramlintide slow releasing preparation and prepares in phospholipid gel, there is not yet the relevant report about Pramlintide PEGization.But, according to relevant knowledge it can be seen that the selection of the suitable molecular weight of PEG and kind is to ensure that effectively to extend is modified the thing half-life, increase and be modified microbic activity, avoid the primary factor being modified thing active part by relatively multi-shielding simultaneously.Meanwhile, PEG modifies and still suffers from many unfavorable factors, for instance reaction condition should not control, and affects modified outcome productivity and purity and increases the factors such as synthesis (production) cost.Therefore, synthesis has longer Half-life in vivo and has the Pramlintide polyethyleneglycol derivative of good hypoglycemic effect and just have very important realistic meaning aborning.
Summary of the invention
The present invention is directed to the drawbacks described above existed in prior art, it is provided that a kind of Pramlintide polyethyleneglycol derivative that there is longer Half-life in vivo and there is good hypoglycemic effect, its preparation method and application thereof.
For this, one aspect of the present invention provides a kind of Pramlintide polyethyleneglycol derivative, it is made up of Pramlintide and the Polyethylene Glycol covalently bound with Pramlintide, wherein Polyethylene Glycol is modified at the N end of Pramlintide, and the N end of Pramlintide can be connected to an end of Polyethylene Glycol or two ends.
In a preferred embodiment of the invention, the Pramlintide polyethyleneglycol derivative of the present invention has structure shown below,
Wherein, X is the PEG residue modified.
In a preferred embodiment of the present invention, Polyethylene Glycol is at the NH of the N end of Pramlintide2Modify.
In a preferred embodiment of the present invention, the molecular weight of Polyethylene Glycol is 4000Da to 12000Da.
Another aspect of the present invention provides the method preparing Pramlintide polyethyleneglycol derivative of the present invention, it includes the step that preparation Pramlintide and Pramlintide N end are covalently bound with Polyethylene Glycol, the step that wherein Pramlintide N end is covalently bound with Polyethylene Glycol, it is under mildly acidic conditions, with PEG-aldehyde and Pramlintide for substrate, at sodium cyanoborohydride (NaCNBH3) catalysis under, reaction obtain Pramlintide PEGization derivant.
In a preferred embodiment of the present invention, adopting chemical synthesis process, it is more preferable to the method adopting solid phase synthesis, preparation is for the Pramlintide modified.
The method of solid phase synthesis Pramlintide of the present invention is preferably used the aminoacid of Fmoc and Boc protection; including the synthesis carrying out aminoacid sequence by Peptide synthesizer or manual synthetic method; then through excision, high-efficient liquid is separated purification, lyophilizing, it is thus achieved that peptide fragment is as the Pramlintide modified.
Solutions of weak acidity of the present invention is preferably and controls the pH value of reaction at 5.5-6.5.
Further aspect of the present invention provides the Pramlintide polyethyleneglycol derivative of the present invention application in preparing the pharmaceutical preparation for treating diabetes.
In a preferred embodiment of the present invention, the pharmaceutical preparation treating diabetes is durative action preparation.
At present, the preparation (production of Symlin, Amylin company) that Pramlintide invention business produces, its usage is subcutaneous injection, is administered before facing dinner.For diabetic, every day need to be administered 2-3 time, and client need stands the misery of multiple subcutaneous injections.
From description of the invention, compared with existing dosage form, Pramlintide polyethyleneglycol derivative prepared by the present invention with compared with polyethyleneglycol modified Pramlintide, the polyethyleneglycol modified half-life that improve Pramlintide in vivo that molecular weight is suitable, and effect experiment proves, Pramlintide polyethyleneglycol derivative provided by the invention uses blood sugar lowering hold time and compared with the PEG Pramlintide modified, be obviously prolonged with insulin combination.Meanwhile, N end NH provided by the invention2The Pramlintide polyethyleneglycol derivative modified, reaction condition is easy to control, and improves productivity and the purity of synthetic modification product.Additionally, the pointed decoration of N end provided by the invention, the shortcoming that also can overcome the peptides active part of polyethyleneglycol modified the caused shielding molecular weight in uncertain site.
Accompanying drawing explanation
Fig. 1: each change of blood sugar schematic diagram organizing mice in being administered 7 days；
The change of blood sugar schematic diagram of mice is respectively organized in administration in Fig. 2: the 3 day in latter 6 hours.
Detailed description of the invention
The present invention is described in further detail by the examples below, it is intended to is used for illustrating rather than the restriction present invention.It should be pointed out that, to those skilled in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention carries out some improvement and modification, these improve and modify and fall into too within protection scope of the present invention.
The implication of abbreviation used in the present invention is listed in the following table.
| Abbreviation and English | Implication |
| PRA | Pramlintide |
| Fmoc | Fluorenylmethyloxycarbonyl |
| Boc | Tertbutyloxycarbonyl |
| PEG | Polyethylene Glycol |
| mPEG | Mono methoxy polyethylene glycol |
| mPEG-ALD | Polyethylene Glycol-propionic aldehyde |
| Tyr | Tyrosine |
| Asn | Agedoite |
| Thr | Threonine |
[0030]
| Gly | Glycine |
| Gln | Glutamine |
| Phe | Phenylalanine |
| Ala | Alanine |
| Met | Methionine |
| Leu | Leucine |
| Cys | Cysteine |
| Lys | Lysine |
| Arg | Arginine |
Embodiment 1: solid-phase synthesis prepares Pramlintide
Method synthesizing pramlintide polypeptide disclosed in CN101747426B, then according to the Pramlintide of synthesis is purified by method disclosed in CN101525382B, thus obtaining for next step PEG Pramlintide modified.
Embodiment 2: synthesizing pramlintide polyethyleneglycol derivative
1, adopt the solid phase PEG method modified, Pramlintide prepared by embodiment 1 is adsorbed on cation exchange resin.
2, the cation exchange resin solution sodium bicarbonate adjustment pH value having adsorbed Pramlintide that contains step 1 prepared is 5.5, add the mPEG-ALD-4k (Mw4000) of 2-3 equivalent, the N end of Pramlintide is carried out selective reaction by mPEG-ALD, is specifically shown in following formula.
3, desalination: by soluble in water for the synthetic product in step 2, carry out desalination with SephadexG-25, using water as eluent, flow velocity 2ml/min, Ultraviolet Detector (wavelength 214nm) detects, and obtains synthetic product crude product after separating lyophilization.
4, purification: adopt C4 chromatographic column, mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopts linear gradient, and B phase is by 5% to 95% (30min), flow rate of mobile phase is 5ml/min, and detection wavelength is 214nm.
5, the Analysis and Identification of synthetic product:
1) HPLC Analysis and Identification:
Adopt KromasilC4 analytical type chromatographic column.Testing conditions: mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopt linear gradient, B phase is by 5% to 95% (30min), and flow rate of mobile phase is 1ml/min, and detection wavelength is 214nm.
Testing result: the Pramlintide PEG derivant purity after purification is 96.3%, and yield is 65%.
2) MS Analysis and Identification:
Adopt ground substance assistant laser to inhale ionizing flight time mass spectrum (MALDI-TOF-MS) sample after synthesis, purification is analyzed.
Testing result:Molecular weight is 7929, is consistent with expection.
Embodiment 3: synthesizing pramlintide polyethyleneglycol derivative
1, adopt the solid phase PEG method modified, Pramlintide prepared by embodiment 1 is adsorbed on cation exchange resin.
2, the cation exchange resin solution sodium bicarbonate adjustment pH value having adsorbed Pramlintide that contains step 1 prepared is 6.0, and the N end of Pramlintide is carried out selective reaction by the mPEG-ALD-1k (Mw1000), the mPEG-ALD that add 2-3 equivalent.
3, desalination: by soluble in water for the synthetic product in step 2, carry out desalination with SephadexG-25, using water as eluent, flow velocity 2ml/min, Ultraviolet Detector (wavelength 214nm) detects, and obtains synthetic product crude product after separating lyophilization.
4, purification: adopt C8 chromatographic column, mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopts linear gradient, and B phase is by 5% to 95% (30min), flow rate of mobile phase is 5ml/min, and detection wavelength is 214nm.
5, the Analysis and Identification of synthetic product:
1) HPLC Analysis and Identification:
Adopt KromasilC8 analytical type chromatographic column.Testing conditions: mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopt linear gradient, B phase is by 5% to 95% (30min), and flow rate of mobile phase is 1ml/min, and detection wavelength is 214nm.
Testing result: the Pramlintide PEG derivant purity after purification is 97.5%, and yield is 70.2%.
2) MS Analysis and Identification:
Adopt ground substance assistant laser to inhale ionizing flight time mass spectrum (MALDI-TOF-MS) sample after synthesis, purification is analyzed.
Testing result:Molecular weight is 4932, is consistent with expection.
3) MS Analysis and Identification is carried out after 4h:
Sample sampling employing MALDI-TOF-MS detection again after physiological saline solution 4h, testing result display molecular weight is 3949, and namely PEGization derivant decays, and the molecular weight of prompting mPEG-ALD-1k is too small, is unfavorable for the long-acting release of Pramlintide.
Embodiment 4: synthesizing pramlintide polyethyleneglycol derivative
1, adopt the solid phase PEG method modified, Pramlintide prepared by embodiment 1 is adsorbed on cation exchange resin.
2, the cation exchange resin solution sodium bicarbonate adjustment pH value having adsorbed Pramlintide that contains step 1 prepared is 6.0, and the N end of Pramlintide is carried out selective reaction by the mPEG-ALD-20k (Mw20000), the mPEG-ALD that add 2-3 equivalent.
3, desalination: by soluble in water for the synthetic product in step 2, carry out desalination with SephadexG-25, using water as eluent, flow velocity 2ml/min, Ultraviolet Detector (wavelength 214nm) detects, and obtains synthetic product crude product after separating lyophilization.
4, purification: adopt C4 chromatographic column, mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopts linear gradient, and B phase is by 5% to 95% (30min), flow rate of mobile phase is 5ml/min, and detection wavelength is 214nm.
5, the Analysis and Identification of synthetic product:
1) HPLC Analysis and Identification:
Adopt KromasilC4 analytical type chromatographic column.Testing conditions: mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopt linear gradient, B phase is by 5% to 95% (30min), and flow rate of mobile phase is 1ml/min, and detection wavelength is 214nm.
Testing result: the Pramlintide PEG derivant purity after purification is 65.2%, and yield is 30.4%.
2) MS Analysis and Identification:
Adopt ground substance assistant laser to inhale ionizing flight time mass spectrum (MALDI-TOF-MS) sample after synthesis, purification is analyzed.
Testing result:Molecular weight is 23925, is consistent with expection.
It follows that when mPEG-ALD molecular weight is excessive, the purity of Pramlintide PEG derivant and yield are affected by very big impact, it are primarily due to molecule and cross caused by senior general's reaction site covers.
Embodiment 5: synthesizing pramlintide polyethyleneglycol derivative
1, adopt the solid phase PEG method modified, Pramlintide prepared by embodiment 1 is adsorbed on cation exchange resin.
2, the cation exchange resin solution sodium bicarbonate adjustment pH value having adsorbed Pramlintide that contains step 1 prepared is 5.0, and the N end of Pramlintide is carried out selective reaction by the mPEG-ALD-4k (Mw4000), the mPEG-ALD that add 2-3 equivalent.
3, desalination: by soluble in water for the synthetic product in step 2, carry out desalination with SephadexG-25, using water as eluent, flow velocity 2ml/min, Ultraviolet Detector (wavelength 214nm) detects, and obtains synthetic product crude product after separating lyophilization.
4, purification: adopt C4 chromatographic column, mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopts linear gradient, and B phase is by 5% to 95% (30min), flow rate of mobile phase is 5ml/min, and detection wavelength is 214nm.
5, the Analysis and Identification of synthetic product:
1) HPLC Analysis and Identification:
Adopt KromasilC4 analytical type chromatographic column.Testing conditions: mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopt linear gradient, B phase is by 5% to 95% (30min), and flow rate of mobile phase is 1ml/min, and detection wavelength is 214nm.
Testing result: the Pramlintide PEG derivant purity after purification is 95.2%, and yield is 25.4%.
2) MS Analysis and Identification:
Adopt ground substance assistant laser to inhale ionizing flight time mass spectrum (MALDI-TOF-MS) sample after synthesis, purification is analyzed.
Testing result:Molecular weight is 7930, is consistent with expection.
The result of embodiment 5 shows, when regulating reduction alkylation reaction liquid pH and being 5.0, the yield of Pramlintide PEG derivant is remarkably decreased.
Embodiment 6: synthesizing pramlintide polyethyleneglycol derivative
1, adopt the solid phase PEG method modified, Pramlintide prepared by embodiment 1 is adsorbed on cation exchange resin.
2, the cation exchange resin solution sodium bicarbonate adjustment pH value having adsorbed Pramlintide that contains step 1 prepared is 7.0, and the N end of Pramlintide is carried out selective reaction by the mPEG-ALD-4k (Mw4000), the mPEG-ALD that add 2-3 equivalent.
3, desalination: by soluble in water for the synthetic product in step 2, carry out desalination with SephadexG-25, using water as eluent, flow velocity 2ml/min, Ultraviolet Detector (wavelength 214nm) detects, and obtains synthetic product crude product after separating lyophilization.
4, purification: adopt C4 chromatographic column, mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopts linear gradient, and B phase is by 5% to 95% (30min), flow rate of mobile phase is 5ml/min, and detection wavelength is 214nm.
5, the Analysis and Identification of synthetic product:
1) HPLC Analysis and Identification:
Adopt KromasilC4 analytical type chromatographic column.Testing conditions: mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopt linear gradient, B phase is by 5% to 95% (30min), and flow rate of mobile phase is 1ml/min, and detection wavelength is 214nm.
Testing result: the Pramlintide PEG derivant purity after purification is 94.3%, and yield is 30.2%.
2) MS Analysis and Identification:
Adopt ground substance assistant laser to inhale ionizing flight time mass spectrum (MALDI-TOF-MS) sample after synthesis, purification is analyzed.
Testing result:Molecular weight is 7932, is consistent with expection.
The result of embodiment 6 shows, when regulating reduction alkylation reaction liquid pH and being 7.0, the yield of Pramlintide PEG derivant is remarkably decreased.
Embodiment 7: synthesizing pramlintide polyethyleneglycol derivative
1, adopt the solid phase PEG method modified, Pramlintide prepared by embodiment 1 is adsorbed on cation exchange resin.
2, the cation exchange resin solution sodium bicarbonate adjustment pH value having adsorbed Pramlintide that contains step 1 prepared is 5.5, and the N end of Pramlintide is carried out selective reaction by the mPEG-ALD-8k (Mw8000), the mPEG-ALD that add 2-3 equivalent.
3, desalination: by soluble in water for the synthetic product in step 2, carry out desalination with SephadexG-25, using water as eluent, flow velocity 2ml/min, Ultraviolet Detector (wavelength 214nm) detects, and obtains synthetic product crude product after separating lyophilization.
4, purification: adopt C4 chromatographic column, mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopts linear gradient, and B phase is by 5% to 95% (30min), flow rate of mobile phase is 5ml/min, and detection wavelength is 214nm.
5, the Analysis and Identification of synthetic product:
1) HPLC Analysis and Identification:
Adopt KromasilC4 analytical type chromatographic column.Testing conditions: mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopt linear gradient, B phase is by 5% to 95% (30min), and flow rate of mobile phase is 1ml/min, and detection wavelength is 214nm.
Testing result: the Pramlintide PEG derivant purity after purification is 96.5%, and yield is 55.8%.
2) MS Analysis and Identification:
Adopt ground substance assistant laser to inhale ionizing flight time mass spectrum (MALDI-TOF-MS) sample after synthesis, purification is analyzed.
Testing result:Molecular weight is 11926, is consistent with expection.
Embodiment 8: synthesizing pramlintide polyethyleneglycol derivative
1, adopt the solid phase PEG method modified, Pramlintide prepared by embodiment 1 is adsorbed on cation exchange resin.
2, the cation exchange resin solution sodium bicarbonate adjustment pH value having adsorbed Pramlintide that contains step 1 prepared is 6.5, and the N end of Pramlintide is carried out selective reaction by the mPEG-ALD-12k (Mw12000), the mPEG-ALD that add 2-3 equivalent.
3, desalination: by soluble in water for the synthetic product in step 2, carry out desalination with SephadexG-25, using water as eluent, flow velocity 2ml/min, Ultraviolet Detector (wavelength 214nm) detects, and obtains synthetic product crude product after separating lyophilization.
4, purification: adopt C4 chromatographic column, mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopts linear gradient, and B phase is by 5% to 95% (30min), flow rate of mobile phase is 5ml/min, and detection wavelength is 214nm.
5, the Analysis and Identification of synthetic product:
1) HPLC Analysis and Identification:
Adopt KromasilC4 analytical type chromatographic column.Testing conditions: mobile phase is A phase (0.1%TFA aqueous solution) and B phase (acetonitrile solution containing 0.1%TFA), adopt linear gradient, B phase is by 5% to 95% (30min), and flow rate of mobile phase is 1ml/min, and detection wavelength is 214nm.
Testing result: the Pramlintide PEG derivant purity after purification is 94.2%, and yield is 50.4%.
2) MS Analysis and Identification:
Adopt ground substance assistant laser to inhale ionizing flight time mass spectrum (MALDI-TOF-MS) sample after synthesis, purification is analyzed.
Testing result:Molecular weight is 7932, is consistent with expection.
Embodiment 9: the blood sugar lowering pharmacology Activity determination effect experiment of Pramlintide PEG derivant
1, animal packet and medication
Body weight 18-20g kunming mice is randomly divided into 4 groups, often group 8, male and female half and half, A group is blank group, B group is model control group, C group is insulin and Pramlintide aqueous solution experimental group, and D group is insulin and Pramlintide PEG derivant (derivant is prepared referring to embodiment 2) experimental group.A group, B group neck every day dorsal sc injection normal saline 2 times, sooner or later respectively once；C group nape every day portion subcutaneous insulin injections and Pramlintide aqueous solution 2 times, every day 3 μ g Kg-1, sooner or later respectively once；D group nape portion disposable subcutaneous insulin injections 3 μ g Kg-1With Pramlintide PEG derivant 5mg Kg-1, insulin injection every day 3 μ g Kg afterwards-1.B, C, D group from experiment start the 1st day, every day lumbar injection streptozotocin (STZ) 30mg/kg (0.1mol L-1Citrate buffer solution configures, pH5.0), continuous 7 days, mice fasting 12h before injection STZ.
2, blood sugar detection
Experiment start the 1st, 3,5,7 day morning measure mouse blood sugar before administration, mice fasting 12h before measurement, tail venous blood sampling, carry out blood sugar detection by three promise blood glucose meter, the change of blood sugar situation within observation mice 7d.3d after experiment starts, measures the blood glucose in 1～6h after mice is administered in the morning, measures once every 1h, mice fasting 12h before administration, observes each group of change of blood sugar situation in 6h upon administration.
Change of blood sugar in each group mice 7d is as shown in Figure 1.A group (blank) mice blood glucose in 7d does not have significant change；B group (model comparison) mice blood glucose after injection STZ presents obvious ascendant trend, and after 3d, glucostasis is at 15mmol L-1Above；Before C group (insulin and Pramlintide aqueous solution) mice, the blood glucose of 3d is in being gradually increasing trend, and after 3d, blood glucose is at 10mmol L-1Fluctuate up and down, and significantly lower than B group；D group (insulin and Pramlintide PEG derivant) mouse blood sugar variation tendency is essentially identical with C group, and has significant difference (P < 0.05) within 7d with B group.D group mouse blood sugar substantially rises at 7d；And the blood glucose 7d change of C group mice is little.
In order to observe the change of blood sugar situation after the administration of insulin and Pramlintide aqueous solution group (C group) mice and the change of blood sugar trend of insulin and Pramlintide PEG derivant group (D group) mice in the same time, experiment start after the change of blood sugar that determines morning after administration in 6h of 3d, result is as shown in Figure 2.Being each fasting glucose (mice is fasting 12h) organizing mice measured before administration when 0, now the blood sugar concentration of C group and D group is close.After A, B group injection injection use water, C group insulin injection and Pramlintide aqueous solution, 1h, C group mouse blood sugar significantly reduces, and D group mouse blood sugar reduction amplitude is less, and the blood glucose difference between two groups of mices is gradually reduced over time, reaches again same level during 6h.
Claims (10)
1. a Pramlintide polyethyleneglycol derivative, it is made up of Pramlintide and the Polyethylene Glycol covalently bound with Pramlintide, it is characterized in that, Polyethylene Glycol is modified at the N end of Pramlintide, and the N end of Pramlintide can be connected to an end of Polyethylene Glycol or two ends.
2. Pramlintide polyethyleneglycol derivative according to claim 1, it has structure as shown below,
Wherein, X is the PEG residue modified.
3. Pramlintide polyethyleneglycol derivative according to claim 1 and 2, wherein Polyethylene Glycol is at the NH of the N end of Pramlintide2Modify.
4. the Pramlintide polyethyleneglycol derivative according to any one of claim 1-3, wherein the molecular weight of Polyethylene Glycol is 4000Da to 12000Da.
5. the method for preparation Pramlintide polyethyleneglycol derivative described in claim 1-4, it includes the step that preparation Pramlintide and Pramlintide N end are covalently bound with Polyethylene Glycol, it is characterized in that, the step that Pramlintide N end is covalently bound with Polyethylene Glycol, carry out under mildly acidic conditions, with PEG-aldehyde and Pramlintide for substrate, at sodium cyanoborohydride (NaCNBH3) catalysis under, reaction obtain Pramlintide PEGization derivant.
6. method according to claim 5, wherein adopts chemical synthesis process, it is preferred to use the method for solid phase synthesis, and preparation is for the Pramlintide modified.
7. method according to claim 6; wherein the method for solid phase synthesis includes using Fmoc and the Boc aminoacid protected, and carries out the synthesis of aminoacid sequence by Peptide synthesizer or manual synthetic method, then through excision; high-efficient liquid is separated purification, lyophilizing, it is thus achieved that peptide fragment is as the Pramlintide modified.
8. the method according to any one of claim 5-7, wherein solutions of weak acidity is control the pH value of reaction at 5.5-6.5.
9. the application in preparing the pharmaceutical preparation for treating diabetes of the Pramlintide polyethyleneglycol derivative according to any one of claim 1-4.
10. application according to claim 9, the pharmaceutical preparation wherein treating diabetes is durative action preparation.
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Citations (11)
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