CN102180943A - Production process of polypeptide medicament for assisting to reduce blood sugar - Google Patents
Production process of polypeptide medicament for assisting to reduce blood sugar Download PDFInfo
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- CN102180943A CN102180943A CN2010105945659A CN201010594565A CN102180943A CN 102180943 A CN102180943 A CN 102180943A CN 2010105945659 A CN2010105945659 A CN 2010105945659A CN 201010594565 A CN201010594565 A CN 201010594565A CN 102180943 A CN102180943 A CN 102180943A
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- tbu
- resin
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 15
- 239000003814 drug Substances 0.000 title abstract description 12
- 229920001184 polypeptide Polymers 0.000 title abstract description 9
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract description 9
- 238000004519 manufacturing process Methods 0.000 title abstract description 5
- 239000008280 blood Substances 0.000 title abstract description 4
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- 108010029667 pramlintide Proteins 0.000 claims abstract description 65
- 229920005989 resin Polymers 0.000 claims abstract description 37
- 239000011347 resin Substances 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000000843 powder Substances 0.000 claims abstract description 20
- 229960003611 pramlintide Drugs 0.000 claims abstract description 9
- 229920003180 amino resin Polymers 0.000 claims abstract description 5
- 239000012634 fragment Substances 0.000 claims description 68
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- BCZXFFBUYPCTSJ-UHFFFAOYSA-L Calcium propionate Chemical compound [Ca+2].CCC([O-])=O.CCC([O-])=O BCZXFFBUYPCTSJ-UHFFFAOYSA-L 0.000 claims description 4
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 4
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- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 claims description 3
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- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 claims description 3
- JDDWRLPTKIOUOF-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-[[4-[2-[bis(4-methylphenyl)methylamino]-2-oxoethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(=O)NC(C=2C=CC(C)=CC=2)C=2C=CC(C)=CC=2)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JDDWRLPTKIOUOF-UHFFFAOYSA-N 0.000 claims description 3
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a production process of a polypeptide medicament for assisting to reduce blood sugar based on Pramlintide as a main medicament. The invention provides a novel synthesis segment division method which comprises the following steps: dividing a segment I-amino resin into AA29-AA37-RinkAimde MBHA; dividing a segment II-CTC resin into AA17-AA28-Chlorotrityl Chloride; and dividing a segment III-CTC-resin into AA1-AA16-Chlorotrityl Chloride. The method has the characteristics of simple reaction operation, easy post-treatment, high yield, low cost and the like. The invention simultaneously discloses a Pramlintide freeze-dried powder injection which mainly comprises the following raw material components: main medicament Pramlintide, an excipient, a pH value regulator and a bacteriostat. The powder injection has the advantages of high dispersibility, good stability and the like.
Description
Technical field:
The present invention relates to a kind of is the production technique of the auxiliary hyperglycemic polypeptide drugs of main ingredient with the tripro-amylin, relates in particular to a kind of solid phase fragment synthesis method and the lyophilized injectable powder preparation process thereof of tripro-amylin.
Background technology:
Tripro-amylin (Pramlintide) is a kind of synthetic analogues of pancreas amyloid polypeptide, also is second medicine of getting permission to be used for the treatment of type i diabetes after Regular Insulin up to now.Clinical study finds, when tripro-amylin and Regular Insulin share, may cause the weight in patients appropriateness to alleviate.
The pancreas amyloid polypeptide is a kind of polypeptide hormone that is made of 37 amino-acid residues, discharging by pancreatic beta cell after the meal, has different physiological roles, as the food that slows down (comprising glucose) is in the absorption rate of small intestine, by suppressing the generation that glucagon reduces glycogen, reduce patient's appetite, assist body blood sugar regulation level or the like.But, natural pancreas amyloid polypeptide is also unstable in solution, and facile hydrolysis has big, the agglutinophilic characteristics of toughness, thereby is not suitable for treatment.Tripro-amylin is a kind of stable pancreas amyloid polypeptide analogue through screening, synthesizing.The aminoacid sequence difference table of tripro-amylin and pancreas amyloid polypeptide now the former the 25th (L-Ala), 28 (Serine) and 29 (Serines) is substituted by proline(Pro).Studies confirm that, the absorption that tripro-amylin can delay glucose, the secretion of glucagon suppression reduces glycogen and generates and discharge, thereby has blood glucose fluctuation frequency and fluctuating range in the diabetic subject of the reduction body, changes the effect of general overall glycemic control.
The structure of tripro-amylin is as follows:
Molecular formula: C
171H
267N
51O
53S
2X C
2H
4O
2, wherein x is a variable.
Molecular weight: 3949.39
For comprising 37 amino acid whose tripro-amylins, this synthetic route of solid phase synthesis sequence protection amino acid is very long successively, and whole technological operation is loaded down with trivial details, and yield is extremely low, and cost is very high, is not suitable for industrialization.Present method provides a kind of productive rate higher, the production technique that environmental pollution is littler and preparation is more stable.
The prescription of the tripro-amylin injection that has gone on the market includes 1.00mg/ml or 0.60mg/ml tripro-amylin, 0.03mmol/ml acetate, and 43.00mg/ml N.F,USP MANNITOL, the 2.25mg/ml meta-cresol is 4.0 with acetic acid and sodium-acetate adjust pH.Its poor stability is only in 2 ℃～8 ℃ low-temperature dark storage a surnames.The tripro-amylin lyophilized injectable powder has overcome the deficiency that there is poor stability in injection, has the dispersity height, advantages such as good stability.The said preparation steady quality, determined curative effect is beneficial to the patient and accepts.
Summary of the invention:
The object of the present invention is to provide a kind of new solid phase fragment division methods and a kind of lyophilized injectable powder of tripro-amylin, to overcome the defective that exists on the prior art and the deficiency of injection poor stability.The invention provides a kind of high yield, low cost, environmental pollution little, help realizing the tripro-amylin solid phase fragment division methods of industrialization, and can improve stability of formulation, extend the expiration date, be convenient to room temperature storage a surname's lyophilized injectable powder.
Some abbreviations commonly used have following implication among the present invention:
Fmoc: fluorenylmethyloxycarbonyl
TBTU:O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid ester
HOBt:1-hydroxybenzene a pair of horses going side by side triazole
DIEA:N, the N-diisopropylethylamine
TFA: trifluoracetic acid
DCM: methylene dichloride
DMF:N, dinethylformamide
EDT: dithioglycol
Water: water
Trt: trityl
TBu: the tertiary butyl
Boc: tertbutyloxycarbonyl
Acm: acetyl aminomethyl
Pbf:2,2,4,6,7-pentamethyl-benzo furans-5-alkylsulfonyl
A kind of method of solid phase fragment synthesizing pramlintide, its fragment division methods is:
Its fragment I-Rink Amide mbha resin is:
29 37
Fmoc-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Ash(Trt)-Thr(tBu)-Tyr(tBu)-Rink
Amide?MBHA
Its fragment II-CTC resin is:
17 28
Fmoc-Val-His(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-Ile-Leu-Pro-
Chlorotrityl?Chloride
Its fragment III-CTC resin is:
A kind of method of solid phase fragment synthesizing pramlintide, its technical scheme may further comprise the steps:
1) with aminoresin be carrier, synthetic fragment I-aminoresin is AA
29-AA
37-Rink Amide mbha resin;
2) with the hydroxy resin be carrier, synthetic fragment II-hydroxy resin is AA
17-AA
28-CTC resin;
3) with the hydroxy resin be carrier, synthetic fragment III-hydroxy resin is AA
1-AA
16-CTC resin, and carry out solid phase cyclization;
4) preparation fragment II-fragment I-RinkAmide mbha resin;
5) preparation fragment III-fragment II-fragment I-Rink Amide mbha resin;
6) cutting obtains the thick peptide of tripro-amylin;
7) refining, obtain the pure product of tripro-amylin.
The invention provides a kind of method of solid phase fragment synthesizing pramlintide, the substitution degree of its RinkAmide mbha resin is 0.2～0.4mmol/g; The substitution degree of its 2-Chlorotrityl Chloride resin (CTC resin) is 0.8～1.2mmol/g; Its protection amino acid is respectively Fmoc-Tyr (tBu), Fmoc-Thr (tBu), Fmoc-Asn (Trt), Fmoc-Ser (tBu), Fmoc-Gly, Fmoc-Val, Fmoc-Pro, Fmoc-Leu, Fmoc-Ile, Fmoc-Phe, Fmoc-His (Trt), Fmoc-Ala, Fmoc-Arg (Pbf), Fmoc-Gln (Trt), Fmoc-Cys (Acm), Fmoc-Lys (Boc); Its condensation reagent is TBTU/HOBt/DIEA; The charging capacity of its protection amino acid and condensation reagent is 2～5 times of equivalents; Its Synthetic 2, the cyclization reagent of the disulfide bridge bond of 7 two halfcystines is I
2, solvent is DMF.
Tripro-amylin preparation method provided by the invention, prepare amino-acid residue fragment I-RinkAmide mbha resin, fragment II-CTC resin and fragment III-CTC resin compound by solid-phase synthesis, then fragment I-RinkAmideMBHA resin and fragment II coupling obtained fragment II-fragment I-Rink Amide mbha resin:
17 28
Fmoc-Val-His(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-Ile-Leu-Pro-
29 37
Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(tBu)-Tyr(tBu)-Rink?Amide?MBHA
Again fragment III and the coupling of fragment II-fragment I-Rink Amide mbha resin are obtained fragment III-fragment II-fragment I-Rink Amide mbha resin:
Last deprotection, purifying obtains tripro-amylin.
The present invention has prepared a kind of lyophilized injectable powder that contains the tripro-amylin medicine simultaneously, and it comprises main ingredient tripro-amylin, vehicle, fungistat, pH value conditioning agent.
The present invention has prepared a kind of lyophilized injectable powder that contains the tripro-amylin medicine simultaneously, and its vehicle is selected from N.F,USP MANNITOL, lactose, glucose, sucrose, sodium-chlor, sorbyl alcohol etc.; Its fungistat is selected from meta-cresol, phenylformic acid, Sorbic Acid, ethanol, glycerine, phenylcarbinol, trichloro-butyl alcohol, calcium propionate, Sodium dehydroacetate, calcium propionate, sodium Diacetate, Sodium.alpha.-hydroxypropionate etc.; Its pH value conditioning agent is selected from acetic acid, sodium-acetate, hydrochloric acid, sulfuric acid, lactic acid, oxysuccinic acid, Citric Acid, phosphoric acid, sodium hydroxide, yellow soda ash, sodium bicarbonate, Sodium phosphate dibasic etc.
Through a large amount of optimization experiment, the present invention has found that most preferably prescription is formed.Wherein, preferred N.F,USP MANNITOL of its vehicle and lactose, preferred meta-cresol of fungistat and trichloro-butyl alcohol, pH value conditioning agent is acetic acid and sodium-acetate most preferably, and the pH value is 3.0-5.0 most preferably.
In addition, the present invention also provides preparation to contain the method for the lyophilized injectable powder of tripro-amylin medicine.Its technology is as follows:
1 precision takes by weighing the above-mentioned material of recipe quantity to sterilising vessel;
2 add an amount of water for injection dissolves fully;
3 usefulness acetic acid and sodium-acetate are regulated pH value to 3.0 between 5.0;
4 water for injection constant volumes;
5 activated carbon adsorption pyrogens;
6 0.45 μ m filtering with microporous membrane;
7 0.22 μ m filtering with microporous membrane;
Can is in cillin bottle after 8 passed examinations, vacuum lyophilization, tamponade, outlet, Zha Gai;
Labeling packing after 9 passed examinations.
Description of drawings:
Fig. 1 is the process flow sheet of solid phase synthesis tripro-amylin of the present invention.
Embodiment:
The present invention is including but not limited to following examples.
Embodiment 1: preparation fragment I-Rink Amide mbha resin
100.00g Fmoc-RinkAmide MBHA resin is joined in the reaction column, and the substitution degree of this resin is 0.3mmol/g.Add the 600mlDMF swelling, add DCM600ml/ washing 3 times then, add DMF600ml/ washing 3 times again.Add the 50% piperidines/DMF solution 600ml for preparing in the glass reaction post, stirring reaction 30min extracts reaction solution, adds DMF600ml/ washing 6 times.The charging capacity of protection amino acid and condensation reagent is 3 times of equivalents; take by weighing Fmoc-Tyr (tBu)-OH 41.36g and TBTU28.90g, HOBt12.16g, DIEA11.63g; add the DMF stirring and dissolving, join in the glass reaction post stirring reaction 24 hours after stirring.Kaiser Test detection reaction degree is finished until reaction.After reaction finishes, take out dereaction liquid,, with DCM600ml/ washing 3 times, drain again, pour out, put into vacuum drying oven dry 12 hours with DMF600ml/ washing 3 times.Taking-up is weighed, and is 104.83g, and the mensuration substitution degree is 0.22mmol/g.
104.83gFmoc-Tyr (tBu)-resin is joined in the reaction column, add DCM600ml/ washing 3 times, add DMF600ml/ washing 3 times again.Add the 50% piperidines/DMF solution 600ml for preparing in the glass reaction post, stirring reaction 30min extracts reaction solution, adds DMF600ml/ washing 6 times.Take by weighing Fmoc-Thr (tBu)-OH 27.43g and TBTU22.15g, HOBt9.32g, DIEA8.92g, add the DMF stirring and dissolving.After the dissolving, the amino acid coupling liquid for preparing is joined in the reaction column stirring reaction 3 hours fully.Sampling, with DMF washing 6 times, Kaiser Test detection reaction degree is finished until reaction.Take out dereaction liquid, with DMF600ml/ washing 6 times.
Repeat above " in the glass reaction post, adding the 50% piperidines/DMF solution 600ml for preparing ... take out dereaction liquid; " step with DMF600ml/ washing 6 times; meet Fmoc-Asn (Trt) successively; Fmoc-Ser (tBu), Fmoc-Gly, Fmoc-Val; Fmoc-Asn (Trt); Fmoc-Thr (tBu)-OH, the acid of Fmoc-Pro protection nitrogen base, synthetic fragment I-Rink Amide mbha resin.
Embodiment 2: preparation fragment II-CTC resin
60.00g 2-Chlorotrityl Chloride resin is joined in the reaction column, and the substitution degree of this resin is 1.0mmol/g.Add the 360mlDMF swelling, add DCM360ml/ washing 3 times then, add DMF360ml/ washing 3 times again.The charging capacity of protection amino acid and condensation reagent is 3 times of equivalents; take by weighing Fmoc-Pro-OH 60.73g and TBTU57.80g, HOBt24.32g, DIEA23.26g; add the DMF stirring and dissolving, join in the glass reaction post stirring reaction 24 hours after stirring.Kaiser Test detection reaction degree is finished until reaction.After reaction finishes, take out dereaction liquid,, with DCM360ml/ washing 3 times, drain again, pour out, put into vacuum drying oven dry 12 hours with DMF360ml/ washing 3 times.Taking-up is weighed, and is 64.78g, and the mensuration substitution degree is 0.82mmol/g.
64.78g Fmoc-Pro-resin is joined in the reaction column, add DCM360ml/ washing 3 times, add DMF360ml/ washing 3 times again.Add the 50% piperidines/DMF solution 360ml for preparing in the glass reaction post, stirring reaction 30min extracts reaction solution, adds DMF360ml/ washing 6 times.Take by weighing Fmoc-Leu-OH56.54g and TBTU51.37g, HOBt21.62g, DIEA20.68g, add the DMF stirring and dissolving.After the dissolving, the amino acid coupling liquid for preparing is joined in the reaction column stirring reaction 3 hours fully.Sampling, with DMF washing 6 times, Kaiser Test detection reaction degree is finished until reaction.Take out dereaction liquid, with DMF360ml/ washing 6 times.
Repeat above " in the glass reaction post, adding the 50% piperidines/DMF solution 360ml for preparing ... take out dereaction liquid, " step, meet Fmoc-Ile successively with DMF360ml/ washing 6 times; Fmoc-Pro; Fmoc-Gly, Fmoc-Phe, Fmoc-Asn (Trt); Fmoc-Asn (Trt); Fmoc-Ser (tBu), Fmoc-Ser (tBu), Fmoc-His (Trt); Fmoc-Val protects amino acid, synthetic fragment II-CTC resin.
Embodiment 3: preparation fragment III-CTC resin
60.00g 2-Chlorotrityl Chloride resin is joined in the reaction column, and the substitution degree of this resin is 1.0mmol/g.Add the 360mlDMF swelling, add DCM360ml/ washing 3 times then, add DMF360ml/ washing 3 times again.The charging capacity of protection amino acid and condensation reagent is 3 times of equivalents, takes by weighing Fmoc-Leu-OH63.61g and TBTU57.80g, HOBt24.32g, DIEA23.26g, adds the DMF stirring and dissolving, joins in the glass reaction post stirring reaction 24 hours after stirring.Kaiser Test detection reaction degree is finished until reaction.After reaction finishes, take out dereaction liquid,, with DCM360ml/ washing 3 times, drain again, pour out, put into vacuum drying oven dry 12 hours with DMF360ml/ washing 3 times.Taking-up is weighed, and is 65.77g, and the mensuration substitution degree is 0.85mmol/g.
65.77g Fmoc-Leu-resin is joined in the reaction column, add DCM360ml/ washing 3 times, add DMF360ml/ washing 3 times again.Add the 50% piperidines/DMF solution 360ml for preparing in the glass reaction post, stirring reaction 30min extracts reaction solution, adds DMF360ml/ washing 6 times.Take by weighing Fmoc-Phe-OH 65.86g and TBTU54.58g, HOBt22.97g, DIEA21.97g, add the DMF stirring and dissolving.After the dissolving, the amino acid coupling liquid for preparing is joined in the reaction column stirring reaction 3 hours fully.Sampling, with DMF washing 6 times, Kaiser Test detection reaction degree is finished until reaction.Take out dereaction liquid, with DMF360ml/ washing 6 times.
Repeat above " in the glass reaction post, adding the 50% piperidines/DMF solution 360ml for preparing ... take out dereaction liquid; " step with DMF360ml/ washing 6 times; meet Fmoc-Asn (Trt) successively; Fmoc-Ala; Fmoc-Leu, Fmoc-Arg (Pbf), Fmoc-Gln (Trt); Fmoc-Thr (tBu); Fmoc-Ala, Fmoc-Cys (Acm), Fmoc-Thr (tBu); Fmoc-Ala; Fmoc-Thr (tBu), Fmoc-Asn (Trt), Fmoc-Cys (Acm); Fmoc-Lys (Boc) protects amino acid, synthetic fragment III-CTC resin.
Take by weighing 43.18g I2 and add stirring and dissolving among the 810mlDMF, join in the reaction column after the dissolving fully, nitrogen blows and stirs reaction 30min, and sampling with DMF washing 6 times, with Ellman reaction detection sulfydryl level of response, is finished until reaction.Take out dereaction liquid,, with DCM810ml/ washing 3 times, drain again, pour out with DMF810ml/ washing 3 times.Thereby Synthetic 2, the disulfide bridge bond of 7 two halfcystines.
Embodiment 4: preparation fragment II-fragment I-Rink Amide mbha resin
To add by the fragment II-CTC resin of embodiment 2 preparations in the glass reaction post, add the 1%TFA/DCM solution 360ml for preparing to the glass reaction post, stirring reaction 30min, collect reaction solution, add 360ml/ washing of 1%TFA/DCM 2 times, merge reaction solution, revolving inspissation contracts, obtain faint yellow oily thing, in water, precipitate, get white meal.
To be added in the glass reaction post by the fragment I-Rink Amide mbha resin of embodiment 1 preparation, and add the 50% piperidines/DMF solution 600ml for preparing to the glass reaction post, stirring reaction 30min extracts reaction solution, adds DMF600ml/ washing 6 times.Take by weighing TBTU22.15g, HOBt9.32g, DIEA8.92g, add the DMF stirring and dissolving, add cracked fragment II compound, be added to the interior stirring reaction of glass reaction post fully after the dissolving 6 hours.Sampling, with DMF washing 6 times, Kaiser Test detection reaction degree is finished until reaction.Take out dereaction liquid, with DMF600ml/ washing 6 times.
Embodiment 5: preparation fragment III-fragment II-fragment I-Rink Amide mbha resin
To add by the fragment III-CTC resin of embodiment 3 preparations in the glass reaction post, add the 1%TFA/DCM solution 360ml for preparing to the glass reaction post, stirring reaction 30min, collect reaction solution, add 360ml/ washing of 1%TFA/DCM 2 times, merge reaction solution, revolving inspissation contracts, obtain faint yellow oily thing, in water, precipitate, get white meal.
To add by the fragment II-fragment I-Rink Amide mbha resin of embodiment 4 preparations in the glass reaction post, add the 50% piperidines/DMF solution 600ml for preparing to the glass reaction post, stirring reaction 30min extracts reaction solution, adds DMF600ml/ washing 6 times.Take by weighing TBTU22.15g, HOBt9.32g, DIEA8.92g, add the DMF stirring and dissolving, add cracked fragment III compound, be added to the interior stirring reaction of glass reaction post fully after the dissolving 6 hours.Sampling, with DMF washing 6 times, Kaiser Test detection reaction degree is finished until reaction.Take out dereaction liquid, with DMF600ml/ washing 6 times.
Embodiment 6: cracking
To join in the round-bottomed flask by the peptide resin of embodiment 5 preparations, add lytic reagent (TFA: EDT: water=95: 2.5: the 2.5) 2400ml for preparing, stirring reaction 120min.Filter, resin washs 3 times with TFA.Merging filtrate slowly is deposited in the anhydrous diethyl ether of 24000ml.It is centrifugal to leave standstill behind the 2h beginning, and the centrifugal back that finishes is put into refrigerator overnight with 2700ml/ centrifuge washing of anhydrous diethyl ether 6 times with the thick peptide that obtains, and lyophilize promptly gets the tripro-amylin crude product.
Embodiment 7: purifying
Chromatographic column: Φ 15cm
Chromatographic column weighting agent: octadecylsilane chemically bonded silica (C18) 10 μ m
Applied sample amount :≤10g purpose peptide/time
Detect wavelength: 230nm
Flow velocity: 400ml/min
Moving phase: A:0.3% aqueous acetic acid; B: acetonitrile
Chromatographic condition: the gradient program sees the following form.
Will add behind the thick peptide drying under reduced pressure that obtain water 8800ml dissolving, solution is with the filtering with microporous membrane of 0.45 μ m.Filtrate is collected purpose peak solution according to sample on the purification condition, and with the solution concentration of collecting, lyophilize gets target product, and yield is 18.5%.
Embodiment 8: preparation tripro-amylin lyophilized injectable powder
0.60mg/ml tripro-amylin, 3%w/v N.F,USP MANNITOL, the 2.25mg/ml meta-cresol, regulating pH with acetic acid and sodium-acetate is 4.0, its preparation technology is as follows:
Under aseptic condition, take by weighing tripro-amylin 3g, N.F,USP MANNITOL 150g and 11.25g meta-cresol place sterilising vessel, add recipe quantity 80% water for injection, stir to make dissolving, transfer pH to 4.0 with acetic acid and sodium-acetate, add the injection water to 5000ml, stir evenly.Add 25g injection gac and stir 30min, the sterilization filter coarse filtration is taken off charcoal, with 0.45 μ m filtering with microporous membrane, uses 0.22 μ m filtering with microporous membrane at last, filtrate detect qualified after, can in 7ml sterilization cillin bottle (5ml/ bottle), vacuum freezedrying, the vacuum tamponade, outlet rolls lid.Make every bottle to be equivalent to the 3mg tripro-amylin.
Make 1000 bottles of tripro-amylin lyophilized injectable powders (containing tripro-amylin 3mg) by present embodiment,, its stability and clinical drug safety are investigated by stable accelerated test and animal blood vessels pungency, muscle irritation, haemolysis and supersensitivity experiment.
The stability accelerated test:
The a collection of trial-product that will go on the market respectively and to put into temperature by a batch sample of commercially available back be that 40 ± 2 ℃, relative humidity are that 75% ± 5% climatic chamber is investigated 0,1,2,3 and sampling and measuring 6 months the time, the results are shown in Table 8-1 and table 8-2 respectively.
Table 8-1 listing reference substance accelerated test result
Table 8-2 self-control sample accelerated test result
By showing 8-1 and table 8-2 as can be seen, investigate 6 months through accelerated test, the tripro-amylin lyophilized injectable powder of the present invention's preparation compares with the tripro-amylin injection liquid that has gone on the market, appearance luster, acidity, clarity of solution do not have considerable change, the impurity of the trial-product of listing increases, content descends obviously, show that the tripro-amylin lyophilized injectable powder that the present invention prepares can preserve under room temperature, stability increases.
Blood vessel irritation, muscle irritation, haemolysis and supersensitivity experiment:
Blood vessel irritation:
Choose 6 of the undamaged healthy rabbits of ears, left side auricular vein injection embodiment 8 solution 1ml, capacity 5% glucose solutions such as auris dextra injection, were injected 7 days continuously at every day 1 time.
During the injection, regularly observe the irritative response of auricular vein every day.Put to death rabbit on the 8th day, and got bilateral auricular vein and surrounding tissue, use formaldehyde fixed, do the conventional organization section at the proximal part of injection site, light microscopic is observed down and is had or not pathological change.Observation index and judging criterion see Table 8-3.
Table 8-3 blood vessel irritation scoring and judging criterion
The result shows that the pungency of rabbit auricular vein injection embodiment 8 solution compares no significant difference with 5% glucose solution.Inflammatory reactions such as the congestion of blood vessel, surrounding tissue oedema are not seen in visual inspection.Tissue slice checks, do not see that blood vessel structure is unusual, endothelial injury, thrombosis and other pathological change.The blood vessel of its naked eyes and om observation, the cumulative score of surrounding tissue show nonirritant all less than 0.5.
Muscle irritation:
Get 6 of healthy rabbits, injection embodiment 8 solution 1m l in every rabbit left side quadriceps muscle of thigh, inject with volume physiological saline on the right side.The injection back is observed injection site muscle and is had or not reactions such as hyperemia, oedema, and (the 3rd day) sacrificed by exsanguination behind the half animal 48h is vertically cut skin, and injection site, visual inspection both sides has or not reactions such as hyperemia, oedema, and gets its tissue and do pathologic finding.Then by the irritant reaction of showing this medicine of standard evaluation among the 8-4.Remaining animal continues to observe 14d, repeats aforesaid operations in the 18th day after the sacrificed by exsanguination, and judgement criteria sees Table 8-4.
Table 8-4 muscular irritation reaction evaluating standard
The result shows, after injecting embodiment 8 solution in the quadriceps muscle of thigh of rabbit left side, visual inspection injection site muscle does not have reactions such as hyperemia, oedema, and palpability irritant reaction such as tissue degeneratiaon or necrosis are not also seen in the pathological tissue inspection, compare no significant difference with the physiological saline side.
Sensitization to cavy:
Choose 6 of healthy guinea pigs, every abdominal injection embodiment 8 solution 0.5ml, the next day, inject 1 time, injects altogether 3 times.Be divided into 2 groups then at random, after the 1st administration 14 or 21 days respectively, quiet notes embodiment 8 solution 1ml.Observe cavy and have or not allergic symptoms such as excited uneasiness, expiratory dyspnea.
Two groups of cavys of result are all movable normal, do not see adnormal respiration etc.
External hemolytic test:
Prepare 2% rabbit red cell suspension.Get 7 in test tube, 8-5 adds various liquid by table.Each test tube is shaken up gently, put in 37 ℃ of waters bath with thermostatic control and hatch, observe 0.5,1,2,3,6 hour result.Red corpuscle agglutination in vitro and hemolytic judging criterion see Table 8-6.
The outer hemolytic test application of sample table of table 8-5 tripro-amylin solution body
Outer haemolysis of table 8-6 red cell body and agglutination test judging criterion
As a result, the distilled water control tube was complete hemolysis in 0.5 hour.Physiological saline and each tripro-amylin solution did not all have haemolysis in 6 hours.Jolting gently, the red corpuscle of physiological saline and each concentration tripro-amylin solution conduit bottom sediments all can disperse fully, shows that tripro-amylin solution does not have red cell agglutination.
Blood vessel irritation, muscle irritation, external hemolytic and supersensitivity experiment show that embodiment 8 solution do not have tangible pungency, supersensitivity, can not cause hemolytic reaction yet.Show that the tripro-amylin lyophilized injectable powder security that the present invention prepares is good, can use for clinical vein injection and intramuscular injection.
Embodiment 9: preparation tripro-amylin lyophilized injectable powder
1.00mg/ml tripro-amylin, 3%w/v N.F,USP MANNITOL, the 0.4%w/v trichloro-butyl alcohol, regulating pH with acetic acid and sodium-acetate is 4.0, its preparation technology is as follows:
Under aseptic condition, take by weighing tripro-amylin 1.5g, N.F,USP MANNITOL 45g and 6g trichloro-butyl alcohol place sterilising vessel, add recipe quantity 80% water for injection, stir to make dissolving, transfer pH to 4.0 with acetic acid and sodium-acetate, add the injection water to 1500ml, stir evenly.Add 25g injection gac and stir 30min, the sterilization filter coarse filtration is taken off charcoal, with 0.45 μ m filtering with microporous membrane, uses 0.22 μ m filtering with microporous membrane at last, filtrate detect qualified after, can in 3ml sterilization cillin bottle (1.5ml/ bottle), vacuum freezedrying, the vacuum tamponade, outlet rolls lid.Make every bottle to be equivalent to the 1.5mg tripro-amylin.
Make 1000 bottles of tripro-amylin lyophilized injectable powders (containing tripro-amylin 1.5mg) by present embodiment, by stable accelerated test and animal blood vessels pungency, muscle irritation, haemolysis and supersensitivity experiment, its stability and clinical drug safety are investigated.
The stability accelerated test:
The a collection of trial-product that will go on the market respectively and to put into temperature by a batch sample of commercially available back be that 40 ± 2 ℃, relative humidity are that 75% ± 5% climatic chamber is investigated 0,1,2,3 and sampling and measuring 6 months the time, the results are shown in Table 9-1 and table 9-2 respectively.
Table 9-1 listing reference substance accelerated test result
Table 9-2 self-control sample accelerated test result
By showing 9-1 and table 9-2 as can be seen, investigate 6 months through accelerated test, the tripro-amylin lyophilized injectable powder of the present invention's preparation compares with the tripro-amylin injection liquid that has gone on the market, appearance luster, acidity, clarity of solution do not have considerable change, the impurity of the trial-product of listing increases, content descends, show that the tripro-amylin lyophilized injectable powder that the present invention prepares can preserve under room temperature, stability increases.
Blood vessel irritation, muscle irritation, haemolysis and supersensitivity experiment:
Blood vessel irritation:
Choose 6 of the undamaged healthy rabbits of ears, left side auricular vein injection embodiment 9 solution 1ml, capacity 5% glucose solutions such as auris dextra injection, are injected 7 big continuously at every day 1 time.
During the injection, regularly observe the irritative response of auricular vein every day.Put to death rabbit on the 8th day, and got bilateral auricular vein and surrounding tissue, use formaldehyde fixed, do the conventional organization section at the injection site proximal part, light microscopic is observed down and is had or not pathological change.Observation index and judging criterion see Table 9-3.
Table 9-3 blood vessel irritation scoring and judging criterion
The result shows that the pungency of rabbit auricular vein injection embodiment 9 solution compares no significant difference with 5% glucose solution.Inflammatory reactions such as the congestion of blood vessel, surrounding tissue oedema are not seen in visual inspection.Tissue slice checks, do not see that blood vessel structure is unusual, endothelial injury, thrombosis and other pathological change.The blood vessel of its naked eyes and om observation, the cumulative score of surrounding tissue show nonirritant all less than 0.5.
Muscle irritation:
Get 6 of healthy rabbits, injection embodiment 9 solution 1ml in every rabbit left side quadriceps muscle of thigh, inject with volume physiological saline on the right side.The injection back is observed injection site muscle and is had or not reactions such as hyperemia, oedema, and (the 3rd day) sacrificed by exsanguination behind the half animal 48h is vertically cut skin, and injection site, visual inspection both sides has or not reactions such as hyperemia, oedema, and gets its tissue and do pathologic finding.Then by the irritant reaction of showing this medicine of standard evaluation among the 9-4.Remaining animal continues to observe 14d, repeats aforesaid operations in the 18th day after the sacrificed by exsanguination, and judgement criteria sees Table 9-4.
Table 9-4 muscular irritation reaction evaluating standard
The result shows, after injecting embodiment 9 solution in the quadriceps muscle of thigh of rabbit left side, visual inspection injection site muscle does not have reactions such as hyperemia, oedema, and palpability irritant reaction such as tissue degeneratiaon or necrosis are not also seen in the pathological tissue inspection, compare no significant difference with the physiological saline side.
Sensitization to cavy:
Choose 6 of healthy guinea pigs, every abdominal injection embodiment 9 solution 0.5ml, the next day, inject 1 time, injects altogether 3 times.Be divided into 2 groups then at random, after the 1st administration 14 or 21 days respectively, quiet notes embodiment 9 solution 1ml.Observe cavy and have or not allergic symptoms such as excited uneasiness, expiratory dyspnea.
Two groups of cavys of result are all movable normal, do not see adnormal respiration etc.
External hemolytic test:
Prepare 2% rabbit red cell suspension.Get 7 in test tube, 9-5 adds various liquid by table.Each test tube is shaken up gently, put in 37 ℃ of waters bath with thermostatic control and hatch, observe 0.5,1,2,3,6 hour result.Red corpuscle agglutination in vitro and hemolytic judging criterion see Table 9-6.
The outer hemolytic test application of sample table of table 9-5 tripro-amylin solution body
Outer haemolysis of table 9-6 red cell body and agglutination test judging criterion
As a result, the distilled water control tube was complete hemolysis in 0.5 hour.Physiological saline and each tripro-amylin solution did not all have haemolysis in 6 hours.Jolting gently, the red corpuscle of physiological saline and each concentration tripro-amylin solution conduit bottom sediments all can disperse fully, shows that tripro-amylin solution does not have red cell agglutination.
Blood vessel irritation, muscle irritation, external hemolytic and supersensitivity experiment show that embodiment 9 solution do not have tangible pungency, supersensitivity, can not cause hemolytic reaction yet.Show that the tripro-amylin lyophilized injectable powder security that the present invention prepares is good, can use for clinical vein injection and intramuscular injection.
Claims (10)
1. the method for a solid phase fragment synthesizing pramlintide, its fragment division methods is:
Its fragment I-Rink Amide mbha resin is:
29 37
Fmoc-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(tBu)-Tyr(tBu)-Rink
Amide?MBHA
Its fragment II-CTC resin is:
17 28
Fmoc-Val-His(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-Ile-Leu-Pro-
Chlorotrityl?Chloride
Its fragment III-CTC resin is:
2. the method for a solid phase fragment synthesizing pramlintide, its technical scheme may further comprise the steps:
1) with aminoresin be carrier, synthetic fragment I-aminoresin is AA
29-AA
37-Rink Amide mbha resin;
2) with the hydroxy resin be carrier, synthetic fragment II-hydroxy resin is AA
17-AA
28-CTC resin;
3) with the hydroxy resin be carrier, synthetic fragment III-hydroxy resin is AA
1-AA
16-CTC resin, and carry out solid phase cyclization;
4) preparation fragment II-fragment I-RinkAmide mbha resin;
5) preparation fragment III-fragment II-fragment I-Rink Amide mbha resin;
6) cutting obtains the thick peptide of tripro-amylin;
7) refining, obtain the pure product of tripro-amylin.
3. the described method of claim 2, the substitution degree of its Rink Amide mbha resin is 0.2～0.4mmol/g; The substitution degree of its 2-Chlorotrityl Chloride resin (CTC resin) is 0.8～1.2mmol/g; Its protection amino acid is respectively Fmoc-Tyr (tBu), Fmoc-Thr (tBu), Fmoc-Asn (Trt), Fmoc-Ser (tBu), Fmoc-Gly, Fmoc-Val, Fmoc-Pro, Fmoc-Leu, Fmoc-Ile, Fmoc-Phe, Fmoc-His (Trt), Fmoc-Ala, Fmoc-Arg (Pbf), Fmoc-Gln (Trt), Fmoc-Cys (Acm), Fmoc-Lys (Boc); Its condensation reagent is TBTU/HOBt/DIEA; The charging capacity of its protection amino acid and condensation reagent is 2～5 times of equivalents.
4. the described method of claim 2, its Synthetic 2, the cyclization reagent of the disulfide bridge bond of 7 two halfcystines is I
2, solvent is DMF.
5. claim 1 or require 2 described methods, its fragment II-fragment I-RinkAmide mbha resin is:
17 28
Fmoc-Val-His(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-Ile-Leu-Pro-
29 37
Pro-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(tBu)-Tyr(tBu)-Rink?Amide?MBHA
Its fragment III-fragment II-fragment I-Rink Amide mbha resin is:
6. the described method of claim 5, the lytic reagent of its CTC resin is 1%TFA/DCM; The lytic reagent of its Rink Amide mbha resin is by TFA: EDT: Water=95: 2.5: 2.5 volume ratio is formulated.
7. tripro-amylin lyophilized injectable powder is characterized in that described preparation is made up of main ingredient tripro-amylin, vehicle, fungistat and pH regulator agent; The preferred 0.1-20mg/ml of the concentration of its main ingredient tripro-amylin.
8. the described preparation of claim 7, its vehicle is selected from N.F,USP MANNITOL, lactose, glucose, sucrose, sodium-chlor, sorbyl alcohol etc.; Preferred 1-10%w/v N.F,USP MANNITOL and 1-10%w/v lactose.
9. the described preparation of claim 7, its sanitas is selected from meta-cresol, trichloro-butyl alcohol, phenylformic acid, Sorbic Acid, ethanol, glycerine, phenylcarbinol, calcium propionate, Sodium dehydroacetate, calcium propionate, sodium Diacetate, Sodium.alpha.-hydroxypropionate etc.; Preferred 0.1-50mg/ml meta-cresol and 0.03-2%w/v trichloro-butyl alcohol
10. the described preparation of claim 7, its pH value conditioning agent is selected from acetic acid, sodium-acetate, hydrochloric acid, sulfuric acid, lactic acid, oxysuccinic acid, Citric Acid, phosphoric acid, sodium hydroxide, yellow soda ash, sodium bicarbonate, Sodium phosphate dibasic etc., and regulating pH is between 2.0 to 6.0; Preferably transferring pH with acetic acid and sodium-acetate is 3.0-5.0.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006045603A1 (en) * | 2004-10-26 | 2006-05-04 | Lonza Ag | S-alkyl-sulphenyl protection groups in solid-phase synthesis |
| US20100081788A1 (en) * | 2008-09-03 | 2010-04-01 | Scinopharm Taiwan Ltd. | Process for the Preparation of Pramlintide |
| CN101790535A (en) * | 2007-06-29 | 2010-07-28 | 隆萨股份公司 | The method for preparing tripro-amylin |
| CN101824086A (en) * | 2009-03-04 | 2010-09-08 | 北京德众万全药物技术开发有限公司 | Preparation method of pramlintide |
| CN101822822A (en) * | 2009-03-04 | 2010-09-08 | 北京德众万全药物技术开发有限公司 | Drug composition of pramlintide and preparation method thereof |
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2010
- 2010-12-16 CN CN2010105945659A patent/CN102180943A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006045603A1 (en) * | 2004-10-26 | 2006-05-04 | Lonza Ag | S-alkyl-sulphenyl protection groups in solid-phase synthesis |
| CN101790535A (en) * | 2007-06-29 | 2010-07-28 | 隆萨股份公司 | The method for preparing tripro-amylin |
| US20100081788A1 (en) * | 2008-09-03 | 2010-04-01 | Scinopharm Taiwan Ltd. | Process for the Preparation of Pramlintide |
| CN101824086A (en) * | 2009-03-04 | 2010-09-08 | 北京德众万全药物技术开发有限公司 | Preparation method of pramlintide |
| CN101822822A (en) * | 2009-03-04 | 2010-09-08 | 北京德众万全药物技术开发有限公司 | Drug composition of pramlintide and preparation method thereof |
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Address after: 518057, room 2, building ten, Shenzhen biological incubation center, No. 412, Nanshan District hi tech, Shenzhen, Guangdong Applicant after: Shenzhen City Jianyuan Pharmaceutical Technology Co., Ltd. Address before: 518000, overseas student Pioneer Building, 29 South Ring Road, Nanshan hi tech Zone, Guangdong, Shenzhen 1702 Applicant before: Shenzhen City Jianyuan Pharmaceutical Technology Co., Ltd. |
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Application publication date: 20110914 |