CN103087179A - Long-acting glucagon-like peptide 1 (GLP-1) analogues and application thereof - Google Patents
Long-acting glucagon-like peptide 1 (GLP-1) analogues and application thereof Download PDFInfo
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Abstract
The invention relates to long-acting glucagon-like peptide 1 (GLP-1) analogues and a synthesis method thereof. GLP-1 analogues with longer pharmacological action time are obtained through modifying the 26th residue of natural GLP-1, the synthesis of target polypeptides is quickly realized through a microwave-promoted solid-phase synthesis method, and crude products are purified and freeze-dried to obtain the GLP-1 analogues.
Description
Technical field
The present invention relates to long-actingization glucagon-like-peptide-1 (GLP-1) analogue and application thereof.
Background technology
Diabetes are Chronic Non-Communicable Diseasess of the third-largest serious threat human health after tumour, cardiovascular disorder.At present, approximately there are 300,000,000 diabetic subjects in the whole world, expects 2025 and will increase to 500,000,000.2010, in state-owned 9,200 ten thousand diabetic subjects, China become diabetes the second big country that is only second to India, wherein diabetes B accounts for 90% of diabetic subject's total number of persons.Now the effective means for the treatment of diabetes B is insulin injection.Adopt clinically the method for insulin intensive treatment to delay the diabetes process, insulinize can reverse the pancreaticβ-cell functional lesion to a certain extent falling the hypoglycemic while.But use Regular Insulin hypoglycemic danger can occur.Be subject to after dosage size, injection site, injecting pathway, the injection of individual diversity XOR the impact of the factor such as not feed, if use Regular Insulin is careless slightly, serious hypoglycemia side effect will occur.
Glucagon-like-peptide-1 (GLP-1) is a kind of dependence on the glucose intestines blood sugar lowing polypeptide hormone, GLP-1 stimulates insulin secretion and hypoglycemia do not occur, the insulin secretion accelerating characteristic of this dependence on the glucose, avoided the danger of the normal generation hypoglycemia that exists in the treating diabetes, these physiological functions make exploitation GLP-1 have broad prospects as a kind of diabetes B medicine.
Glucagon-like-peptide-1 (GLP-1) is main by the secreted dependence on the glucose intestines blood sugar lowing polypeptide hormone of the L cell of end jejunum, ileum and colon, and multiple existence form is arranged in vivo.It is long-armed that the Proglucagon gene is positioned at No. 2 karyomit(e)s, is comprised of 6 exons and 5 introns, at pancreas and enteron aisle L cell inner expression, generates the Proglucagon (proglucagon, PG) that is comprised of 160 amino acid.The product that Proglucagon transforms after cracking in pancreas and enteron aisle is different.PG mainly is cracked in enteron aisle: enteroglucagon (Glicentin:PG1～69), enteroglucagon molecule continue to be cracked into GRPP (PG1～30) and oxyntomodulin (Oxyntomodulin:PG33～69); Insert peptide-2 (IP-2:PG111～123); Glucagon-like-peptide-2 (GLP-2:PG126～158); And GLP-1 (1～37)-OH (PG 72～108).GLP-1 (1～37)-OH is the peptide chain of non-activity, need enzymolysis excision N end 6 peptides, become GLP-1 (7～37) with physiologically active-OH, its C-terminal glycine can be used as the substrate of amidating enzyme, so namely generates GLP-1 (7～36) with high activity-NH after the C-terminal amidation of GLP-1 (7～37)-OH
2, aminoacid sequence is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH
2The aminoacid sequence of GLP-1 (7～37)-OH is HAEGTFTSDVSSLEGQAAKEFIAWLVKGRG-COOH.GLP-1 (7～36)-NH
2Be the main natural form of GLP-1 in human body, account for 80%, it promotes that acting in the GLP-1 peptide of insulin secretion is the strongest.GLP-1 (7～37)-OH accounts for 20% in addition, and both have identical physiological function.
GLP-1 promotes the secretion of Regular Insulin by acting on the acceptor GLP-1 acceptor (GLP-1R) on the pancreaticβ-cell film.
GLP-1R highly expresses on pancreas beta cell film, is comprised of 463 amino acid, belongs to the G-protein linked receptor family of seven cross-films, is combined with the GLP-1 high degree of specificity.Can increase the activity of islet cells adenylate cyclase after GLP-1 and its receptors bind, the increase of the second messenger cAMP in irritation cell causes cytolemma K
+Pathway closure, cell depolarization brings out the Ca of voltage-dependent
2+Channel opener, extracellular Ca
2+Interior stream, cytoplasm Ca
2+Concentration raises and triggers the release of Regular Insulin.The cAMP level raises in addition, activates again protein kinase A and Starch phosphorylase that cAMP relies on, and then stimulates transcribing and translating of beta cell insulin gene, stimulates increment and the differentiation of beta cell.
Glucagon-like-peptide-1 (GLP-1) has the various biological effect.As follows:
1, has the dependent incretin secretion of blood sugar;
2, stop the pancreas beta cell to be degenerated, stimulate increment and the differentiation of beta cell;
3, induce transcribing of proinsulin gene, promote the biosynthesizing of proinsulin;
4, increase the susceptibility of Regular Insulin;
5, increase Somatostatin Secretion, the generation of glucagon suppression (this effect is also the blood sugar dependency);
6, gastric acid secretion inhibiting postpones stomach emptying;
7, by acting on the maincenter depress appetite of hypothalamus, reduce the effects such as food intake.
Yet although natural GLP-1 has above plurality of advantages on the treatment diabetes, it in vivo can be by DPP IV (dipeptidyl peptidase IV, DPP-IV) fast degradation.But N-terminal second L-Ala (Ala) residue of DPP-IV specific recognition GLP-1 excises dipeptides from the 2nd L-Ala of peptide chain N-terminal (Ala), makes its form that changes non-activity into, and its Half-life in vivo is only about 5 minutes.GLP-1 molecule N end is and the combining site of GLP-1 acceptor that its histidine residues is lost, and causes GLP-1 to lose biological activity fully.Natural GLP-1 can be filtered metabolism fast by kidney in addition, so this needs us that natural GLP-1 is transformed, searches out with expectation and can resist the DPP-IV degraded, avoids kidney to filter fast the GLP-1 analogue of metabolism.
Here, we have designed a class GLP-1 analogue, by introducing the small molecules group, increase peptide chain and sero-abluminous combination, have avoided the kidney of GLP-1 to filter fast and the metabolism inactivation, thus the blood sugar reducing function time in significant prolongation transformation period and body.
Summary of the invention
The present invention relates to glucagon element sample peptide-1 (GLP-1) analogue, its sequence is:
Wherein:
Xaa:Ala, Leu, Val, Met, Ile, Tyr, Phe, Arg, Asn, Lys, Thr, Asp, His, Trp, Gln, Glu, Ser or Gly;
N is selected from 1～20.
In one embodiment, the present invention relates to have the GLP-1 analogue of following sequence:
In one embodiment, the present invention relates to have the GLP-1 analogue of following sequence:
In one embodiment, the present invention relates to have the GLP-1 analogue of following sequence:
In one embodiment, the present invention relates to have the GLP-1 analogue of following sequence:
The present invention also provides a kind of pharmaceutical composition, comprises at least a above-claimed cpd and its pharmacy acceptable salt for the treatment of significant quantity, or pharmaceutically acceptable carrier or thinner.
The present invention further provides and stated compound and its pharmacy acceptable salt, or pharmaceutically acceptable carrier or thinner are for the preparation of the utilization in the medicine of diabetes.
Above-claimed cpd stable chemical nature provided by the invention, be difficult for DPP IV (DPP-IV) degraded in body, more than all compounds has reached 42h plasma half-life, increase significantly than prototype GLP-1 (transformation period 2min), must instil or continue the defective that subcutaneous injection could produce curative effect by during continuous intravenous infusion thereby overcome GLP-1.In addition, for reducing in body during blood sugar concentration, existing very long plasma half-life, (more than 42 hours), had again significant hypoglycemic effect as the pharmaceutical composition of effective constituent preparation for above-claimed cpd provided by the invention or compound.
The present invention also provides the preparation method of above-claimed cpd, and the present invention adopts microwave to promote Fmoc/tBu orthogonally protect solid phase synthesis strategy efficiently to synthesize rapidly and obtains above-mentioned target compound.
Below hypoglycemic pharmacological experimental method and result in the body of glucagon-like-peptide-1 (GLP-1) analogue that relates in the present invention:
(1) the next day hypoglycemic experiment of GLP-1 and glucagon-like-peptide-1 (GLP-1) analogue
Normal kunming mice is divided into 8 groups, and 6 every group, mouse is raised in the stdn Animal House.During the experiment beginning, 24h gives GLP-1 and GLP-1 analogue, control group injecting normal saline in advance.Normal diet drinking-water 12h, then fasting 12h, after compound injection 24h, carry out the abdominal cavity glucose tolerance experiment of mouse single.Each group is decided to be 0min according to the glucose solution (concentration 20%) of every kilogram of abdominal injection 18mmol of Mouse Weight during injectable dextrose monohydrate, measure glucose level at 0,15,30,45,60,120min with blood glucose meter.
Hypoglycemic effect next day of table 1 GLP-1 and glucagon-like-peptide-1 (GLP-1) analogue
n＝6，
*P＜0.05，
**P＜0.01，
***P＜0.001vs?saline?as?control
As shown in table 1, because glucagon-like-peptide-1 (GLP-1) analogue after modifying has resistance to enzymolysis and anti-kidney filtration, more than all having reached 42h its biological half-life, so in vivo after metabolism 24h, hypoglycemic experiment shows that its promoting insulin secretion still keeps, and lose activity already without the natural GLP-1 of transformation, illustrate that our interior blood sugar reducing function time of GLP-1 similar object extends significantly.
(2) the repeatedly abdominal cavity glucose tolerance experiment of GLP-1 and glucagon-like-peptide-1 (GLP-1) analogue
Choose 8 age in week the db/db diabetic mice, random packet, 8 every group, adaptability was raised after 7 days, in experiment beginning fasting in front 12 hours, only drank water.Every group of mouse surveyed initial blood glucose value before giving GLP-1 and GLP-1 analogue, be decided to be-30min, then the abdominal cavity gives the GLP-1 analogue, 30min pneumoretroperitoneum injectable dextrose monohydrate (every kilogram of 18mmol) is decided to be 0min, physiological saline and the GLP-1 of control group injection equal volume.0min, 15min, 30min, 60min, 90min, 120min afterbody respectively get blood, measure blood glucose concentration.And in 180min, 360min, 540min, 720min, 900min, 1080min, 1260min is injectable dextrose monohydrate again, continues to measure blood glucose value, repeats altogether to give glucose 8 times, the long-actingization hypoglycemic activity of detection compound.
Table 2GLP-1 and glucagon-like-peptide-1 (GLP-1) analogue be abdominal cavity glucose tolerance effect repeatedly
n＝8，
*P＜0.05，
**P＜0.01，
***P＜0.001vs?saline?as?control
As shown in table 2, GLP-1 has lost activity when abdominal cavity sugar tolerance experiment for the first time, and all GLP-1 analogues (pass by 22h apart from giving compound this moment) when giving glucose the 8th time, appoint and so kept hypoglycemic activity preferably, after illustrating that compound is through transformation, its long-actingization blood sugar reducing function is remarkable, more than having reached 24h.
The invention has the advantages that:
1. a kind of glucagon-like-peptide-1 (GLP-1) analogue that proposes can be on the basis that keeps hypoglycemic activity, have anti-kidney and filter elimination and anti-DPP-IV enzymolysis, biological half-life is than GLP-1 prototype significant prolongation, all reached more than 42 hours, improve stability, extended the blood sugar reducing function time.
2. microwave promotes glucagon-like-peptide-1 (GLP-1) analogue of solid phase synthesis to improve greatly coupled reaction speed, amino acid of the conventional abundant coupling of solid phase synthesis process goes to resin, often needed do not wait by 20 hours in 2 hours, even longer.Microwave promotes that average needs about 10 minutes; conventional solid phase synthesis process takes off the Fmoc protecting group, often needs do not wait by 1 hour in 30 minutes, and microwave promotes that average needs about 5 minutes; this has improved the synthetic efficient of polypeptide greatly, has shortened synthesis cycle.
3. the crude product purity of microwave promotion solid phase synthesis glucagon-like-peptide-1 (GLP-1) analogue is greater than 80%, and more conventional solid phase synthesis process improves greatly, and this has facilitated follow-up purifying work.
4. microwave promotes synthetic glucagon-like-peptide-1 (GLP-1) analogue of solid phase method, and its cost is low, because coupling efficiency is higher, required protected amino acid is average only need 2 times excessive, more conventional solid phase synthesis process needs 4 to 5 times excessively greatly to reduce.
5. microwave promotes that the method for solid phase synthesis glucagon-like-peptide-1 (GLP-1) analogue easily is automated, large-scale, and this makes it be more suitable for suitability for industrialized production.
Therefore promote with microwave provided by the invention glucagon-like-peptide-1 (GLP-1) analogue that solid phase synthesis technique prepares, yield is high, synthesis cycle is short, purifying crude is easy, and production cost is low, be easy to industrial automation production.The glucagon-like-peptide-1 for preparing (GLP-1) analogue, more stable than natural GLP-1, the hypoglycemic activity time is long, is suitable as the activeconstituents for the treatment of diabetes medicament.
Description of drawings
Above the present invention has been done general description, below accompanying drawing be used for illustrating specific embodiments of the present invention.Wherein:
That Fig. 1 shows is GLP-1 (7～36)-NH
2Incubate the HPLC analysis of spectra of 0h with DPP IV temperature;
That Fig. 2 shows is GLP-1 (7～36)-NH
2Incubate 4h with the human plasma temperature and be degraded into GLP-1 (9～36)-NH
2The HPLC analysis of spectra;
What Fig. 3 showed is the HPLC analysis of spectra that GLP-1 analogue of the present invention and DPP-IV temperature are incubated 4h;
What Fig. 4 showed is that prototype GLP-1 and GLP-1 analogue blood plasma temperature of the present invention are incubated the degraded figure after 96 hours;
Embodiment
Adopt in this specification following abbreviation:
Et
3N: triethylamine; The NMM:N-methylmorpholine; DIEA:N, N '-diisopropylethylamine; DMF: dimethyl formamide; DMSO: methyl-sulphoxide; DCM: methylene dichloride; The Fmoc:N-9-fluorenylmethyloxycarbonyl; DIC:N, N '-DIC; CDI:N, N '-carbonyl dimidazoles; The DMAP:4-Dimethylamino pyridine; The HOSU:N-N-Hydroxysuccinimide; EDC.HCl:1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; HATU:2-(7-azo benzotriazole)-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester; HBTU: benzotriazole-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester; HCTU:6-Chloro-Benzotriazole-1,1,3,3-tetramethyl-urea phosphofluoric acid ester; HOAT:1-hydroxyl-7-azo benzotriazole; HOBT:1-hydroxyl-benzotriazole; PyBOP: phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus; HPLC: high performance liquid chromatography; ESI-MS: electrospray ionization mass spectrum; Gly: glycine; Ser: Serine; Ala: L-Ala; Thr: Threonine; Val: α-amino-isovaleric acid; Ile: Isoleucine; Leu: leucine; Tyr: tyrosine; Phe: phenylalanine; His: Histidine; Pro: proline(Pro); Asp: aspartic acid; Met: methionine(Met); Glu: L-glutamic acid; Trp: tryptophane; Lys: Methionin; Arg: arginine.Asn: l-asparagine; Gln: glutamine.
The present invention describes by the following example, but these embodiment do not do any restriction explanation of the present invention.
Microwave promote solid phase synthesis
(1) it is synthetic that side chain changes the halfcystine of structure
Take Fmoc-Cys-OH 0.21g, be dissolved in DCM, add 0.29g 3,3 '-(4-(6-dimaleoyl imino propionamido-) α-tolylene)-two-4 hydroxy coumarin, 4ul DIEA is as catalyzer, and under room temperature, stirring reaction is 4 hours, after the thin layer plate monitoring reaction is complete, reaction solution concentrating under reduced pressure, column chromatography for separation get product 0.4g, yield 74%.
MS(70eV)m/z：986.5([M+Na]
+)。
(2) swelling of resin
Take Fmoc-Rink amide-MBHA Resin 50mg (replacement amount 0.4mmol/g), through 7mL DCM swelling 30min, suction filtration removes DCM, then uses 10mLNMP swelling 30min, uses respectively at last NMP, DCM, and NMP 7mL rinses well.
(3) microwave promotes removing of Fmoc protecting group
The resin that swelling is good is put into reactor, add 7mL to contain 25% piperidines of 0.1M HOBT/NMP (V/V) solution, react 1min in microwave reactor, microwave power is 15W, temperature of reaction is controlled in 50 ℃, use the air compressor pressure-air cooling, elimination solution after reaction finishes; Add 7mL to contain 25% piperidines of 0.1M HOBT/NMP (V/V) solution again and react 4min again in microwave reactor, microwave power is 25W, and temperature of reaction is controlled at 50 ℃, uses the air compressor pressure-air cooling.Elimination solution after reaction finishes is used the NMP washes clean.Obtain sloughing the resin of the Fmoc protecting group of initial connection.
(4) microwave promotes synthesizing of Fmoc-Arg (Pbf)-Rink amide-MBHA Resin
With Fmoc-Arg (Pbf)-OH (0.04mmol), HBTU (0.04mmol), HOBT (0.04mmol) and DIPEA (0.08mmol) are dissolved in 10mL NMP, again this solution is added in top resin, react 7min in microwave reactor, microwave power is 25W, and temperature of reaction is controlled at 50 ℃, uses the air compressor pressure-air cooling.Filtering reaction solution after reaction finishes is used DCM and each 7mL washing resin of NMP 3 times.
(5) detection of coupling efficiency
With the coupling efficiency of ninhydrin method or bromjophenol blue method qualitative detection resin, color reaction is negative can enter next coupling circulation.
Ninhydrin method: the resin particle washing with alcohol takes a morsel, put into transparent bottle and add respectively 2 of 5% triketohydrindene hydrate ethanol, KCN pyridine solution (2ml 0.001M KCN is diluted in the 98ml pyridine), 80% phenol ethanolic solns, in 100 ℃ of heating 5 minutes, if the aobvious blueness of resin is namely positive.
The bromjophenol blue method: the resin particle that takes a morsel washs with two formyl ethanamides, puts into the tetrabromophenol sulfonphthalein dimethylacetamide solution that transparent bottle adds 3 1%, and under normal temperature, jolting is 3 minutes, if the aobvious blueness of resin is namely positive.
(6) prolongation of peptide chain
Sequence according to peptide chain; repeating above-mentioned deprotection is connected step and connects successively upper corresponding amino acid with coupling; when being synthesized to 26 of peptide chains; add side chain to change the halfcystine of structure; coupling 45min; then continue to repeat deprotection and be connected with coupling that step connects upper corresponding amino acid successively until peptide chain is synthetic complete, obtain being connected with the resin of compound.
(7) cracking of polypeptide on resin
The resin that is connected with compound obtained above is put into reaction flask, and (TFA/ thioanisole/water/phenol/EDT, 82.5: 5: 5: 5: 2.5, V/V) 10mL first at 0 ℃ of lower jolting 30min, then reacted 3h at normal temperatures respectively to add cracking agent Reagent K.Suction filtration after reaction finishes adds a small amount of TFA and DCM washing three times, merging filtrate.Filtrate is added in a large amount of ice ether separate out white flocks, frozen centrifugation obtains the crude product of target polypeptides.Finally obtain the crude product 63.2mg of compound, yield is 94.3%.
(7) purifying of polypeptide
The crude product polypeptide is dissolved in 50% acetonitrile/water, uses the preparative liquid chromatography purifying, chromatographic condition is: C18 reversed-phase column (320mm * 28mm, 5 μ m); Mobile phase A: 0.1%TFA/ water (V/V), Mobile phase B: 0.1%TFA/ acetonitrile (V/V); Eluent gradient: Mobile phase B 40%～90%, 20min; Flow velocity is that 6mL/min detection wavelength is 214nm.The solution freeze-drying of collecting gets sterling 30mg.Theoretical relative molecular mass is 3892.8.ESI-MS?m/z：found[M+4H]
4+974.2，[M+5H]
5+779.6；calu[M+4H]
4+973.9，[M+5H]
5+779.1。
Embodiment 2～4
Method described according to embodiment 1, glucagon-like-peptide-1 (GLP-1) analogue that obtain embodiment 2～4 synthetic according to corresponding sequence is by electrospray ionization mass spectrum (ESI-MS) conclusive evidence molecular weight separately.
Embodiment 2
Theoretical relative molecular mass is 3879.8.ESI-MS?m/z：found[M+4H]
4+970.9，[M+5H]
5+776.9；calu[M+4H]
4+970.3，[M+5H]
5+776.2
Embodiment 3
Theoretical relative molecular mass is 3976.9.ESI-MS?m/z：found[M+4H]
4+995.2，[M+5H]
5+796.4；calu[M+4H]
4+994.9，[M+5H]
5+796.1。
Theoretical relative molecular mass is 3963.8.ESI-MS?m/z：found[M+4H]
4+991.9，[M+5H]
5+793.7；calu[M+4H]
4+991.2，[M+5H]
5+793.2。
Embodiment 5
The stability experiment of GLP-1 and glucagon-like-peptide-1 (GLP-1) analogue to DPP-IV
Be in the Tris-HCL buffered soln of 50mM through the GLP-1 after purifying and the DPP IV of glucagon-like-peptide-1 (GLP-1) analogue 5nmol and 5mU in 200 μ L concentration, 37 ℃ of temperature are incubated 4h, and pH 7.4.The acetonitrile/water solution termination reaction that adds at last 10 μ L20%.Get respectively 0h, the temperature that 4h is ordered is incubated solution, and is centrifugal, gets supernatant liquor, advances HPLC and analyzes; The post tail is collected degraded product GLP-1 (9～36)-NH
2Analyze and adopt C18 reversed-phase column (150mm * 4.6mm, 5 μ m); Mobile phase A: 0.1%TFA/ water (V/V), Mobile phase B: 0.1%TFA/ acetonitrile (V/V); Eluent gradient: Mobile phase B 10%～45%, 22min; Flow velocity is 1mL/min; Column temperature is 40 ℃; The detection wavelength is 214nm.
As Fig. 1, shown in Figure 2, result shows natural GLP-1 without transformation after incubating 4h with the DPP-IV temperature, basically all is hydrolyzed to the GLP-1 (9～36) of non-activity-NH
2, complete peptide chain is less than 10%.And Fig. 3 shows and still keeps prototype after glucagon-like-peptide-1 (GLP-1) analogue and DPP-IV temperature are incubated 4h, and there are no degraded, complete peptide chain is greater than 95%.Result shows that glucagon-like-peptide-1 (GLP-1) analogue that we design has the enzymolysis of resisting DPP-IV.
Embodiment 6
Determination experiment plasma half-life of GLP-1 and glucagon-like-peptide-1 (GLP-1) analogue
Rat eye is got blood, and blood is packed into and contained in the centrifuge tube of heparin, and centrifugal 10 minutes of 3000rpm gets supernatant blood plasma and incubates blood plasma as temperature, utilizes LC-MS to come the response signal of detection compound.The blood plasma of the GLP-1 of 100ul and GLP-1 analogue solution and 100ul is inserted after vortex mixed in 37 ℃ of water-baths, and temperature was incubated 96 hours, 0,0.5,1,2,4,8,12,24,36,48,72,96h time point is got 10ul, add the 20ul acetonitrile precipitation, 14000rpm is centrifugal, gets supernatant liquor and advances LC-MS, calculate the peak area of each time point, make extinction curve, calculate the transformation period.As shown in Figure 4, through the transformation the prototype GLP-1 transformation period do not only have 15min, and the transformation period of GLP-1 analogue all more than 42 hours, the longest transformation period has reached 62 hours.
Claims (7)
1. glucagon-like-peptide-1 (GLP-1) analogue that contains formula I (SEQ.ID NO:1) structure, its sequence is:
Wherein:
Xaa:Ala, Leu, Val, Met, Ile, Tyr, Phe, Arg, Asn, Lys, Thr, Asp, His, Trp, Gln, Glu, Ser or Gly;
N is selected from 1～20.
2. glucagon-like-peptide-1 according to claim 1 (GLP-1) analogue, preferred sequence is:
3. a pharmaceutical composition, comprise glucagon-like-peptide-1 (GLP-1) analogue described at least a claim 1 for the treatment of significant quantity and its pharmacy acceptable salt.
4. a pharmaceutical composition, comprise glucagon-like-peptide-1 (GLP-1) analogue and pharmaceutically acceptable carrier or the thinner described at least a claim 1 for the treatment of significant quantity.
5. the glucagon-like-peptide-1 described in claim 1 (GLP-1) analogue and its pharmacy acceptable salt are for the preparation of the utilization in the medicine of diabetes.
6. (GLP-1) analogue of the glucagon-like-peptide-1 described in claim 1 and pharmaceutically acceptable carrier or thinner are for the preparation of the utilization in the medicine of diabetes.
7. the preparation method of the glucagon-like-peptide-1 described in claim 1 (GLP-1) analogue comprises biological expression, liquid phase is synthetic and solid phase synthesis preparation method thereof.
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| CN118465154A (en) * | 2024-07-12 | 2024-08-09 | 军科正源（北京）药物研究有限责任公司 | Analysis method for detecting benalundin and plasma sample treatment method thereof |
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Application publication date: 20130508 |