HK1095045B - Modified exendins and uses thereof - Google Patents
Modified exendins and uses thereof Download PDFInfo
- Publication number
- HK1095045B HK1095045B HK07102543.1A HK07102543A HK1095045B HK 1095045 B HK1095045 B HK 1095045B HK 07102543 A HK07102543 A HK 07102543A HK 1095045 B HK1095045 B HK 1095045B
- Authority
- HK
- Hong Kong
- Prior art keywords
- glu
- gly
- ser
- ala
- pro
- Prior art date
Links
Abstract
Provided are new Exendin-4 and its polyethylene glycol derivatives modified at one or more of the following positions: 2, 14, 27, 28. These compounds are used as GLP-1 receptor agonists in the treatment of type II diabetes.
Description
Technical Field
The invention relates to a long-acting insulinotropic Peptide derivative and a pharmaceutically acceptable salt thereof, in particular to an insulinotropic Peptide compound modified by polyethylene glycol and a pharmaceutically acceptable salt thereof, and also relates to a preparation method thereof, and application of the compound in preventing and treating type II diabetes by acting on Glucagon-like Peptide-1 (GLP-1) receptors to stimulate beta-cells to secrete insulin to regulate blood sugar.
Technical Field
In recent years, with the improvement of living standard, the modernization of life style and the aging of society, the prevalence of diabetes is increasing year by year around the world, and particularly, the rising of developing countries that are getting from poverty to richness is more obvious. Diabetes mellitus is a third serious chronic non-infectious disease after tumor and cardiovascular and cerebrovascular diseases, and is one of the main causes of death and disability. In 1997 WHO reported that there were 1.35 million diabetics worldwide and an estimated 1.75 million people by 2000. The latest survey report in China shows that the prevalence rate of diabetes in natural population over 20 years old is 3.21%, and the initial estimation shows that Chinese diabetes patients are at least over 2000 ten thousand, wherein more than 95% of the Chinese diabetes patients are II diabetes patients. The cost of spending in the united states, directly or indirectly for diabetes, increased from $ 10 million to $ 920 million each year from 1987 to 1992 on a statistical basis. The cost of diabetes mellitus in China has also increased at a surprising rate in recent years, and from the statistical analysis of the relevant data in 1993, the cost of direct treatment for diabetes mellitus in the year is as high as 22.16 billion yuan, and the cost does not include the cost of treatment of complications caused by diabetes mellitus, the cost of treatment and health care outside hospitals and indirect socioeconomic loss.
Current methods of managing type II diabetes include diet modification, exercise attention, and drug administration to regulate blood glucose levels. The most commonly used drugs include insulin, sulfonylureas, biguanides and glitazones. These drugs emphasize that they promote the normalization of blood sugar in vivo and fail to correct complications caused by diabetes, especially damage to the kidney, cardiovascular system, vision and nervous system. These complications are directly related to the increased mortality caused by diabetes. The major side effects of the first generation of diabetes-treating drugs included hypoglycemia, weight gain, and edema. The mechanisms of action of these drugs may differ, but none of them has the effect of protecting beta-cells having the function of secreting insulin, and thus it is impossible to maintain normal blood glucose metabolism and endocrine regulation in vivo. In many cases, the single drug is used and loses effect slowly, so that a compound treatment method is forced, and patients often use the blood pressure-lowering and cholesterol-lowering drugs at the same time, so that the long-term effects of the scheme are different. Therefore, research and development of new drugs for controlling blood sugar, together with the current drugs, emphasize the functions of protecting and repairing beta cells, and regulating the response of the endocrine system to food intake, will revolutionize and advance the treatment of diabetes.
The research prospect in the aspect of the agonist aiming at the glucagon-like peptide-1 (GLP-1) receptor is considerable, and the research and development in the field are possible to open up a new chapter in the field of treating the type II diabetes. Glucagon-like peptide-1 was found in 1984 to be an enteroendocrine hormone. Blood glucose levels can be regulated to normal levels if a type II diabetic is administered this hormone by infusion (Nathan, DM, et al diabetes Care 1992; 15: 270-6; Zander, M, et al Lancet 2002; 359: 824-30). Research shows that the action of glucagon-like peptide and its receptor agonist is mainly caused by activating glucagon analogue-1 receptor on pancreatic beta-cell surface to secrete insulin. Because this effect is determined by the level of intrinsic blood glucose concentration in the body, it does not produce hypoglycemic shock which can cause severe hypoglycemia and thus life-threatening hypoglycemia shock even in the presence of glucagon-like peptide-1 and its receptor agonists, as in conventional drugs. Specifically, glucagon-like peptide-1 can have a significant insulin secretion promoting effect when the blood glucose concentration in vivo is higher than 6mmol/L, and no longer acts when the blood glucose level in vivo approaches a normal level. In addition, such agonists also have the effect of stimulating the growth of beta-cells of the islets of Langerhans of rodents (rats) and increasing the tissue of the beta-cells. This repair of islet beta-cell function offers the prospect of curing type II diabetes, at least delaying the progression from type II to type I. Furthermore, glucagon-like peptide-1 and its receptor agonists simultaneously inhibit glucagon secretion and thus reduce the potential for hepatic glucose output. More significantly, such agonists effectively inhibit gastrointestinal motility and gastric emptying resulting in reduced food intake and weight loss. This helps to control the weight of type II diabetics.
Disclosure of Invention
The invention aims to provide a long-acting polyethylene glycol modified insulinotropic peptide compound and a pharmaceutically acceptable salt thereof. They have the effects of activating glucagon-like peptide-1 (GLP-1) receptor to promote insulin secretion and reduce blood sugar, and thus can be used for treating type II diabetes or preventing type II diabetes. The compound has longer stay time in vivo to play a role, not only the elimination time of the compound through the kidney is delayed after the compound is modified by polyethylene glycol, but also the polypeptide skeleton has better enzyme-mediated and chemical stability in vivo, thereby ensuring the long-acting effect brought by the modification of the polyethylene glycol, reducing the injection and use times of patients, really facilitating the patients and improving the treatment quality and benefit.
In particular, the present invention relates to all of the precursor polypeptides listed in the sequence listing as modified by polyethylene glycol, as well as compounds formed after modification with polyethylene glycol of different molecular weights and their pharmaceutically acceptable salts.
Another object of the present invention is to provide a method for preparing a long-acting insulinotropic peptide compound modified with polyethylene glycol and a pharmaceutically acceptable salt thereof.
The invention also aims to provide the application of the long-acting insulinotropic peptide derivative and/or the pharmaceutically acceptable salt as a glucagon-like peptide-1 (GLP-1) receptor agonist in preventing and treating type II diabetes.
The invention aims to achieve the following technical scheme, and relates to insulinotropic hormone secretion peptides and pharmaceutically acceptable salts thereof, wherein the polypeptide skeleton of the insulinotropic hormone secretion peptides has optimized in-vivo enzyme-mediated and chemical stability. In particular, the present invention relates to an insulinotropic peptide comprising (A) an amino acid sequence represented by SEQ ID NO. 4 to 141, and (B) an amino acid sequence having substantially the same sequence as the amino acid sequence represented by SEQ ID NO. 4 to 141.
The invention also relates to the insulinotropic peptide and the medicinal salt thereof which are obtained by modifying and deriving one or more than one polyethylene glycol in the amino acid sequence of the insulinotropic peptide shown in the sequence numbers 4 to 141, particularly in the 2 position, the 14 position, the 27 position and the 28 position, wherein the molecular weight of the polyethylene glycol is 5,000-80,000, and preferably 20,000-60,000. The amino acid compounds of these insulinotropic peptides of the present invention have key modification sites including the amino acid sequence of insulinotropic peptide at positions 2, 14, 27 and 28.
The invention also relates to a method for preparing the insulinotropic hormone secretion peptide and the medicinal salt thereof, which comprises a solid phase and liquid phase synthesis method, a purification method through reversed phase high performance liquid phase, ion exchange and gel filtration, and freeze drying.
The invention further provides application of the polyethylene glycol modified insulinotropic peptide derivative and the pharmaceutically acceptable salt thereof in treating and/or preventing type II diabetes.
Clinical experimental data indicate that fasting plasma glucose levels return to normal when treated with glucagon-like peptide-1 (GLP-1) in type II diabetics with poorly controlled plasma glucose levels (Gutniak, et al, New Eng.J.Med.326: 1316-. The long-term use of glucagon-like peptide-1 (GLP-1) enables normal human beta-cell function (Rachman, et al, Diabetes 45: 1524-. Glucagon-like peptide-1 (GLP-1) is able to respond to improved glucose function in beta cells of patients with impaired glucose tolerance (Byrne, et al., Diabetes 47: 1259-1265, 1998). However, glucagon-like peptide-1 (GLP-1) is easily destroyed and inactivated by a dipeptidyl endopeptidase (DPP IV) in vivo, and at the same time, glucagon-like peptide-1 (GLP-1) has a cleavage point by other endopeptidases (such as NEP24.11), so that the action of glucagon-like peptide-1 (GLP-1) in vivo is short-lived. Only by the continuous administration method can the real therapeutic effect be achieved. Many laboratories are therefore concerned with the development of more stable glucagon-like peptide-1 (GLP-1) receptor agonists, such compounds being formed primarily by modification of glucagon-like peptide-1 (GLP-1) itself. Significantly, Exendin-4 was isolated by Eng et al in the late 80 s, early 90 s from the salivary excretory organs of the Indiana (Gila Monster, Heloderma Sespecum) in the southwest U.S.A. (Eng, J., et al, J.biol. chem., 265: 20259-62, 1990, Eng, J., et al, J.biol. chem., 267: 7402-05, 1992). Exendin-4 is a 39 amino acid polypeptide that shares 53% homology with glucagon-like peptide-1 (GLP-1). It has affinity with glucagon-like peptide-1 (GLP-1) receptor and its ability is stronger than glucagon-like peptide-1 (GLP-1) itself. Exendin-4 has a better ability to regulate sugar metabolism in vivo than glucagon-like peptide-1 (GLP-1), requires a lower concentration to produce the insulinotropic effect than glucagon-like peptide-1 (GLP-1), and has a longer half-life in vivo than glucagon-like peptide-1 (GLP-1) (Kudsen, L.B.J.Med.chem.47: 4128) 4134, 2004). This is mainly due to the unique enzyme-mediated stability of Exendin-4, which is mainly due to the elimination of the cleavage site of endopeptidases such as NEP 24.11.
At present, a great deal of literature reports compounds having glucagon-like peptide-1 (GLP-1) receptor agonist functions, such as GLP-1(7-36), GLP-1(7-37), Exendin-4 and other GLP-1 and Exendin-4 derivatives. These documents include WO98/43658, WO00/15224, WO00/66629, WO01/98331, WO01/04156, US Pateat No.5,545,618, US Patent No.5,118, WO03/058203, US Patent application Serial No.60/395,738, WO04/022004 and the references cited therein, etc.
The naturally occurring GLP-1 receptor agonists are controlled as follows
| Name of peptide | Sequence of | SEQ ID NO |
| GLP-1(7-36) | HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2 | 1 |
| GLP-1(7-37) | HAEGTFTSDV SSYLEGQAAK EFIAWLVKGRG | 2 |
| Exendin-4 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG | 3 |
| PSSGAPPPS-NH2 |
The abbreviations H (His) histidine, A (Ala) alanine, E (Glu) glutamic acid, G (Gly) glycine, T (Thr) threonine, F (Phe) phenylalanine, S (Ser) serine, D (Asp) aspartic acid, V (Val) valine, Y (Tyr) tyrosine, L (Leu) leucine, Q (Gln) glutaminase, K (Lys) lysine, I (Ile) isoleucine, R (Arg) arginine, M (Met) methionine, N (Asn) asparagine, P (Pro) proline.
Although many laboratories develop more stable glucagon-like peptide-1 (GLP-1) receptor agonists, their effects in vivo are still short lived, and the development of long acting, insulinotropic peptide derivatives based on the mechanism of glucagon-like peptide-1 (GLP-1) receptor agonism is imminent. The therapeutic effect of these compounds and the window of side effects (vomiting and nausea) are narrow, so there is less chance of success with the approach of depot sustained release formulations. The solution to obtaining long acting glucagon-like peptide-1 (GLP-1) receptor agonists is achieved only by generating a compound that is stable with sufficient residence time in vivo.
The introduction of polyethylene glycol into the active protein and polypeptide will prolong the residence time of the active protein and polypeptide. This technique has been successfully applied to many protein-based biopharmaceuticals, such as PEG-Intron, PEGASYS, Neulasta, Somavert, and others. Methods and chemistry for introducing polyethylene glycol into a polypeptide or protein scaffold can be found in the literature, for example, in Veronese for review (Veronese, FM, biomaterials 200122: 405-. In view of the fact that glucagon-like peptide-1 (GLP-1) and Exendin-4 are both glucagon-like peptide-1 (GLP-1) receptors, U.S. Pat. No. 4,054286 and world patent WO98/05351 disclose comparative experiments on the in vivo insulinotropic action of glucagon-like peptide-1 (GLP-1) and Exendin-4. Experiments show that Exendin-4 has stronger in vivo effect than glucagon-like peptide-1 (GLP-1) and longer time than glucagon-like peptide-1 (GLP-1), and the reason is that Exendin-4 is more stable to polypeptide aqueous enzyme in vivo (DPP IV, NEP24.11 and the like). World patent application 2004/022004 discloses that glucagon-like peptide-1 (GLP-1) receptor agonists modified with polyethylene glycol are disclosed and suggests that when polyethylene glycol is used with a molecular weight above 30000 daltons, the resulting derivatives may no longer have nausea and vomiting side effects due to activation of the glucagon-like peptide-1 (GLP-1) receptor in the brain. This suggests that polyethylene glycol modified glucagon-like peptide-1 (GLP-1) receptor agonists not only prolong the duration of action in vivo, but also have the potential to reduce their side effects. However, in addition to the limitations of in vivo and in vitro activity, the in vivo enzymatic and chemical stability of the polypeptide backbone of these disclosed compounds is not improved, which limits the possibility of these compounds to be long acting formulations. The reduced in vivo and in vitro activity will increase the production cost. Therefore, the possibility of success is greater when the Exendin-4 skeleton with better enzyme-mediated stability is used as the starting point for modifying the precursor with polyethylene glycol. Although the world patent application WO00/66629 discloses compounds and methods for modifying precursors directly with Exendin-4 as polyethylene glycol, there is a distance from actually obtaining a long-lasting, stable, low-cost drug product. The reason for this is as follows: although the stay time of Exendin-4 in vivo is prolonged from the original several hours to several tens of hours or even longer, the possibility that His-Gly at the N terminal is still cut by dipeptide incision enzyme (such as DPP IV) leads to the inactivation of the activation activity function of glucagon-like peptide-1 (GLP-1) receptor; similarly, the long-acting polyethylene glycol modified glucagon-like peptide-1 (GLP-1) receptor agonist also needs to have good chemical stability, especially under the condition of 37 ℃ in vivo, while methionine at the 14-position in the Exendin-4 skeleton is easily oxidized to cause the change of biological activity, and simultaneously, the preparation is troublesome; hydrolysis of asparagine at position 28 is the major cause of drug inactivation and formulation difficulties. The hydrolysis mechanism is as follows:
as can be seen from the hydrolysis mechanism, due to the presence of asparagine, the resulting five-membered ring hydrolysis not only reduces the activity of the modified glucagon-like peptide-1 (GLP-1) receptor agonist, but also results in the separation of polyethylene glycol from the polypeptide backbone, thereby affecting the residence time of the long-acting compound in vivo. Therefore, modification of glycine at position 2 can improve enzyme-mediated stability and chemical stability of the Exendin-4-based polypeptide skeleton; modification of methionine at position 14 and asparagine at position 28 will improve the chemical stability of the Exendin-4 based polypeptide backbone. WO00/66629 strongly calls polyethylene glycol to modify the amino group of the introduced lysine side chain in Exendin-4 to perform acylation reaction to form polyethylene glycol conjugate. Since Exendin-4 itself has lysine, the reaction selectivity of such modification is achieved only by using a protecting group, and thus the production cost is inevitably affected. The arrangement of the joint for modifying and connecting the polyethylene glycol and the special reaction group at the carboxyl terminal (C-terminal) of the polypeptide does not reduce the influence of the polyethylene glycol on the action of the polypeptide and a receptor, and has special reaction and obviously reduced production cost.
The invention discloses a series of Exendin-4 derivatives modified by polyethylene glycol at 2, 14, 27 or 28 positions, and insulinotropic peptide obtained by modifying the polypeptide skeleton with polyethylene glycol. The polyethylene glycol modified insulinotropic peptide has long-acting effect in vivo, and can be prepared into long-acting injection for use.
The insulinotropic peptide compound disclosed by the present invention has a function of activating a GLP-1 receptor located on the surface of a beta-cell in vivo and in vitro to secrete insulin, thereby lowering blood glucose concentration. Examples of such insulinotropic peptides include, but are not limited to, the polypeptide sequences listed in Table 1 and compounds thereof modified with polyethylene glycol. As the modification point of polyethylene glycol, the serine at position 39 can be replaced by cysteine or other synthetic amino acid containing sulfhydryl; also, the multi-point polyethylene glycol modification can be accomplished in the following manner, that isAdding two or more sulfhydryl-containing amino acids (such as cysteine) at carboxyl terminal, and using the extended polypeptide derivatives as polyethylene glycol modified precursor, which is used as two-point modified Cys(39)-(Xaa)(n-1)-Cys(n+39)Wherein n is 1-10 and Xaa is any amino acid.
These polypeptides can be prepared by chemical synthesis methods, including solution phase fragment synthesis, solid phase synthesis (see, e.g., Merrifield, J.Am.chem.Soc.1963, 85: 2149-2154) or a combination of solid and liquid phase methods; polypeptide synthesis includes manual and automated methods, and if automated, an Applied Biosystems 431A polypeptide synthesizer, a Csbio polypeptide synthesizer, etc. can be used; in addition, the polypeptide synthesis can also adopt a combined synthesis method.
The polypeptide prepared by chemical synthesis is purified by high performance liquid phase preparation method, and usually uses a reverse phase material as column packing (such as C)4，C8，C18). The obtained polypeptide can be analyzed and identified (such as High Performance Liquid Chromatography (HPLC), Mass Spectrum (MS) and Amino Acid Analysis (AAA)) to perform in vitro and in vivo drug effect research. The compounds are purified by a high performance liquid phase preparation method and then freeze-dried to obtain the product.
Polyethylene glycols can be purchased from different commercial sources or synthesized in a known manner. The molecular weight of the polyethylene glycol is in the range of 5000-.
The polyethylene glycol and the polypeptide should be linked at the carboxy-terminal end of the polypeptide so as to minimize interference of the polyethylene glycol chain with the action of the polypeptide itself on its receptor. That is to say that polyethylene glycol can be attached anywhere from residue 29 to residue 39. This requires the substitution of any one or several amino acids simultaneously with a thiol-containing amino acid, such as cysteine. If a single point polyethylene glycol modification is used, it is preferred to replace the serine at the carboxy-terminal 39 position with cysteine; similarly, if a two-point modification is made, the most preferred method is to replace serine at position 39 with cysteine, and then add another cysteine at position 40, or at position 39+ n (n-1-10).
Methods for attaching polyethylene glycol using cysteine or sulfhydryl groups are described in many publications (see Veronese, Biomaterials 2001, 22: 405-. The polyethylene glycol can be linked to the sulfhydryl group-containing insulinotropic peptide by a chemically trained technician.
Specifically, the linking reaction using a thiol group can be carried out in the following manner:
1) the sulfhydryl group is derived from a polypeptide chain by introducing an amino acid as described by
n＝1-10
In this case, the polyethylene glycol should contain double chains in Michael addition acceptors such as maleimide, halogen and sulfonate substituted groups, and the polypeptide and polyethylene glycol should be linked by forming a thioether chain.
2) The sulfhydryl group is derived from the side chain of an amino acid in the modified polypeptide, such as a sulfhydryl group attached to the side chain amino group of a lysine. Side chain modified amino acids are of the general formula:
n＝1-10；m＝1-10
in this case, the polyethylene glycol should contain double chains in Michael addition acceptors such as maleimide, halogen and sulfonate substituted groups, and the polypeptide and polyethylene glycol should be linked by forming a thioether chain.
3) The sulfhydryl group is derived from polyethylene glycol, and the linkage point in the polypeptide should contain double-chain, halogen and sulfonate-substituted groups in Michael addition acceptors such as maleimide, and the polypeptide and polyethylene glycol form a thioether chain interface.
4) If both the polyethylene glycol and the polypeptide contain a thiol group, they may be linked in such a manner as to form an asymmetric disulfide bond.
Preferably, the polyethylene glycol forms a covalent linkage with the polypeptide disclosed in the present invention by forming a thioether. The attachment of polyethylene glycol to these disclosed polypeptide sequences is not limited to this chemical attachment method, and other attachment methods such as by acylation, reductive amination, oxime formation, and the like are included in the present invention.
Suitable polyethylene glycol-modified precursor polypeptide derivatives of the present invention are shown in Table 1, which are only examples of the present invention, and the present invention is not limited to these sequences. In the sequence listing, preferred polypeptide sequences are selected from SEQ ID NO80 through SEQ ID NO 141.
These polyethylene glycol-modified insulinotropic peptides and their polypeptide precursors are themselves amphoteric compounds and can react with acidic or basic substances to form salts. The acids usually used for salt formation are selected from: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, trifluoroacetic acid and the like. Examples of such salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, hydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, hydroiodide, acetate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, p-methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, r-hydroxybutyrate, glycerate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like. Preferred acid addition salts are the hydrochloride, sulfate, acetate, trifluoroacetate salts; the base used for the salt formation is generally selected from sodium hydroxide, potassium hydroxide, aqueous ammonia, potassium carbonate, and the like.
The insulinotropic peptide compound disclosed by the invention, particularly the insulinotropic peptide compound modified by polyethylene glycol, can be used for preventing and treating type II diabetes, particularly for patients with abnormal secretion caused by overweight and even obesity, because the insulinotropic peptide compound has the potential of repairing beta-cell functions.
Accordingly, the present invention also relates to a method for the treatment and/or prevention of type II diabetes, wherein a pharmaceutically effective dose of an insulinotropic peptide according to the present invention is administered to a human in need of treatment and/or prevention of type II diabetes.
The insulinotropic peptide compounds disclosed herein can be used alone or in combination with other antidiabetic agents (e.g., PPAR agonists, sulfonylurea drugs, non-sulfonylurea analogs (Secretagues), alpha-glucosidase inhibitors, insulin sensitizers, insulin Seeretagues, glycogen release inhibitors, insulin, and other antiobesity agents) to treat diabetes.
The clinical amount will depend on the potency of the particular compound. It is in the range of 0.0001mg/kg to about 200mg/kg body weight, preferably in the range of 0.001mg/kg to 20mg/kg body weight, and most preferably in the range of 0.01mg/kg to 1mg/kg body weight. The administration may be by injection (including intravenous, intramuscular, subcutaneous) or other continuous injection. The compounds may also be formulated for administration by any conventional route, such as orally, transdermally, pulmonarily, nasally, buccally, by suppositories, and the like.
Drawings
FIG. 1 is a LC-MS diagram of SEQ ID NO. 95.
FIG. 2 shows the effect on glucose tolerance of db/db mice on the day of subcutaneous injection of PEG-EX-4 analogues.
FIG. 3 shows the effect on glucose tolerance on day 3 following subcutaneous injection of PEG-EX-4 analogs in db/db mice.
FIG. 4 shows the effect on glucose tolerance on day 6 following subcutaneous injection of PEG-EX-4 analogs in db/db mice.
FIG. 5 shows the effect on glucose tolerance on day 9 following subcutaneous injection of PEG-EX-4 analogs in db/db mice.
FIG. 6 shows the blood glucose lowering effect of mice injected with PEG-EX-4 analogue 1100. mu.g/kg subcutaneously.
FIG. 7 the blood sugar lowering effect of mice injected with 3300 μ g/kg of PEG-EX-4 analogues subcutaneously.
Detailed Description
In order to illustrate the present invention in more detail, the following examples are given. The scope of the invention is not limited thereto.
Example one
Solid-phase synthesis preparation of compound SEQ ID NO.95 obtained by the invention
(1) Amino acid monomers used for synthesis
Fmoc-His(Trt)-OH，Fmoc-dAla-OH，Fmoc-Gly-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Thr(tBu)-OH，Fmoc-Phe-OH，Fmoc-Ser(tBu)-OH，Fmoc-Asp(OtBu)-OH，
Fmoc-Leu-OH，Fmoc-Lys(Boc)-OH，Fmoc-Gln(Trt)-OH，Fmoc-Nle-OH，
Fmoc-Ala-OH，Fmoc-Val-OH，Fmoc-Arg(pbf)-OH，Fmoc-Ile-OH，Fmoc-Trp(Boc)-OH，Fmoc-Asn(Trt)-OH，Fmoc-Pro-OH，Fmoc-Cys(Trt)-OH
The above abbreviations are: fmoc: 9-fluorenylmethoxycarbonyl; boc: a tert-butoxycarbonyl group; trt: a trityl group; OtBu: tert-butyloxy; tBu: a tertiary butyl group.
(2) The used medicines are as follows: the reagent is as follows: n, N-diisopropylethylamine, Diisopropylcarbodiimide (DIC), N, N-Dimethylformamide (DMF), dichloromethane, piperidine, 1-hydroxybenzotriazole, Rinkamide resin, ninhydrin, methanol, anisole, triisopropylsilane, trifluoroacetic acid.
(3) Operation of
A. Synthesizing: taking 0.25mmol as an example, 0.5g Rink amide resin is weighed and placed in a reactor, 1mmol of amino acid is weighed and activated by a DIC/HOBt method, and the synthesis is carried out in sequence from the C end to the N end according to a polypeptide sequence. The reaction was carried out at 25 ℃ and room temperature, and was carried out as follows:
1. deprotecting the Fmoc group with 20% piperidine in DMF for 10 min each time;
2. washing with 10 ml DMF for three times, and pumping to dry;
3. weighing protected amino acid (1mmol) and HOBt (1mmol), dissolving in 10 ml DMF, and adding DIC (1mmol) for activation for 10 min;
4. adding the activated amino acid solution into a reactor, and shaking for reaction for 1 hour;
5. washing the resin with DMF for three times, and pumping to dry;
6. if the ninhydrin reaction is negative, performing the next cycle, and repeating the steps 1 to 5;
if the ninhydrin reaction is positive, steps 3 to 5 are repeated.
After the polypeptide chain is gradually synthesized, the resin is washed with methanol and dried.
B. Deprotection and cleavage of polypeptides
1 g of the resin with the polypeptide is placed in a reactor, and lysis solution is added. For example, the following table:
| solvent(s) | Dosage (ml) |
| Phenylmethyl ether | 2 |
| Methanol | 2 |
| Tri-isopropyl silane | 2 |
| Trifluoroacetic acid | 6 |
Shaking for 2 hr at room temperature, filtering, collecting filtrate, washing resin with small amount of acetic acid, mixing the collected solutions, concentrating, adding diethyl ether to generate precipitate, filtering the precipitate, and washing with small amount of diethyl ether to obtain crude product.
C. Separating and purifying with high performance liquid phase, and lyophilizing
Dissolving the obtained crude product in a small amount of 10% acetic acid solution, loading on a column, separating and purifying by high performance liquid chromatography, and lyophilizing to obtain the product, wherein the obtained polypeptide is proved to be the required compound by chromatography-mass spectrometry detection.
A chromatographic column: luna C18(2), 5u,
separation wavelength: 220nm lambda, Waters preparation system
Gradient: (TFA: trifluoroacetic acid)
| T (minute) | A：(0.05％TFA)CHCN | B：(0.05TFA)HO |
| 0 | 10％ | 90％ |
| 20 | 45％ | 55％ |
| 30 | 45％ | 55％ |
| 30.1 | 10％ | 90％ |
Molecular weight of the resulting compound: 4212.6 g/mol; theoretical molecular weight: 4213 g/mol.
FIG. 1: SEQ ID NO95LC-MS diagram
Example two
Method for modifying insulinotropic hormone secretion peptide by polyethylene glycol
Modification of the polypeptide with polyethylene glycol can be carried out in a conventional manner. The present invention addresses the problem of combining polyethylene glycol with polypeptides based on a method of modifying sulfhydryl groups to form thioethers. Specifically, one or more cysteine(s) are added at the carboxyl end of an optimized Exendin-4 derivative, then polyethylene glycol containing a functional group of maleimide is adopted for carrying out polyethylene glycol modification reaction, and a thioether chain is formed after Michael addition reaction so as to enable the polypeptide and the polyethylene glycol to be covalently bonded. Typically, the desired polypeptide is dissolved in 0.1M phosphate buffer and, under deoxygenation, polyethylene glycol is added. The molar ratio of polyethylene glycol to polypeptide is 1: 1. The pH value of the reaction is 6-7.5, and the oxidation of sulfydryl can be reduced by adding EDTA into the reaction liquid. After reacting for 2 hours, the reaction liquid is separated and purified by a reverse phase high performance liquid phase method, and the surplus and unreacted polyethylene glycol can be removed by an ion exchange method. The product obtained was confirmed by mass spectrometry for molecular weight and analyzed for purity by RP-HPLC and gel chromatography. Taking the modification of SEQ ID NO95 as an example, when modified with a 43KD PEG, the yield is 70-90%. (in terms of polyethylene glycol)
EXAMPLE III
Polypeptide stability assay
The Exendin-4 derivative disclosed by the invention has optimized enzymatic or chemical stability. The following method was used to examine the chemical stability of some of the polypeptides disclosed in the present invention.
Dissolving 1mg of each test sample in a buffer solution of 150mM sodium chloride and 20mM sodium phosphate to prepare a solution with the concentration of 4mg/ml, wherein the pH value is 8.0, placing the test sample solution in a thermostat at 40 ℃, and checking the purity of the polypeptide by a liquid chromatography-mass spectrometry method, wherein the chemical stability of the polypeptide is expressed by the relation between the disappearance ratio of the main peak area and the time.
| Purity (%) | Day 0 | 5 days | 10 days | 15 days |
| Test article 1 | 98.2 | 88.0 | 81.6 | 76.2 |
| Sample 2 | 98 | 93.1 | 90.4 | 88.2 |
| Test article 3 | 98.9 | 98.8 | 98.8 | 98.8 |
| Test article 4 | 99.7 | 99.4 | 99.0 | 99.3 |
TABLE 2 stability determination table for Exendin-4 compounds
Wherein, the sample 1: exendin-4 control. The sequence is as follows:
His-Gly2-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met14-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn28-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser39。
sample 2: 2# Gly was changed to d-Ala and 39# to Cys in sample 1. Sample 3: the sample 1 was modified to have d-Ala for 2# Gly, Nle for 14# Met, and Gln for 28# Asn. Sample 4: test 2 and PEG40K were covalently linked through a C-terminal Cys, where the C-terminals of test 1-4 were all amides.
And (4) conclusion:
the sequence of the test sample 1 is a polypeptide sequence separated from the nature, but His-Gly at the N terminal is a proper substrate of dipeptidase, Met contained in the test sample is easy to oxidize, Asn is easy to react with the substrate, and the test sample 1 is not stable; the stability of the peptide is greatly improved by replacing three points of the 2-position, the 14-position and the 28-position, and the stability of the peptide is improved by replacing the three points, which greatly exceeds the stability of the peptide when only His-Gly is replaced by His-dAla; sample 2, although not very stable, became very stable after attachment to PEG40K (i.e., sample 4), indicating that PEG has a great role in improving polypeptide stability.
Example four
Preparation
PEG-EX-4 analog 5g
M-cresol 0.04g
Appropriate amount of glacial acetic acid
Appropriate amount of glacial sodium acetate
Proper amount of water for injection
100ml/100 pieces package
Sterile preparation
Example one
Oral glucose tolerance test with polypeptide
After the polypeptide with the sequence number of SEQ ID NO 25 is modified by polyethylene glycol with the molecular weight of about 40000 (the number is that of a test article 5), an oral glucose tolerance experiment is carried out on normal mice, and the results are shown in the following table:
TABLE 1. different doses of test article 5 injected subcutaneously on normal mice, the effect of first and third day oral glucose tolerance and area under the blood glucose curve can be seen
(day 1)
v.s Con ***P＜0.001；n＝10
(day 3)
v.s Con，*P＜0.05，**P＜0.01，***P＜0.001；n＝10
Experimental example two
Effect of PEG-EXENDIN-4(PEG-EX-4) analogues on type II diabetic db/db mice
1. The test animals were: species and strain: db/db mice, origin: the model animal center of Nanjing university provides, the weight of the mice: 35-50g, half male and half female. Number of animals: 45, 5-6 per group. Feeding conditions are as follows: SPF-level animal house feeding, temperature: 22-24 ℃, humidity: 45-80%, illumination: 150-300Lx, alternating day and night for 12 hours.
2. The test method comprises the following steps:
dose setting: 5 dosing groups were set: 0.03, 0.1, 0.3, 1 and 3 mg/kg; simultaneously establishing a blank control group; the administration route is as follows: subcutaneous injection; the administration volume: 0.05ml/kg body weight.
(1) Effect on blood glucose in db/db mice in non-fasted State
db/db mice were divided into 6 groups of non-fasting blood glucose and body weight, and each group was a placebo and 5 PEG-EX-4 analogue administration groups. The test drug and physiological saline were administered to each group of animals by single subcutaneous injection, and blood glucose was measured before and after administration at 1, 2, 4, 8, and 24 hours, and non-fasting blood glucose was measured every 24 hours thereafter, and the duration of the hypoglycemic effect of the test substance was observed, and the change in food intake and body weight of each group of animals after administration was measured.
(2) Effect on blood glucose in db/db mice in fasted State
db/db mice were divided into 6 groups with non-fasting, fasting blood glucose and body weight, and the groups were hermaphrodite, and were blank control and 5 PEG-EX-4 analogue administration groups. The animals of each group were administered the test drug and physiological saline by single subcutaneous injection after 5 hours of fasting, and blood glucose was measured before administration and 1 and 2 hours after administration, and thereafter non-fasting and fasting blood glucose was measured every 24 hours, and the duration of the hypoglycemic action of the test substance was observed, and the change in food intake and body weight of the animals of each group after administration was measured.
(3) Effect on glucose tolerance in db/db mice in fasted State
db/db mice were divided into 5 groups of 5 mice per group based on fasting blood glucose and body weight, and were a blank control and 5 groups administered with PEG-EX-4 analogues. The test drug and physiological saline were administered separately by single subcutaneous injection after 5 hours of fasting, glucose was orally administered at 2.5g/kg at 15 minutes after administration, and blood glucose values were measured at 0, 30, 60 and 120 minutes after administration of glucose. On days 3, 6 and 9 after administration, oral glucose tolerance tests were again performed, respectively. The effect of the test substance on db/db glucose tolerance and the duration of its action were observed, and the change in food intake and body weight after administration was determined for each group of animals.
3. And (3) test results: referring to FIGS. 2-5 and tables 1-6, the effects of PEG-EX-4 analogs on blood glucose in db/db mice
(1) Effect on blood glucose in db/db mice in fasted and non-fasted states
Table 1: effect of subcutaneous PEG-EX-4 analogues in db/db mice on fasting plasma glucose (mean. + -. SD, n ═ 6)
Table 2: effect of subcutaneous PEG-EX-4 analogues in db/db mice on daily non-fasting blood glucose (mean. + -. SD, n ═ 6)
(2) Effect on glucose tolerance in db/db mice in fasted State
Table 3: effect of subcutaneous injection of PEG-EX-4 analogs on glucose tolerance in db/db mice on the day (mean. + -. SD, n ═ 5)
Table 4: effect on glucose tolerance on day 3 following subcutaneous injection of PEG-EX-4 analogs in db/db mice (mean. + -. SD, n ═ 5)
Table 5: effect on glucose tolerance on day 6 following subcutaneous injection of PEG-EX-4 analogs in db/db mice (mean. + -. SD, n ═ 5)
Table 6: effect on glucose tolerance on day 9 following subcutaneous injection of PEG-EX-4 analogs in db/db mice (mean. + -. SD, n ═ 5)
*，P＜0.05；**，P＜0.01；***P is less than 0.001, compared with the blank control group
Experimental example III
Effect of PEG-EXENDIN-4(PEG-EX-4) analogues on blood glucose in KKAY mice preliminary test results
1. The test method comprises the following steps:
KKAY mice were tested for changes in blood glucose at different times following a single subcutaneous injection of various doses of PEG-EX-4 analogs.
2. And (3) test results:
(1) referring to FIG. 6, after 1100 μ g/kg of PEG-EX-4 analog is injected subcutaneously once into KKAY mice, the blood sugar lowering effect can be maintained for about 3-4 days.
(2) Referring to fig. 7, after one-time subcutaneous injection of 3300 μ g/kg of PEG-EX-4 analog in KKAy mice, the blood glucose lowering effect was maintained for about 3-4 days.
The amino acid sequence of the long-acting insulinotropic peptide of the present invention can be seen in table 7 below:
| numbering | Sequence of | SEOIDNO |
| HR1 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS | 4 |
| HR2 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPC | 5 |
| HR3 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPCC | 6 |
| HR4 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhC | 7 |
| HR5 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhChC | 8 |
| HR6 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK | 9 |
| HR7 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPKK | 10 |
| HR8 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPC-NH | 11 |
| HR9 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPCC-NH | 12 |
| HR10 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhC-NH | 13 |
| HR11 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPhChC-NH | 14 |
| HR12 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK-NH | 15 |
| HR13 | HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPKK-NH | 16 |
| HR14 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS | 17 |
| HR15 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPC | 18 |
| HR16 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPCC | 19 |
| HR17 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhC | 20 |
| HR18 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhChC | 21 |
| HR19 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK | 22 |
| HR20 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPKK | 23 |
| HR21 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS-NH | 24 |
| HR22 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPC-NH | 25 |
| HR23 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPCC-NH | 26 |
| HR24 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSS GAPPPhC-NH | 27 |
| HR25 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPhChC-NH | 28 |
| HR26 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK-NH | 29 |
| HR27 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPKK-NH2 | 30 |
| HR28 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPS | 31 |
| HR29 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPC | 32 |
| HR30 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPCC | 33 |
| HR31 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhC | 34 |
| HR32 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhChC | 35 |
| HR33 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPK | 36 |
| HR34 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPKK | 37 |
| HR35 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPS-NH | 38 |
| HR36 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPC-NH | 39 |
| HR37 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PS SGAPPPCC-NH | 40 |
| HR38 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhC-NH | 41 |
| HR39 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPhChC-NH | 42 |
| HR40 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPK-NH | 43 |
| HR41 | HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPKK-NH | 44 |
| HR42 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPS | 45 |
| HR43 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPC | 46 |
| HR44 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPCC | 47 |
| HR45 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPhC | 48 |
| HR46 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPhChC | 49 |
| HR47 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK | 50 |
| HR48 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPKK | 51 |
| HR49 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPS-NH | 52 |
| HR50 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPC-NH | 53 |
| HR51 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PS SGAPPPCC-NH | 54 |
| HR52 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PS SGAPPPhC-NH | 55 |
| HR53 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGGPSSGAPPPhChC-NH | 56 |
| HR54 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK-NH | 57 |
| HR55 | HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPKK-NH | 58 |
| HR56 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPS | 59 |
| HR57 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPC | 60 |
| HR58 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPCC | 61 |
| HR59 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhC | 62 |
| HR60 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhChC | 63 |
| HR61 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPK | 64 |
| HR62 | HdAEGTFTSDL SKQNleEEEAVRLFIEWLKNGG PSSGAPPPKK | 65 |
| HR63 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPS-NH | 66 |
| HR64 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPC-NH | 67 |
| HR65 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPS SGAPPPCC-NH | 68 |
| HR66 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhC-NH | 69 |
| HR67 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPhChC-NH | 70 |
| HR68 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPK-NH | 71 |
| HR69 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPKK-NH | 72 |
| HR70 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPS | 73 |
| HR71 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPC | 74 |
| HR72 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPCC | 75 |
| HR73 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PS SGAPPPhC | 76 |
| HR74 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPhChC | 77 |
| HR75 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK | 78 |
| HR76 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPKK | 79 |
| HR77 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPS-NH | 80 |
| HR78 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPC-NH | 81 |
| HR79 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPCC-NH | 82 |
| HR80 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPhC-NH | 83 |
| HR81 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGGPS SGAPPPhChC-NH | 84 |
| HR82 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK-NH | 85 |
| HR83 | HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGGPSSGAPPPKK-NH | 86 |
| HR84 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPS | 87 |
| HR85 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPC | 88 |
| HR86 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPCC | 89 |
| HR87 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPhC | 90 |
| HR88 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPhChC | 91 |
| HR89 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPK | 92 |
| HR90 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPKK | 93 |
| HR91 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPS-NH | 94 |
| HR92 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPC-NH | 95 |
| HR93 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGGPSSGAPPPCC-NH | 96 |
| HR94 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGGPSSGAPPPhChC-NH | 97 |
| HR95 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPK-NH | 98 |
| HR96 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGGPSSGAPPPKK-NH | 99 |
| HR97 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPS | 100 |
| HR98 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPC | 101 |
| HR99 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPCC | 102 |
| HR100 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPhC | 103 |
| HR101 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPhChC | 104 |
| HR102 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSS GAPPPK | 105 |
| HR103 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPKK | 106 |
| HR104 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPS-NH | 107 |
| HR105 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPC-NH | 108 |
| HR106 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGGPSSGAPPPCC-NH | 109 |
| HR107 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPhC-NH | 110 |
| HR108 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGGPSSGAPPPhChC-NH | 111 |
| HR109 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPK-NH | 112 |
| HR110 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGGPSSGAPPPKK-NH | 113 |
| HR111 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPS | 114 |
| HR112 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPC | 115 |
| HR113 | HdAEGTFTSDL SKQMEEEAVRLFIEWLVKGG PSSGAPPPCC | 116 |
| HR114 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPhC | 117 |
| HR115 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPhChC | 118 |
| HR116 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPK | 119 |
| HR117 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPKK | 120 |
| HR118 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPS-NH | 121 |
| HR119 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPC-NH | 122 |
| HR120 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPCC-NH | 123 |
| HR121 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPhC-NH | 124 |
| HR122 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG | 125 |
| PSSGAPPPhChC-NH | ||
| HR123 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPK-NH | 126 |
| HR124 | HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGGPSSGAPPPKK-NH | 127 |
| HR125 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPS | 128 |
| HR126 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPC | 129 |
| HR127 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPCC | 130 |
| HR128 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPhC | 131 |
| HR129 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPhChC | 132 |
| HR130 | HdAEGTFTSDL SKQNleEEEAVRLFIEWLKGG PSSGAPPPK | 133 |
| HR131 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPKK | 134 |
| HR132 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPS-NH | 135 |
| HR133 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPC-NH | 136 |
| HR134 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPCC-NH | 137 |
| HR135 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPhC-NH | 138 |
| HR136 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPhChC-NH | 139 |
| HR137 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPK-NH | 140 |
| HR138 | HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPKK-NH | 141 |
In Table 7, C, hC, K*Is a polyethylene glycol modification point. C is cysteine, hC is higher cysteine, K*Lysine modified for side chain, such as mercaptopropionic acid, is attached to the side chain amino group. If two CC, hCHC, or K*K*Appearing in the sequence, means two polyethylene glycol modification points. Nle is norleucine, dAla is D-alanine, -NH2Is a C-terminal amide.
Sequence listing
<110> Wuxi HongChuang pharmaceutical science and technology Limited
<120> long-acting insulinotropic peptide derivative, its medicinal salt, preparation method and application
<160>141
<170>PatentIn version 3.3
<210>1
<211>30
<212>PRT
<213> Artificial sequence
<400>1
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210>2
<211>31
<212>PRT
<213> Artificial sequence
<400>2
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210>3
<211>39
<212>PRT
<213> Artificial sequence
<400>3
His Gly Gly Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>4
<211>39
<212>PRT
<213> Artificial sequence
<400>4
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>5
<211>39
<212>PRT
<213> Artificial sequence
<400>5
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>6
<211>40
<212>PRT
<213> Artificial sequence
<400>6
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>7
<211>39
<212>PRT
<213> Artificial sequence
<400>7
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>8
<211>40
<212>PRT
<213> Artificial sequence
<400>8
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>9
<211>39
<212>PRT
<213> Artificial sequence
<400>9
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>10
<211>40
<212>PRT
<213> Artificial sequence
<400>10
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>11
<211>39
<212>PRT
<213> Artificial sequence
<400>11
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>12
<211>40
<212>PRT
<213> Artificial sequence
<400>12
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>13
<211>39
<212>PRT
<213> Artificial sequence
<400>13
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>14
<211>40
<212>PRT
<213> Artificial sequence
<400>14
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>15
<211>39
<212>PRT
<213> Artificial sequence
<400>15
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>16
<211>40
<212>PRT
<213> Artificial sequence
<400>16
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>17
<211>39
<212>PRT
<213> Artificial sequence
<400>17
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>18
<211>39
<212>PRT
<213> Artificial sequence
<400>18
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>19
<211>40
<212>PRT
<213> Artificial sequence
<400>19
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>20
<211>39
<212>PRT
<213> Artificial sequence
<400>20
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>21
<211>40
<212>PRT
<213> Artificial sequence
<400>21
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>22
<211>39
<212>PRT
<213> Artificial sequence
<400>22
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>23
<211>40
<212>PRT
<213> Artificial sequence
<400>23
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>24
<211>39
<212>PRT
<213> Artificial sequence
<400>24
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>25
<211>39
<212>PRT
<213> Artificial sequence
<400>25
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>26
<211>40
<212>PRT
<213> Artificial sequence
<400>26
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>27
<211>39
<212>PRT
<213> Artificial sequence
<400>27
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>28
<211>40
<212>PRT
<213> Artificial sequence
<400>28
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>29
<211>39
<212>PRT
<213> Artificial sequence
<400>29
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>30
<211>40
<212>PRT
<213> Artificial sequence
<400>30
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>31
<211>39
<212>PRT
<213> Artificial sequence
<400>31
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>32
<211>39
<212>PRT
<213> Artificial sequence
<400>32
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>33
<211>40
<212>PRT
<213> Artificial sequence
<400>33
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>34
<211>39
<212>PRT
<213> Artificial sequence
<400>34
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>35
<211>40
<212>PRT
<213> Artificial sequence
<400>35
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>36
<211>39
<212>PRT
<213> Artificial sequence
<400>36
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>37
<211>40
<212>PRT
<213> Artificial sequence
<400>37
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>38
<211>39
<212>PRT
<213> Artificial sequence
<400>38
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>39
<211>39
<212>PRT
<213> Artificial sequence
<400>39
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>40
<211>40
<212>PRT
<213> Artificial sequence
<400>40
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>41
<211>39
<212>PRT
<213> Artificial sequence
<400>41
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>42
<211>40
<212>PRT
<213> Artificial sequence
<400>42
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>43
<211>39
<212>PRT
<213> Artificial sequence
<400>43
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>44
<211>40
<212>PRT
<213> Artificial sequence
<400>44
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>45
<211>39
<212>PRT
<213> Artificial sequence
<400>45
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>46
<211>39
<212>PRT
<213> Artificial sequence
<400>46
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>47
<211>40
<212>PRT
<213> Artificial sequence
<400>47
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>48
<211>39
<212>PRT
<213> Artificial sequence
<400>48
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>49
<211>39
<212>PRT
<213> Artificial sequence
<400>49
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Cys Cys
35
<210>50
<211>39
<212>PRT
<213> Artificial sequence
<400>50
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>51
<211>40
<212>PRT
<213> Artificial sequence
<400>51
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>52
<211>39
<212>PRT
<213> Artificial sequence
<400>52
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>53
<211>39
<212>PRT
<213> Artificial sequence
<400>53
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>54
<211>40
<212>PRT
<213> Artificial sequence
<400>54
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>55
<211>39
<212>PRT
<213> Artificial sequence
<400>55
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>56
<211>40
<212>PRT
<213> Artificial sequence
<400>56
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>57
<211>39
<212>PRT
<213> Artificial sequence
<400>57
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>58
<211>40
<212>PRT
<213> Artificial sequence
<400>58
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>59
<211>39
<212>PRT
<213> Artificial sequence
<400>59
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>60
<211>39
<212>PRT
<213> Artificial sequence
<400>60
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>61
<211>40
<212>PRT
<213> Artificial sequence
<400>61
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>62
<211>39
<212>PRT
<213> Artificial sequence
<400>62
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>63
<211>40
<212>PRT
<213> Artificial sequence
<400>63
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>64
<211>39
<212>PRT
<213> Artificial sequence
<400>64
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>65
<211>40
<212>PRT
<213> Artificial sequence
<400>65
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>66
<211>39
<212>PRT
<213> Artificial sequence
<400>66
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>67
<211>39
<212>PRT
<213> Artificial sequence
<400>67
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro ProPro Cys
35
<210>68
<211>40
<212>PRT
<213> Artificial sequence
<400>68
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>69
<211>39
<212>PRT
<213> Artificial sequence
<400>69
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>70
<211>40
<212>PRT
<213> Artificial sequence
<400>70
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>71
<211>39
<212>PRT
<213> Artificial sequence
<400>71
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>72
<211>40
<212>PRT
<213> Artificial sequence
<400>72
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>73
<211>39
<212>PRT
<213> Artificial sequence
<400>73
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>74
<211>39
<212>PRT
<213> Artificial sequence
<400>74
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>75
<211>40
<212>PRT
<213> Artificial sequence
<400>75
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>76
<211>39
<212>PRT
<213> Artificial sequence
<400>76
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>77
<211>40
<212>PRT
<213> Artificial sequence
<400>77
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>78
<211>39
<212>PRT
<213> Artificial sequence
<400>78
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>79
<211>40
<212>PRT
<213> Artificial sequence
<400>79
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>80
<211>39
<212>PRT
<213> Artificial sequence
<400>80
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>81
<211>39
<212>PRT
<213> Artificial sequence
<400>81
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>82
<211>40
<212>PRT
<213> Artificial sequence
<400>82
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>83
<211>39
<212>PRT
<213> Artificial sequence
<400>83
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>84
<211>40
<212>PRT
<213> Artificial sequence
<400>84
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>85
<211>39
<212>PRT
<213> Artificial sequence
<400>85
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>86
<211>40
<212>PRT
<213> Artificial sequence
<400>86
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>87
<211>39
<212>PRT
<213> Artificial sequence
<400>87
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>88
<211>39
<212>PRT
<213> Artificial sequence
<400>88
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>89
<211>40
<212>PRT
<213> Artificial sequence
<400>89
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>90
<211>39
<212>PRT
<213> Artificial sequence
<400>90
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>91
<211>40
<212>PRT
<213> Artificial sequence
<400>91
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>92
<211>39
<212>PRT
<213> Artificial sequence
<400>92
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>93
<211>40
<212>PRT
<213> Artificial sequence
<400>93
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>94
<211>39
<212>PRT
<213> Artificial sequence
<400>94
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>95
<211>39
<212>PRT
<213> Artificial sequence
<400>95
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>96
<211>40
<212>PRT
<213> Artificial sequence
<400>96
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>97
<211>40
<212>PRT
<213> Artificial sequence
<400>97
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>98
<211>39
<212>PRT
<213> Artificial sequence
<400>98
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>99
<211>40
<212>PRT
<213> Artificial sequence
<400>99
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Gln Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>100
<211>39
<212>PRT
<213> Artificial sequence
<400>100
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu PheIle Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>101
<211>39
<212>PRT
<213> Artificial sequence
<400>101
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>102
<211>40
<212>PRT
<213> Artificial sequence
<400>102
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>103
<211>39
<212>PRT
<213> Artificial sequence
<400>103
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>104
<211>40
<212>PRT
<213> Artificial sequence
<400>104
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>105
<211>39
<212>PRT
<213> Artificial sequence
<400>105
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>106
<211>40
<212>PRT
<213> Artificial sequence
<400>106
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>107
<211>39
<212>PRT
<213> Artificial sequence
<400>107
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>108
<211>39
<212>PRT
<213> Artificial sequence
<400>108
His Ala Glu GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>109
<211>40
<212>PRT
<213> Artificial sequence
<400>109
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>110
<211>39
<212>PRT
<213> Artificial sequence
<400>110
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>111
<211>40
<212>PRT
<213> Artificial sequence
<400>111
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>112
<211>39
<212>PRT
<213> Artificial sequence
<400>112
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>113
<211>40
<212>PRT
<213> Artificial sequence
<400>113
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Gln Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>114
<211>39
<212>PRT
<213> Artificial sequence
<400>114
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>115
<211>39
<212>PRT
<213> Artificial sequence
<400>115
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>116
<211>40
<212>PRT
<213> Artificial sequence
<400>116
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>117
<211>39
<212>PRT
<213> Artificial sequence
<400>117
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>118
<211>40
<212>PRT
<213> Artificial sequence
<400>118
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>119
<211>39
<212>PRT
<213> Artificial sequence
<400>119
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>120
<211>40
<212>PRT
<213> Artificial sequence
<400>120
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>121
<211>39
<212>PRT
<213> Artificial sequence
<400>121
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>122
<211>39
<212>PRT
<213> Artificial sequence
<400>122
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>123
<211>40
<212>PRT
<213> Artificial sequence
<400>123
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu PheIle Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>124
<211>39
<212>PRT
<213> Artificial sequence
<400>124
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>125
<211>40
<212>PRT
<213> Artificial sequence
<400>125
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>126
<211>39
<212>PRT
<213> Artificial sequence
<400>126
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>127
<211>40
<212>PRT
<213> Artificial sequence
<400>127
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>128
<211>39
<212>PRT
<213> Artificial sequence
<400>128
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>129
<211>39
<212>PRT
<213> Artificial sequence
<400>129
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>130
<211>40
<212>PRT
<213> Artificial sequence
<400>130
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>131
<211>39
<212>PRT
<213> Artificial sequence
<400>131
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>132
<211>40
<212>PRT
<213> Artificial sequence
<400>132
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>133
<211>39
<212>PRT
<213> Artificial sequence
<400>133
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>134
<211>40
<212>PRT
<213> Artificial sequence
<400>134
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
<210>135
<211>39
<212>PRT
<213> Artificial sequence
<400>135
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>136
<211>39
<212>PRT
<213> Artificial sequence
<400>136
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>137
<211>40
<212>PRT
<213> Artificial sequence
<400>137
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>138
<211>39
<212>PRT
<213> Artificial sequence
<400>138
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys
35
<210>139
<211>40
<212>PRT
<213> Artificial sequence
<400>139
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Cys Cys
35 40
<210>140
<211>39
<212>PRT
<213> Artificial sequence
<400>140
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys
35
<210>141
<211>40
<212>PRT
<213> Artificial sequence
<400>141
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Nle Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Val Lys Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Lys Lys
35 40
Claims (9)
1. An insulinotropic peptide, wherein the amino acid sequence of the insulinotropic peptide is selected from the amino acid sequences represented by SEQ ID Nos. 5 to 141.
2. The insulinotropic peptide according to claim 1, wherein the insulinotropic peptide is an amino acid sequence represented by SEQ ID nos. 5 to 141 modified with polyethylene glycol.
3. The insulinotropic peptide according to claim 2, wherein the insulinotropic peptide is the amino acid sequence of SEQ ID NO.95 modified with polyethylene glycol.
4. The insulinotropic peptide according to claim 3, wherein the linker and the reaction-specific group which are modified to link to polyethylene glycol are arranged at the carboxy terminus of the insulinotropic peptide when modified with polyethylene glycol.
5. The insulinotropic peptide according to claim 2, wherein the molecular weight of the polyethylene glycol is in the range of 5,000-80,000 daltons.
6. The insulinotropic peptide according to claim 2, wherein the molecular weight of the polyethylene glycol is in the range of 20,000-60,000 daltons.
7. The insulinotropic peptide according to any of the preceding claims, comprising a pharmaceutically acceptable salt of the insulinotropic peptide.
8. A process for preparing the insulinotropic peptide of claim 1 or 2, which comprises solid and liquid phase synthesis methods, reversed phase high performance liquid phase, ion exchange and gel filtration purification methods, and freeze-drying.
9. Use of the insulinotropic peptide according to claim 1 or 2 in the manufacture of a medicament for the treatment and/or prevention of type II diabetes.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200510038102 | 2005-01-14 | ||
| CN200510038102.3 | 2005-01-14 | ||
| PCT/CN2006/000029 WO2006074600A1 (en) | 2005-01-14 | 2006-01-10 | Modified exendins and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1095045A1 HK1095045A1 (en) | 2007-04-20 |
| HK1095045B true HK1095045B (en) | 2011-01-21 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100540565C (en) | Modified Exendins and their applications | |
| JP4716641B2 (en) | Glucagon-like peptide-1 analog | |
| US8716221B2 (en) | Modified exendins and uses thereof | |
| CN102397558B (en) | Positioning pegylation modified compound of Exendin-4 analog and application thereof | |
| US8450266B2 (en) | Analogues of glucose-dependent insulinotropic polypeptide | |
| US20110136733A1 (en) | Analogues of glucose-dependent insulinotropic polypeptide | |
| CN1192689A (en) | Appetite regulating compositions | |
| CN106661096B (en) | New exenatide analogs and their uses | |
| KR20150005904A (en) | Novel gh-rh analogs with potent agonistic effects | |
| CN108676084B (en) | Exenatide modifier and application thereof | |
| CN109280083B (en) | GLP-1 polypeptide having GLP-1 receptor agonist activity and use thereof | |
| EP3647319A1 (en) | Peptide compound, application thereof and composition containing same | |
| HK1095045B (en) | Modified exendins and uses thereof | |
| CN102241757B (en) | Polypeptide analogue of human osteocalcin | |
| CN112898404A (en) | Long-acting modified glucagon peptide analogue or salt thereof and application thereof | |
| CN1683408A (en) | Glucagon-like peptide analog, its composition and its using method | |
| WO2024213022A1 (en) | Incretin analogues and preparation method therefor, and application | |
| JP2012513981A (en) | GLP-1 analog and use thereof | |
| EP1152011A1 (en) | Peptides inhibiting vascular endothelial cell migration | |
| JPWO1996014336A1 (en) | A novel oxyntomodulin | |
| HK1033324A1 (en) | Glucagon-like peptide-2 analogs | |
| HK1033324B (en) | Glucagon-like peptide-2 analogs |