Patent Application
20240150423
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy is named GBKY076_Sequence_Listing.txt, created on Apr. 11, 2022, and is 44,098 bytes in size.
The present disclosure belongs to the field of biopharmaceutics, particularly the preparations and application of a variety of human novel GLP-1 analogue monomers or homodimers in treatment of diabetes.
Glucagon-like peptide 1 (GLP-1) derived from glucagon proprotein is an incretin analogue having 30 amino acid residues, and is released by intestine L cells during the intake of nutrients. It enhances insulin secretion from pancreas β cells, increases expression of insulin and utilization of peripheral glucose, inhibits apoptosis of β cells, promotes satiety and regeneration of β cells, reduces secretion of glucagon and delays gastric emptying. Through these multiple effects, GLP-1 receptor agonists have significance in treatment of type II diabetes. So far, GLP-1 analogues approved by FDA include Liraglutide administered once a day, Exenatide administered twice a day and Albiglutide, Dulaglutide, Exenatide LAR, Lixisenatide, Semaglutide and Taspoglutide administered once a week.
Exendin-4 is an incretin analogue isolated from the saliva of Heloderma suspectum, with 39 amino acids and 53% sequence homology with GLP-1. Exenatide, a synthetic Exendin-4 molecule, has a long half-life (3.3-4.0 h) in anti-hyperglycemic effect and is administered twice a day.
Liraglutide is a GLP-1 analogue, and has 97% homology with natural human GLP-1. It contains Arg→34Lys substitution and a glutamylpalmitoyl chain at 26Lys. After subcutaneous injection, it has an average elimination half-life of 13 h, and allows to be administered once a day, and its pharmacokinetic characteristics are not affected by ages, genders, kidney or liver functions.
PB-105 is produced by replacing cysteine at position 39 of Exenatide and is modified at the cysteine through specific PEGylation to prepare PB-110 (PEG5kd), PB-106 (PEG20kd), PB-107 (PEG30kd) and PB-108 (PEG40kd). PB-106 has a plasma T1/2 that is about 10 times higher than that of PB-105 and therefore exhibits better hypoglycemic activity, but the hypoglycemic activity per milligram (specific activity) is reduced above 90%.
Lixisenatide, a novel long-acting GLP-1R agonist which contains 44 amino acids, is similar to Exendin-4 in structure, except that there is no proline at position 38, and 6 lysine residues are added at position 39. In clinic trial for 24 weeks, Lixisenatide significantly reduced glucagon activity level after being administered once a day, the incidence of side reaction in the Lixisenatide group was similar to that in control group (Lixisenatide 2.5% vs placebo 1.9%), and the symptomatic hypoglycemia rate was 3.4% for Lixisenatide and 1.2% for placebo.
BPI-3016 is produced by structurally modifying the linkage (DIM) between position 8 (Ala) and positions 8-9 (Glu) of human GLP-1, wherein the —CH3 side chain in 8Ala is replaced with —CF3, the carbonyl in the linkage is converted into methyl, a palmitoylated Lys→26Arg substitution is used and a C-terminal Gly is added. BPI-3016 showed a half-life of over 95 hours in Diabetic cynomolgus monkeys after a single administration. Obviously after medication for one week, FPG, postprandial blood glucose (PPG), body mass index (BMI), and body fat were reduced, meanwhile the glucose tolerance was improved and the insulin-increasing effect was presented.
Albiglutide, which is a recombinant fusion protein, expressed from two linked copies of the human GLP-1 gene in tandem with the human albumin gene. It is endowed resistance to DPP-4 hydrolysis by using Gly→8Ala substitution, and allows to be administered once a week.
Research showed that Albiglutide can reduce blood glucose parameters (HbA1c, PPG, and FPG) by enhancing glucose-dependent insulin secretion and alleviating gastric emptying.
Dulaglutide, which is a GLP-1 analogue fused to a Fc fragment, has a structure of Gly8Glu22Gly36-GLP-1(7-37)-(Gly4Ser)3-Ala-Ala234,235Pro228-IgG4-Fc. It is administered once a week. Compared with placebo, metformin, insulin glargine, sitagliptin, and Exenatide, Dulaglutide showed a higher HbAlc reduction. Dulaglutide showed various curative effects in the treatment of T2D such as reducing body weight, alleviating nephropathy progression, decreasing the incidence of myocardial infarction, and reducing blood pressure.
Semaglutide, which is a long-acting GLP-1 analogue, has an Aib→8Ala substitution and a longer 26Lys linker (2xAEEAC-δ-glutamyl-α-oleic diacid). It maintains 94% homology with GLP-1. Compared with Liraglutide, Semaglutide showed 3 times of hypoglycemic activity reduction and an increasing binding ability with albumin. It was speculated that Semaglutide has a half-life of 165-184 hours (7 days). Semaglutide showed a significant reduction in HbAlc and body weight.
Taspoglutide contains α-aminoisobutyric acid (α-Aib)→8Ala and 35Gly with hGLP-1(7-36)NH2. Taspoglutide has a strong affinity with GLP-1R and a complete resistance to aminodipeptidase. In a clinic trial for 24 weeks, Taspoglutide showed a significant reduction of HbA1c, FPG, and body weight with obvious side effects.
There is still a need for the optimization of GLP-1 analogues, because it has been proven that the current long-acting activators are less effective than Liraglutide or natural GLP-1 in specific activity (hypoglycemic effect per milligram), administration dosage, body weight reduction, and side effects. For example, in a trial for 26 weeks, the body weight was reduced by 0.6 kg for Albiglutide group, by 2.2 kg for Liraglutide group, by 2.9 kg for Dulaglutide group, and by 3.6 kg for Liraglutide group. In rodents, Semaglutide can cause thyroid C-cell tumor on a dose-dependent and time-dependent manners. Clinical trial indicated that renal function was normal in 57.2%, mildly impaired in 35.9%, and moderately impaired in 6.9% of patients. Among patients receiving Semaglutide, gastrointestinal adverse reactions such as nausea, vomiting, diarrhea, abdominal pain, and constipation occurred more frequently than placebo (15.3% for placebo group, 32.7% and 36.4% for Semaglutide 0.5 mg and 1 mg groups). When Semaglutide was used in combination with sulfonylurea, severe hypoglycemia occurred in 0.8-1.2% of patients, injection-site discomfort and erythema occurred in 0.2% of patients. The patients had a mean increase in amylase of 13% and lipase of 22%. Cholelithiasis was occurred in 1.5% and 0.4%, respectively.
The present disclosure intends to provide a glucagon-like peptide 1 analogue monomer and homodimers thereof to overcome the above defects in the prior art.
The first purpose of the present disclosure is to provide a glucagon-like peptide 1 analogue monomer having an amino acid sequence that is any one of the following four sequences:
Preferably, when the X26 is lysine modified with alkanoylglutamyl [γ-Glu(N-α-alkanoyl)] on the side chain ε-amino, it has the structural formula in Formula 1; when the X26 is lysine modified with an alkanoyl on the side chain ε-amino, it has the structural formula in Formula 2, in Formulas 1 and 2, n is equal to 14 or 16:
The second purpose of the present disclosure is to provide a glucagon-like peptide 1 analogue homodimer. The dimers are formed by two identical monomers as described above connection through a disulfide bond between cysteines, constitute an H or U-like glucagon-like peptide 1 analogues.
Preferably, the amino acid sequence of the dimer is any one of the following four sequences:
wherein, X8 is L-α-alanine (Ala), β-alanine (β-ALa), or α-/β-amino isobutyric acid (α- or β-Aib);
X26 is lysine, lysine modified with alkanoylglutamyl on the side chain ε-amino, or lysine modified with an alkanoyl on the side chain ε-amino;
X34 is Arg, Lys, or lysine modified with alkanoylglutamyl on the side chain ε-amino;
X35 is Gly, Ala, β-alanine, or α-/β-amino isobutyric acid (Aib);
X37 is a moiety of Gly-COOH (C-terminal glycine with carboxyl group), Gly-NH2 (C-terminal glycine with amidation), NH2 (36arginine with amidation), or OH (36arginine with carboxyl terminus, or an allosteric amino acid sequence of the first 7-36 positions as provided in the first purpose which formed in a copy of one similar repeat sequence, wherein the 8alanine (X8) in the repeat sequence is replaced with a glycine, or α-/β-amino isobutyric acid (Aib), the cysteine is replaced with serine or glycine, and the X26 in the repeat sequence is arginine or a PEG-modification by combining the C-terminal amido with a polyethylene glycol molecule, wherein the molecular weight of the PEG is 0.5-30 KD.
Preferably, when the X26 is lysine modified with alkanoylglutamyl [γ-Glu(N-α-alkanoyl)] on the side chain ε-amino, it has the structural formula in Formula 1; when the X26 is lysine modified with an alkanoyl on the side chain ε-amino, it has the structural formula in Formula 2. In Formulas 1 and 2, n is equal to 14 or 16.
The third purpose of the present disclosure is to provide application of the above monomeric GLP-1 analogue or above dimeric GLP-1 analogue in preparation and of the pancreas protection or/and hypoglycemic drug for treating type II diabetes.
The fourth purpose of the present disclosure is to provide a drug for protecting pancreas or treating type II diabetes, which uses the above monomeric GLP-1 analogue or dimeric GLP-1 analogue as an active content.
The present disclosure has the beneficial effects that the H-like GLP-1 analogue homodimer significantly increases 2-4 times of hypoglycemic time than its single chain GLP 1 analogue i.e. the dimeric peptide had obviously increased specific activity and significantly prolonged the effect of the single chain of GLP-1R agonists approved by FDA. The in-vivo activity duration of the provided GLP-1 analogue homodimer is up to 19 days, which is obviously longer than that of the positive drug Liraglutide. Obviously a technological upgrade is achieved and it will be more benefit in clinical application and market sale. The U-like dimer does not affect blood glucose level, but can obviously protect pancreatic exocrine cells such as pancreas acini and ducts to protect pancreas functions, and thus can be used in treatment of pancreas-related diseases.
To more clearly illustrate the technical solution, objectives, and advantages of the present disclosure, the present disclosure will be described in detail in combination with specific examples and figures below.
Method b: 3 times of FMOC-AA and 1-hydroxybenzotriazole (HOBt, Suzhou Tianma Pharmaceutical Group Fine Chemicals Co., Ltd.) were dissolved with as little DMF as possible, and then added into a reaction tube. N,N′-diisopropylcarbodiimide (DIC) in 3 times was instantly added to react for 30 min. Detection showed negative.
8. The operations in steps 2-6 were repeated to successively combine correct amino acids as GLP-1 peptide sequence without or with side chain modification shown in Table 1 from the right to the left. The peptides with K26 or K34 modification were synthesized according to the method in 9 below.
Some monomeric peptides claimed herein can be synthesized in the above solid-phase synthesis, or also done through the genetic recombination combining with chemical modification as the examples of G3 and G9 syntheses below.
The identification method is following:
Some GLP-1 analogue monomers and the dimers were synthesized in our laboratory and some peptides were done by commercial companies. The inventor confirmed the peptide structure by using HPLC purity, EIMS, and cysteine oxidation. The amino acid sequences of the GLP-1 analogue monomers and the homodime were showed in the present disclosure as shown in Tables 1 and 2.
34Lys[N-ε-(N-α-Palmitoyl)] and 34Lys[N-ε-(N-α-oleoyl)] represent 34lysine modified with an alkanoyl on the ε-amino of the side chain;
C57B16/J mice were placed in an SPF-level environment with a standard diet. All experimental operations were performed in accordance with the guidelines of the ethics and uses of experimental animals. After feeding several days according to the standard diet, 5-week-old C57B16/J male mice were grouped: NaCl-PB, T2D model control, Liraglutiade, low, medium and high dose dimeric peptide 2G3 or 2G1 groups. The NaCl-PB group served as blank control and the model control group served as the T2D model control. They were injected with NaCl-PB solution. The T2D model group was fed with 60 kcal % high-fat diet (D12492, Changzhou SYSE Bio-tech. Co., Ltd., China) until the end of the experiment. Meanwhile the blank control group maintained standard diet. The preparation method of the diabetic models: After 4 weeks of high-fat feeding, the mice were injected intraperitoneally with 75 mg/kg dose of streptozotocin (STZ, Sigma Chemical Company, USA), and re-injected intraperitoneally with 50 mg/kg dose of STZ after 3 days, mice with blood glucose value equal to or higher than 11 mM after 3 weeks were considered as diabetic mice. These groups were subjected to a 35-day treatment study on the basis of the high-fat diet.
The 2G1 groups showed a dose-dependent decrease in insulin (P>0.05). The ALT level in the L-2G1 group was higher than those in the NaCl-PB group and the Liraglutide group, and the ALT level in the M-2G1 group was lower than those in the model control group and the L-2G1 group (P<0.05 or 0.01). The AST level in the M-2G1 group was significantly lower than that in the L-2G1 group (P<0.05), and the AST level in the H-2G1 group was significantly higher than that in the M-2G1 group (P<0.05). Compared with the NaCl-PB group or the L-2G1 group, the ALP level in the M-2G1 group was lower (P<0.05). The 2G1 groups showed a dose-dependent decrease in albumin (P<0.05, 0.01 or 0.001), and the albumin in the model control group was significantly lower than that in the NaCl-PB group (P<0.05). The serum creatinine level in the 2G1 groups was lower than that in the NaCl-PB or Liraglutide group, and had a dose-dependent decrease (P<0.05, 0.01 or 0.001). The total cholesterol (T-CHO) or HDL-CHO in the 2G1 groups had an dose-dependent decrease, whereas the T-CHO or/and HDL-CHO and LDL-CHO levels in the Liraglutide group or the 2G1 groups were significantly higher than those in the NaCl-PB group (P<0.01 or 0.001). The T-CHO and HDL-C-CHO levels in the L- and M-2G1 groups were significantly higher than those in the Liraglutide group (P<0.05 or 0.01). As 2G3, 2G1 significantly promoted synthesis of HDL. The HDL-CHO level in the H-2G1 group was significantly lower than that in the model control group (P<0.05). There was no significant difference in triglyceride (TG) between the groups. Interestingly, compared with the NaCl-PB group, the amylase in the 2G1 groups had a dose-dependent-decrease (P<0.05 or 0.01), indicating an obvious protective effect on pancreatic exocrine cells (Table 4).
The Ki67 protein in the model control, the Liraglutide, and the H-2G1 groups was significantly higher than that in the NaCl-PB group (P<0.05 or 0.01). Compared with the model control or the M-2G1 group, the Liraglutide and the H-2G1 group had significant differences (P<0.05). The Ki67 expression in the M-2G1 group was lower than that in the Liraglutide group (P<0.01). These results revealed that 2G1 significantly promoted the proliferation of pancreatic cells (
Distribution and location of insulin in T2D pancreatic islets were observed by using an anti-insulin antibody (
Conclusions: From the above examples, concluded that the classification based on the effective duration clearly distinguishes the characteristics of long-acting and short-acting moleculars. Obviously, the homodimeric 2G3 and 2G6 series developed belong to the longest-acting molecules. The 2G3 peptide as a representative in dimeric peptides induces the synthesis of insulin by binding with GLP-1R and generate the hypoglycemic effect in T2D model. The biological effects of active GLP-1 homodimers were evaluated in various assays. These studies suggest that the dimeric peptides show the most promising application for T2D in rodent models, such as the longest-lasting hypoglycemic effects and other effects in weight loss and organ protection.
The structure-activity relationship reveals that the dimers without Aib have the best solubility in water and the dimers with Aib or even an amidated moiety at the C-terminus have a poor solubility in water. The individual peptide of them can maintain a long activity. These properties suggest that in the 2G3 peptide, the N-terminal moiety containing 8Ala sequence may be wrapped by the symmetrical 26Lys-glutamyl fatty acid chain in the dimer to form a hydrophobic core, which in turn is surrounded by a hydrophilic polypeptide chain and is not easily hydrolyzed by DPP-4, so that a longer effect is maintained. For the sequences containing Aib or even having an amidated moiety at the C-terminus, the Aib and the amidated moiety may be exposed, resulting in low solubility in water. Since Aib is not a substrate for DDP-4, it can maintain a longer activity. Aminoisobutyric acid (Aib) and β-Ala are similar to L-α-Ala or Gly, β-Aib and β-Ala are also normal metabolites of human pyrimidine nucleotides, and can be highly tolerated in human body. Therefore, the toxic reaction of these compounds should be extremely low, and thus, the present disclosure uses these amino acids substitution to significantly prolong the hypoglycemic activity.
In the hypoglycemic effect of normal mice, the results of single OGTT experiment showed that the dimer has a long hypoglycemic effect through slow absorption in the blood. The results of the multiple OGTT experiment showed that the long duration effect involves the amino acid at position 8 of the polypeptide, the position of the disulfide bond in the dimer, the symmetrical 26Lys fatty acid modification and the C-terminal amidation, and is independent of the Lys modification at multiple sites in the same molecule. Table 2 shows that the long active structure contains 8Aib, 18Cys-Cys disulfide bond, symmetrical oleoyl-1-γ-glutamoyl-26Lys, and C-terminal amidated moiety. These modifications are characterized in that (1) α-/β-Aib or β-Ala→8Ala substitution produces longer activity, in which α-Aib substitution generates the best effect; (2) compared with other fatty acid modifications, the mono-oleoyl-L-γ-glutamyl-26Lys structure achieves the best results; (3) C-terminal amidation significantly prolongs the activity: (4) the disulfide bond moiety at the position 18 in the dimer molecule shows the best activity; (5) PEG modification significantly shortens the specific activity (hypoglycemic duration per mg) while prolonging the half-life; (6) the activity of the monomeric peptide is only ½-¼ of that of the corresponding dimer.
In the T2D treatment experiments, the HbA1c reduction (−8, −23, −32%) or FPG reduction (−26.3, −46.9 and −47.3%) in the 2G3 groups and the fasting HbA1c reduction (−29%) or FPG reduction (−50.2%) in the Liraglutide group obviously showed hypoglycemic effects, indicating that 2G3 peptides and Liraglutide in the same molar concentration have similar effects on PPG, FPG and HbA1c.
The body weight of 2G3 groups had a dose-dependent decrease. The H-2G3 group had similar weight curve in body weight or adipose tissue to that in Liraglutide group, suggesting that it had less influence on diet and fat metabolism compared with Liraglutide. This was also confirmed by statistic data in drinking water or food during the preparation of T2D animals. However, the weights of some organs such as left kidney, right testis, and adipose tissue increased, indicating that compared with Liraglutide, this dimer has less influence on diet and fat metabolism. 2G3 caused the increase of liver weight and alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase showed a dose-dependent decrease, indicating that the drug has a strong protective effect on liver and heart. But 2G3 led to a higher alkaline phosphatase level than Liraglutide, indicating stronger stimulation to liver. The increase in platelet quantity and spleen weight showed that 2G3 can promote hemostasis to protect the integrity of the vascular wall in T2D models. The Albumin in the 2G3 groups had a dose-dependent increase, suggesting that 2G3 may be transported by binding to albumin like Liraglutide. However, compared with the normal NaCl-PB group, the T2D model groups had significant decrease in albumin, showing that the hypertension, hyperlipidemia, hyperglycemia, or STZ reagent caused less albumin. 2G3 induced more total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol, showing that it increase the synthesis of cholesterol. Compared with the Liraglutide group, the total cholesterols were higher in the L- and M-2G3 groups, and the high-density lipoproteins was higher in the M- and H-2G3 groups, indicating that 2G3 promotes the retrograde transport of cholesterol by increasing the high-density lipoprotein. The significant pancreatic enlargement and higher amylase level in the M- and H-2G3 groups indicated that 2G3 had a certain promoting effect on pancreatic exocrine. 2G3 had no effects on kidney and lung function as well as white blood cells, red blood cells, hemoglobin, creatinine, and triglyceride.
The 2G1 groups showed significant increase in weights of liver and spleen, higher alanine aminotransferase, and aspartate aminotransferase levels, and less alkaline phosphatase and albumin levels, indicating that it significantly affects the functions of liver and spleen.
In the T2D treatment experiment with 2G3, the NaCl-PB mice (HbA1c 7.3±2.45 mm and FPG 5.171±4.24 mm) had the normal insulin level (1.411±3.01 ng/ml) and T2D control mice (HbA1c 20±5.03 mm and FPG 14.149±5.95 mm) had the T2D insulin value (0.625±0.23 ng/ml), but the Liraglutide group (HbA1c 14.2±2.20 mm and FPG 7.042±1.63 mm) induced the insulin value of 0.595±0.21 ng/ml, showing that T2D showed significant increase in insulin tolerance. Meanwhile Liraglutide induced a lower insulin level by the inhibition of diet. The insulin content (0.626±0.23, 1.141±0.66, 1.568±1.79 ng/ml) in the 2G3 groups had a dose-dependent increase. The percentage increments of these insulin values corresponding to the Liraglutide group are +5.2, +91.8, and +163.5%, indicating that 2G3 has stronger insulin release than Liraglutide, and therefore 2G3 has a better hypoglycemic effect. If the hypoglycemic effect was evaluated according to the secretion amount of insulin, the L-2G3 group should have a bioequivalent relationship with the Liraglutide group, and the hypoglycemic effect of the M- or H-2G3 group should be doubled or higher, but the M-2G3 group actually shows similar hypoglycemic effect with the Liraglutide group, which reflects that higher dose of 2G3 does not induce a greater hypoglycemic effect, or even hypoglycemia when the blood glucose level was normal. In this experiment, 8 and 68 times of low dose of 2G3 did not induce any one of 6 KM mice fasted for 13 hour to produce hypoglycemia within 3 hours after the administration, indicating that the dimeric peptides do not induce hypoglycemia.
The results of H-E staining showed that compared with the NaCl-PB group, 2G3 or 2G1 strongly protected pancreatic acinar cells and rescued pathological damages in T2D models, such as sparse acini, pathological vacuoles, pancreatic islet cell deformation, atrophy or pyknosis. 2G3 induced a dose-dependent increase in Ki67, suggesting that 2G3 promotes the proliferation of pancreatic cells. The expression of Ki67 in the M-2G1 group was lower than that in the Liraglutide group, indicating that 2G1 group had a weaker proliferation of pancreatic cells than the Liraglutide group. The TUNEL positive rate in the 2G1 groups had a dose-dependent decrease, and the TUNEL positive rate in the H-2G1 group was lower than that in the Liraglutide group or the M-2G1 group, indicating that 2G1 significantly protects pancreatic cells such as acinus and ducts against STZ toxicity or pathological damage. 2G3 induced significant increase in GLP-1R expression, the insulin staining intensity, and the number of pancreatic islet, suggesting that the hypoglycemic effect of 2G3 was mediated by GLP-1R, resulting in more insulin release and more pancreatic islets.
Our conclusion is that the monomeric or dimeric peptides claimed in the present disclosure can induce more insulin release by binding to GLP-1R, thereby resulting in different hypoglycemic or pancreatic protective effects.
The above embodiments only represent several embodiments of the present disclosures, The descriptions are specific and detailed, but cannot be understood as a limitation in the scope of the present disclosures. It was noted that the persons skilled in the arts can make development and improvements in the concept of the present disclosures. These development and improvements fall into the protection scope of the present disclosures. Therefore, the protection scope of the present application shall be determined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910969964.X | Oct 2019 | CN | national |
| 201911142332.2 | Nov 2019 | CN | national |
This application is the national phase entry of International Application No. PCT/CN2020/127422, filed on Nov. 9, 2020, which is based upon and claims priority to Chinese Patent Application No. 201910969964.X, filed on Oct. 12, 2019, and Chinese Patent Application No. 201911142332.2, filed on Nov. 20, 2019, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/127422 | 11/9/2020 | WO |
