CN118146350A - Preparation method and application of GLP-2 peptide analogue - Google Patents

Preparation method and application of GLP-2 peptide analogue Download PDF

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CN118146350A
CN118146350A CN202410283735.3A CN202410283735A CN118146350A CN 118146350 A CN118146350 A CN 118146350A CN 202410283735 A CN202410283735 A CN 202410283735A CN 118146350 A CN118146350 A CN 118146350A
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pharmaceutically acceptable
acceptable salt
peptide
reaction
resin
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朱玉珍
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Beijing Zeqin Biomedical Co ltd
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Beijing Zeqin Biomedical Co ltd
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Abstract

The invention discloses a preparation method and application of GLP-2 peptide analogues, wherein the GLP-2 peptide analogues are all reported for the first time, and the invention proves the effectiveness of the GLP-2 peptide analogues in the treatment of gastrointestinal related diseases through experiments, and further proves that the effect of the GLP-2 peptide analogues is obviously better than that of commercial GLP-2 analogues Apraglutide, and has good clinical application prospect.

Description

Preparation method and application of GLP-2 peptide analogue
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a preparation method and application of GLP-2 peptide analogues.
Background
Glucagon-like peptides (Glucagon-LIKE PEPTIDE), including glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2), both derived from glucagon-like peptide (Proglucagon). GLP-1 has the pharmacological effects of promoting insulin secretion, protecting islet beta cells, inhibiting glucagon secretion, inhibiting gastric emptying and reducing appetite, and can be clinically used for treating type II diabetes and obesity; GLP-2 is a nutritional factor, has pharmacological effects of promoting small intestine growth, inhibiting apoptosis, promoting gastric emptying and stimulating appetite, and can be clinically used for treating gastrointestinal related diseases.
Glucagon and its hyperglycemic effect were found earlier than 1922, and its precise amino acid sequence was determined as early as 1957. The most important milestone in this process is probably the identification of pre-glucagon, the precursor protein, early in the 80 s of the 20 th century, which paves the way for the characterization of the pre-glucagon derived peptide (PGDP) family.
The glucagon gene (GCG) is located on chromosome 2, and its expression results in the synthesis of glucagon, which is subsequently cleaved into glucagon. Depending on the tissue of expression, different groups PGDPs are obtained by further enzymatic treatment with Prohormone Convertases (PCs); cleavage of PC2 (most abundant in pancreatic alpha cells) results in the production of glucagon and major glucagon pre-fragment (MPFG), while the action of PC1/3 in L enteroendocrine cells results in the formation of glucagon-like peptide-1 and glucagon-like peptide-2 (GLP-1 and GLP-2, respectively), oxymodulin and glucagon. PGDPs exert unique physiological effects in metabolism and energy regulation by binding to specific G protein-coupled membrane receptors, which makes them promising candidates for the treatment of a variety of clinical entities. In particular, GLP-1 receptor agonists have been approved for the treatment of type 2 diabetes (T2 DM) since 2005, and GLP-2 analogues are being investigated for their potential use in various gastrointestinal disorders.
The GLP-2 receptor is a G protein coupled transmembrane receptor, as is the GLP-1 receptor. GLP-2 binding to its receptor in the gastrointestinal tract results in increased production of intracellular cyclic adenosine monophosphate (c-AMP), which in turn stimulates intestinal cell proliferation while inhibiting apoptosis. Thus, GLP-2 exhibits an enteral nutritional effect. GLP-2 and the remaining PC1/3-CLEAVED PGDPS and PEPTIDE YY are released from enteroendocrine L cells after ingestion of nutrients. Although there is a continuing interest in the potential use of GLP-2 in the treatment of diabetes, there is growing evidence that the primary role of GLP-2 is to regulate the growth and proliferation of gastrointestinal cells, and its key properties in increasing intestinal and portal venous blood flow and decreasing gastrointestinal motility. These GI attributes of GLP-2 make it an effective drug for the treatment of several debilitating GI diseases, especially short bowel syndrome-bowel failure (SBS-IF), acute graft versus host disease (aGVHD).
Short Bowel Syndrome (SBS) refers to a syndrome that after extensive small intestine excision or relaxation, the effective absorption area of the intestine is significantly reduced, the remaining functional intestinal canal cannot sustain the nutrition or growth needs of the patient, and diarrhea, acid-base/water/electrolyte disorders, and various nutrient absorption and metabolism disorders are predominant. The severity and prognosis of the disease depends on the primary disease, the length and location of the remaining small intestine, whether the ileocecal valve and colon remain, and whether the intestinal adaptation process is good, etc. The domestic morbidity has a tendency to rise year by year according to the statistics of the data of the participation centers of the Chinese short bowel syndrome treatment cooperative group, but no exact statistical data of the morbidity is available nationwide. Common primary causes of adult SBS are mesenteric vascular disease, crohn's disease, radiation enteritis, trauma, and intestinal fistulae. The consensus of Chinese short bowel syndrome diagnosis and treatment (2016 edition, nanjing) indicates that it is difficult for SBS patients in China to effectively develop long-term home PN (parenteral nutrition) at present, and it is recommended that such patients should be hospitalized for standard nutritional support and related medication.
Acute graft versus host disease (aGVHD) is a life threatening complication following allogeneic hematopoietic cell transplantation, and the gastrointestinal tract (GI, aGVHD) is one of the major causes of morbidity and mortality. Acute graft versus host disease (aGVHD) is a serious and life threatening complication that is common after allogeneic hematopoietic cell transplantation (allogeneic hematopoietic cell transplantation, alloHCT). In the united states, europe and japan, approximately 26000 patients receive allogeneic HCT therapy each year. Although prevented by immunosuppressive therapy, approximately 20-80% of patients receiving allogeneic HCT therapy develop aGVHD. For initial treatment of aGVHD, the consensus suggests support for the use of large doses of systemic steroids. Patients who develop Steroid Refractory (SR) -aGVHD suffer from impaired survival and about 50% mortality. At the same time, there is no consensus on standard treatment methods, which is one of the reasons for limited progress in the last decades.
Of the approximately 70% of aGVHD patients, gastrointestinal (GI) tract-associated aGVHD is the leading cause of associated morbidity and mortality, and is one of the most difficult gastrointestinal diseases to treat. The major tissue system of aGVHD is damaged, leading to impaired mucosal barriers, protein loss and overall malabsorption. Strategies other than adding immunosuppression are being explored to overcome current therapeutic challenges to promote healing of aGVHD-related tissue lesions, aimed at restoring tissue homeostasis. Glucagon-like peptide (GLP-2) is an enteroendocrine hormone enterol cell produced by enterol cells to maintain normal gastrointestinal integrity, a key injury target for cytotoxic opsonic therapy and aGVHD. In both mouse and human aGVHD patients, both the number of L cells and the level of GLP-2 showed a decrease, and preclinical studies indicate that GLP-2 analogues have regenerating and protecting effects on the gastrointestinal tract, suggesting a role in the prevention or treatment of gastrointestinal aGVHD.
However, natural GLP-2 is easily hydrolyzed and inactivated by dipeptidyl peptidase 4 (DPP-4), and has a half-life (t 1/2) of only a few minutes, thus having no clinical medicinal value. The structure of the DPP-4 enzyme is modified, the binding site of the DPP-4 enzyme is covered, the half life period of the DPP-4 enzyme is prolonged, and the clinical application frequency of patients is reduced, so that the DPP-4 enzyme is a main strategy for developing the DPP-4 enzyme. The global sales of the unique GLP-2 drug Tidolutetin (Gattex) in 2022 is 7.5 hundred million dollars, and the market prospect is wide.
Disclosure of Invention
In view of the above, the invention modifies the structure of natural GLP-2 with short half-life and no clinical pharmaceutical value, which is the technical problem in the prior art, and not only can achieve the effect of sealing DPP-4, but also can more stably play a role in vivo. The GLP-2 peptide analogues A1-A6 prepared by the invention are all reported for the first time, the effectiveness of the GLP-2 peptide analogues in the treatment of gastrointestinal related diseases is proved by experiments, and the effect of the GLP-2 peptide analogues is further proved to be obviously better than that of a commercial GLP-2 analogue Apraglutide (apaglalutide), so that the GLP-2 peptide analogues have good clinical application prospect.
The above object of the present invention is achieved by the following technical solutions:
In a first aspect, the invention provides an agonistic peptide of a GLP2R target or a pharmaceutically acceptable salt thereof.
Further, the agonist peptide or a pharmaceutically acceptable salt thereof comprises or consists of an amino acid sequence of formula I:
general formula (VI) Ⅰ：X1-X2-DGSFSDE-Nle-DPhe-TILD-X16-LAARDFINWLIQT-X30-ITD-XX1-R1;
Wherein,
X 1 is H or Y;
X 2 is Aib or Mfe-Gly or other unnatural amino acid;
x 16 is N or L;
x 30 is ψ, ψ is Lys with side chains modified;
XX 1 is GGPSSGAPPPS(SEQ ID NO:1)、GGGPSSGAPPPS(SEQ ID NO:2)、GGPSSGAdPPPS(SEQ ID NO:3)、GGGPSSGAdPPPS(SEQ ID NO:4)、GGPSSGAPPPSKVSRA(SEQ ID NO:5)、GGGPSSGAPPPSKVSRA(SEQ ID NO:6)、GGPSSGAdPPPSKVSRA(SEQ ID NO:7) or GGGPSSGA d PPPSKVSRA (SEQ ID NO: 8);
R 1 is NH 2 or OH, or a pharmaceutically acceptable salt and/or ester thereof.
Further, the ψ is Lys having a side chain modified by a structure having the following general formula ii;
General formula II: B-Z;
Wherein B is (AEEA or Glu) a - (AEEA or Glu) b - (AEEA or Glu) c, wherein a, B, c are each independently 0 or 1, and a, B, c are not simultaneously 0; the carboxyl end of the B is connected with epsilon-amino of a side chain of Lys, Z is-CO- (CH 2)m-R2), m is an integer between 6 and 24, and R 2 is selected from-COOH.
Further, the general formula II is AEEA-gamma G1u-CO (CH 2)18 COOH;
preferably, the agonistic peptide or a pharmaceutically acceptable salt thereof is chemically modified at least one site by a fatty acid side chain group.
Further, the agonistic peptide or pharmaceutically acceptable salt thereof is selected from any one of the following:
H-Aib-DGSFSDE-Nle-DPhe-TILD-L-LAARDFINWLIQT-K (AEEA-K-C18) -ITD-GGPSSGAPPPS-NH 2, designated A1;
H-Aib-DGSFSDE-Nle-DPhe-TILD-D-LAARDFINWLIQT-K (AEEA-K-C18) -ITD-GGGPSSGAPPPS-NH 2, designated A2;
H-Aib-DGSFSDE-Nle-DPhe-TILD-D-LAARDFINWLIQT-K (AEEA-K-C18) -ITD-GGPSSGAPPPS-NH 2, designated A3;
H-Aib-DGSFSDE-Nle-DPhe-TILD-L-LAARDFINWLIQT-K (AEEA-K-C20) -ITD-GGPSSGAPPPS-NH 2, designated A4;
H-Aib-DGSFSDE-Nle-DPhe-TILD-D-LAARDFINWLIQT-K (AEEA-K-C20) -ITD-GGPSSGAPPPS-NH 2, designated A5;
H-Aib-DGSFSDE-Nle-DPhe-TILD-D-LAARDFINWLIQT-K (AEEA-K-C20) -ITD-GGGPSSGAPPPS-NH 2, designated A6.
Further, the agonistic peptide or pharmaceutically acceptable salt thereof can significantly enhance the small intestine quality;
Preferably, the agonistic peptide or pharmaceutically acceptable salt thereof is capable of significantly enhancing the cellular activity of small intestine epithelial cells;
Preferably, the agonistic peptide or pharmaceutically acceptable salt thereof is capable of significantly increasing the small intestine length;
Preferably, the agonistic peptide or pharmaceutically acceptable salt thereof is capable of significantly increasing the weight of the small intestine;
preferably, the agonistic peptide or pharmaceutically acceptable salt thereof is capable of significantly increasing the amount of small intestinal villi;
Preferably, the agonistic peptide or a pharmaceutically acceptable salt thereof is capable of significantly increasing crypt depth.
In a specific embodiment of the present invention, the A0 is Apraglutide (aparaglutide), the sequence is HGDGSFSDE-Nle-f-TILDLLAARDFINWLIQTKITD (molecular formula: C 172H263N43O52, molecular weight: 3765.25).
In a second aspect, the invention provides a pharmaceutical composition.
Further, the pharmaceutical composition comprises an agonistic peptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof;
preferably, the pharmaceutical composition may further comprise other drugs which can be used for the treatment and/or prevention of stomach and/or intestine related diseases;
More preferably, the stomach and/or intestine related diseases comprise short bowel syndrome, gastrointestinal acute graft versus host disease, malabsorption syndrome, cullet syndrome, inflammatory bowel disease, abdominal type site-prednisole diarrhea, tropical type site-prednisole diarrhea, low-propylene globulinemia type site-prednisole diarrhea, mucositis and diarrhea caused by chemotherapy and/or radiation therapy, ulcers, digestive disorders, enteritis, crohn's disease, small intestine injury, metabolic syndrome, obesity and/or diabetes.
In the present invention, the stomach and/or intestine-related diseases are not limited to the specific diseases listed above, and any diseases that can be improved, assisted by promotion of small intestine growth, enhanced nutrient absorption, assisted by treatment, assisted by therapy, prevention and/or assisted by prevention are within the scope of the present invention.
In a third aspect the invention provides a pharmaceutical formulation.
Further, the pharmaceutical formulation comprises a pharmaceutical composition according to the second aspect of the invention;
Preferably, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier and/or adjuvant;
More preferably, the pharmaceutically acceptable carrier and/or adjuvant comprises a buffer, a non-ionic tonicity modifier;
More preferably, the buffer comprises a histidine buffer, a mesylate buffer, an acetate buffer, a glycine buffer, a lysine buffer, a TRIS buffer, a Bis-TRIS buffer and/or a MOPS buffer;
More preferably, the nonionic tonicity modifier comprises mannitol, sucrose, glycerol, sorbitol and/or trehalose;
preferably, the dosage form of the pharmaceutical preparation comprises a liquid preparation, a solid preparation, a semisolid preparation and a gas preparation;
more preferably, the liquid formulation comprises injection, solution, suspension;
More preferably, the solid formulation comprises a tablet, capsule, powder, granule;
more preferably, the semi-solid formulation comprises a gel, cream, suppository;
more preferably, the gas formulation comprises an aerosol, a spray, a powder spray.
In some embodiments, the buffer represents a pharmaceutically acceptable excipient that stabilizes the pH of the pharmaceutical formulation. Suitable buffers are well known in the art and can be found in the literature. In particular, the buffering agents include, but are not limited to: histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, bis-TRIS buffer and MOPS buffer, as these buffers provide stable formulations in which the GLP-2 analogue dissolves and does not become sticky, turbid or precipitate the peptide drug. In some preferred embodiments, the buffer is a histidine buffer, such as L-histidine. Typically, the buffer will be present at a concentration of about 5mM to about 50mM, more preferably at a concentration of about 5mM to about 25mM, and most preferably at a concentration of about 15 mM.
In some embodiments, the tonicity modifying agent represents a pharmaceutically acceptable tonicity agent for modifying the tonicity of a formulation. The formulation of the invention is preferably isotonic, i.e. it has substantially the same osmotic pressure as human serum. The tonicity modifier used in the formulation is preferably a non-ionic tonicity modifier and is preferably selected from mannitol, sucrose, glycerol, sorbitol and trehalose. The preferred nonionic tonicity modifier is mannitol, such as D-mannitol. The concentration of tonicity adjusting agent will depend on the concentration of the other components of the formulation, especially if the formulation is intended to be isotonic. Typically, the nonionic tonicity modifier will be used at a concentration of about 90mM to about 360mM, more preferably at a concentration of about 150mM to about 250mM, and most preferably at a concentration of about 230 mM.
A fourth aspect of the invention provides any one of the following products:
(1) A kit comprising a pharmaceutical composition according to the second aspect of the invention or a pharmaceutical formulation according to the third aspect of the invention, and a container containing the pharmaceutical composition or pharmaceutical formulation;
(2) A delivery device comprising a liquid formulation of an agonistic peptide or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention;
preferably, the delivery device comprises a prefilled syringe, a syringe device, an injection pen, a dispensable automatic syringe, a disposable automatic syringe, a wearable syringe, an infusion pump.
In some embodiments, the delivery device is intended for parenteral administration of a liquid formulation of an agonistic peptide according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, to a subject in need thereof, examples of which include, but are not limited to: prefilled syringes, syringe devices, injection pens, adjustable dose autoinjectors, disposable autoinjectors, wearable syringes, and infusion pumps.
In a fifth aspect the present invention provides a method of preparing an agonistic peptide according to the first aspect of the invention or a pharmaceutically acceptable salt thereof.
Further, the preparation method comprises the following steps:
(1) Pretreatment of resin: adding the resin into a reaction kettle, adding DMF, introducing nitrogen, stirring for swelling, filtering to remove the solvent, and washing;
(2) Preparing an amino acid solution: fmoc-Ser (tBu) -OH and HOBt are dissolved by DMF, DIC is added, and the mixture is uniformly mixed;
(3) Deprotection: adding 20% piperidine/DMF solvent into a reaction kettle, introducing nitrogen, stirring for reaction, and washing the obtained resin after the reaction is completed;
(4) Coupling reaction: adding the amino acid solution prepared in the step (2) into a reaction kettle, controlling the temperature, introducing nitrogen, stirring and reacting, washing the obtained resin after the reaction is completed, and repeating the operations, wherein the amino acid sequence of the agonistic peptide or the pharmaceutically acceptable salt thereof is sequentially coupled with corresponding protective amino acids until the backbone of the agonistic peptide is synthesized;
(5) And removing the Lys side chain protecting group iLEde: adding 8% hydrazine hydrate/DMF solution into a reaction kettle, introducing nitrogen, stirring for reaction, filtering to remove solvent after the reaction is completed, and washing;
(6) Modification of Lys side chain: dissolving Eicosanedioic acid (mon-tBu) -gamma Glu (alpha-OtBu) -AEEA-OH and HOBt with DMF, adding DIC, mixing uniformly, adding into a reaction kettle, controlling temperature, introducing nitrogen, stirring for reaction, washing the obtained resin after the reaction is completed to obtain peptide resin containing the agonistic peptide or the pharmaceutically acceptable salt thereof according to the first aspect of the invention, and drying;
preferably, the method further comprises the steps of:
(a) Peptide resin cleavage: preparing cutting fluid, adding the peptide resin obtained by drying in the step (6) into the cutting fluid, stirring for reaction, filtering after the reaction is completed, adding filtrate into ice methyl tertiary butyl ether for precipitation, carrying out suction filtration, washing a filter cake with methyl tertiary butyl ether slurry, and carrying out vacuum drying to obtain a crude product;
(b) Purifying and refining: refining and purifying the crude product by a C18 reversed phase preparation chromatographic system to obtain a purified product;
Preferably, the resin in step (1) is RINK AMIDE AM RESIN resin;
Preferably, the resin in step (1) is used in a dose of 26.30g;
preferably, the DMF used in step (1) is used in a dosage of 200mL;
preferably, the swelling time in step (1) is 30min;
Preferably, the conditions of the washing in step (1) are 3 washes with DMF, 200mL each time;
preferably, the Fmoc-Ser (tBu) -OH, HOBt, DMF, DIC in step (2) is used in a dose of 11.50g, 4.04g, 150mL, 3.78g, respectively;
Preferably, the 20% piperidine/DMF solvent of step (3) is used in a dosage of 200mL;
preferably, the time of the reaction in step (3) is 35min;
preferably, the conditions of the washing in step (3) are 6 washes with DMF 200mL each;
preferably, the step (3) further comprises detecting the washed resin with ninhydrin reagent, wherein the resin is positive;
Preferably, the temperature control condition in the step (4) is 25-35 ℃;
preferably, step (4) further comprises monitoring the progress of the reaction with ninhydrin reagent during the reaction until the resin is negative indicating that the reaction is complete;
preferably, the conditions of the washing in step (4) are 3 washes with DMF 200mL each;
preferably, the 8% hydrazine hydrate/DMF solution in step (5) is used in an amount of 200mL;
preferably, the time of the reaction in step (5) is 1h;
preferably, the conditions of the washing in step (5) are 12 washes with DMF, 200mL each time;
Preferably, the step (5) further comprises taking resin and detecting the resin by ninhydrin reagent after the washing is finished, wherein the resin is positive;
Preferably, the dosage of Eicosanedioic acid (mon-tBu) - γGlu (α -OtBu) -AEEA-OH, HOBt, DMF, DIC used in step (6) is 14.58g, 2.70g, 300mL, 2.60g, respectively;
preferably, the temperature control condition in the step (6) is 25-35 ℃;
Preferably, step (6) further comprises monitoring the progress of the reaction with ninhydrin reagent until the resin is negative indicating that the reaction is complete;
preferably, the conditions of the washing in step (6) are 4 times with DMF, 2 times with DCM, 3 times with methyl tert-butyl ether, 200mL each;
More preferably, the cutting fluid in step (a) is formulated according to TFA/TIS/DTT/H 2 o=180/5/5/10;
more preferably, the time of the reaction in step (a) is 3h;
more preferably, the ice methyl tertiary butyl ether in step (a) is 5 volumes of ice methyl tertiary butyl ether;
More preferably, the sizing conditions in step (a) are 3 times with methyl tert-butyl ether.
A sixth aspect of the invention provides the use of any one of the following:
(1) The use of an agonistic peptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and/or prevention of gastric and/or intestinal related diseases;
(2) Use of an agonistic peptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof and/or a pharmaceutical composition according to the second aspect of the present invention for the preparation of a pharmaceutical formulation for the treatment and/or prevention of a gastric and/or intestinal related disorder;
(3) Use of an agonistic peptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, a pharmaceutical composition according to the second aspect of the present invention and/or a pharmaceutical formulation according to the third aspect of the present invention for the manufacture of a kit for the treatment and/or prophylaxis of a gastric and/or intestinal related disorder;
(4) Use of an agonistic peptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, a pharmaceutical composition according to the second aspect of the present invention and/or a pharmaceutical formulation according to the third aspect of the present invention for the manufacture of a delivery device for the treatment and/or prevention of a gastric and/or intestinal related disorder;
(5) Use of an agonistic peptide according to the first aspect of the invention or a pharmaceutically acceptable salt thereof, a pharmaceutical composition according to the second aspect of the invention, a pharmaceutical formulation according to the third aspect of the invention and/or a delivery device according to the fourth aspect of the invention for the manufacture of an agent or medicament for increasing the mass of the small intestine, increasing the cellular activity of the epithelial cells of the small intestine, increasing the length of the small intestine, increasing the weight of the small intestine, increasing the number of villi of the small intestine and/or increasing the depth of the crypt;
Preferably, the stomach and/or intestine related diseases comprise short bowel syndrome, gastrointestinal acute graft versus host disease, malabsorption syndrome, cullet syndrome, inflammatory bowel disease, abdominal type site-prednisole diarrhea, tropical type site-prednisole diarrhea, low-propisochlainemia type site-prednisole diarrhea, mucositis and diarrhea caused by chemotherapy and/or radiation therapy, ulcers, digestive disorders, enteritis, crohn's disease, small intestine injury, metabolic syndrome, obesity and/or diabetes.
Furthermore, the present invention provides a method for treating and/or preventing stomach and/or intestine related diseases, the method comprising the steps of: administering to a subject in need thereof a therapeutically effective amount of an agonistic peptide according to the first aspect of the invention or a pharmaceutically acceptable salt thereof, a pharmaceutical composition according to the second aspect of the invention and/or a pharmaceutical formulation according to the third aspect of the invention.
Further, the stomach and/or intestine-related diseases include short bowel syndrome, gastrointestinal acute graft versus host disease, malabsorption syndrome, cullet syndrome, inflammatory bowel disease, abdominal type site-oriented diarrhea, tropical type site-oriented diarrhea, low-propisochlainic type site-oriented diarrhea, mucositis and diarrhea caused by chemotherapy and/or radiation therapy, ulcers, digestive disorders, enteritis, crohn's disease, small intestine injury, metabolic syndrome, obesity and/or diabetes.
Further, the subject includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. In a specific embodiment of the invention, the subject is preferably a human.
Drawings
Fig. 1: results of the effect of peptide compounds on mouse Mode-k cell activity;
fig. 2: results of peptide compounds on mouse plasma Cit content;
fig. 3: a graph of the effect of peptide compounds on small intestine length changes;
fig. 4: HE staining (40X) results plot of small intestine;
fig. 5: a graph of small intestine villus relative length statistics;
Fig. 6: results of peptide compound effect on mouse serum ALT/AST levels.
Detailed Description
The inventor creatively prepares GLP-2 peptide analogues A1-A6 for the first time, and experiments prove that A1-A6 can remarkably improve the quality of small intestine, improve the cell activity of small intestine epithelial cells, increase the length of small intestine, increase the weight of small intestine, increase the villus quantity of small intestine and/or increase the depth of crypt, thus having good application prospect in the technical field of gastrointestinal related disease treatment, and in addition, the invention also proves that the effect of the GLP-2 peptide analogues A1-A6 is remarkably better than that of commercial GLP-2 analogues Apraglutide (apaglalutide) through comparison experiments.
In the present invention, abbreviations used have the following meanings:
GLP-2: glucagon-like peptide-2;
nle: norleucine;
DPhe: d-phenylalanine;
Aib: a-amino isobutyric (α -aminoisobutyric acid);
Mfe-Gly: mfe-esterified glycine;
a d: d-alanine (D-alanine);
γg1u: gamma glutamic acid;
AEEA: [2- (2-amino-ethoxy) -ethoxy ] -ethyl-phenyl;
cAMP: adenosine cyclophosphate;
Fmoc: fluorenylmethoxycarbonyl;
boc: t-butoxycarbonyl;
DMF: dimethylformamide;
Trt: a trityl group;
iLEde:1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) -3-methyl-butyl;
tBu: a tertiary butyl group;
OtBu: an oxy-t-butyl group;
TFA: trifluoroacetic acid;
TIS: triisopropylsilane;
DCM: dichloromethane.
The amino acids in the sequences of the compounds of the invention are derived from natural amino acids or related amino acid variants and/or derivatives. The abbreviations and codes for the natural amino acids employ common rules well known to those skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In order to facilitate an understanding of the present invention, the following terms referred to in the present invention are explained herein:
As used herein, the terms "comprises" or "comprising" are intended to include the stated element or component without excluding other elements or other components.
As used herein, the term "GLP-2 peptide analogue" refers to a GLP-2 peptide analogue having one or more amino acid substitutions, deletions, inversions, additions or other alterations compared to the native GLP-2 peptide. Analogs provided herein may additionally have chemical modifications of one or more of their amino acid side groups, alpha-carbon atoms, terminal amino groups, or terminal carboxylic acid groups.
In some embodiments, the chemical modification includes, but is not limited to: adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications at the amino acid side groups include, but are not limited to: acylation of lysine epsilon-amino groups, N-alkylation of arginine, histidine or lysine, alkylation of glutamic acid or aspartic acid carboxylic acid groups, and deamidation of glutamine or asparagine. Modification of the terminal amino group includes, but is not limited to: deamination, N-lower alkyl, N-di-lower alkyl and N-acyl modification. Modification of the terminal carboxyl group includes, but is not limited to: amide, lower alkylamide, dialkylamide and lower alkyl ester modifications.
It will be appreciated that the GLP-2 peptide analogues (pharmaceutical substances) provided by the present invention may also be provided in the form of salts or other derivatives. Salts include pharmaceutically acceptable salts, such as acid addition salts and basic salts. Some examples of acid addition salts include hydrochloride, citrate, chloride and acetate salts. Some examples of basic salts include salts wherein the cation is selected from the group consisting of: alkali metals (e.g., sodium and potassium); alkaline earth metals (e.g., calcium); and ammonium +n (R 3)3(R4), wherein R 3 and R 4 independently represent an optionally substituted C 1-6 alkyl group, an optionally substituted C 2-6 alkenyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. Further examples of pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17 th edition, ed. Alfonso r. Gennaro (Ed.), mark PublishingCompany, easton, PA, u.s.a.,1985 and recent versions, and Encyclopaedia of PharmaceuticalTechnology.
Other derivatives of GLP-2 peptide analogs provided by the invention include: complexes, esters, e.g. in vivo hydrolysable esters, free acids or bases, hydrates, prodrugs or lipids, with metal ions such as Mn 2+ and Zn 2+. Esters can be formed between hydroxyl or carboxylic acid groups present in the compounds and suitable carboxylic acid or alcohol reaction partners using techniques well known in the art. Derivatives as prodrugs of the compounds may be converted to one of the parent compounds in vivo or in vitro. Typically, at least one biological activity of a compound will be reduced in a prodrug form of the compound, and may be activated by converting the prodrug to release the compound or a metabolite thereof. Examples of prodrugs include the use of protecting groups that are removable in situ to release the active compound or for inhibiting drug clearance in vivo.
As used herein, the term "amino acid" or "amino acid residue" refers to naturally occurring amino acids, unnatural amino acids, amino acid analogs, and/or amino acid mimics that function in a manner similar to naturally occurring amino acids, in their D and L stereoisomer forms, if their structures allow such stereoisomer forms. Amino acids are referred to herein by their name, by their three-letter symbols known in the art, or by the one-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee.
As used herein, the term "naturally occurring" or "natural" refers to materials found in nature and not artificially manipulated. The term "naturally occurring" or "natural" when used in connection with an amino acid refers to 20 conventional amino acids (i.e., alanine (Ala or a), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y)), selenocysteine, pyrrolysine (PYL) and pyrroline-carboxylysine (PCL).
As used herein, the term "unnatural amino acid" refers to an amino acid that is not naturally encoded or found in the genetic code of any organism. It may for example be a pure synthetic compound. Examples of unnatural amino acids include, but are not limited to: hydroxyproline, gamma-carboxyglutamic acid, O-phosphoserine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, t-butylglycine, 2, 4-diaminoisobutyric acid, desmin, 2-diaminopimelic acid, 2, 3-diaminopropionic acid, N-ethylglycine, N-methylglycine, N-ethylaspargine, homoproline, hydroxylysine, allophanate, 3-hydroxyproline, 4-hydroxyproline, isodesmin, allo-leucine, N-methylalanine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline, norleucine, ornithine, D-arginine, D-alanine, p-aminophenylalanine, pentylglycine, piperidinic acid and thioproline.
As used herein, the term "pharmaceutical composition" or "pharmaceutical formulation" may have any formulation selected from the group consisting of: tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Furthermore, one or more administrations may be performed. At this time, the pharmaceutical composition and/or pharmaceutical preparation is administered in the form of a liquid preparation, powder, aerosol, capsule or suppository.
In some embodiments, the dosage of the "pharmaceutical composition" or "pharmaceutical formulation" is not particularly limited and may be used in a once or twice daily dosage regimen. In some cases, the "pharmaceutical compositions" or "pharmaceutical formulations" provided herein may be used on a once-weekly or twice-weekly dosage regimen. Alternatively or additionally, the dosing regimen of a "pharmaceutical composition" or "pharmaceutical formulation" provided herein may comprise several doses or courses of doses separated in time by 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days or 12 days. In a preferred embodiment, the doses are separated in time by 3 days, 3.5 days, 4 days, 5 days, 6 days, 7 days or 8 days. In a preferred embodiment, the doses are separated in time by 3 days, 3.5 days, 4 days or 7 days. As will be appreciated in the art, the time between doses may be somewhat different such that each and every dose is not separated by exactly the same time. This will typically be guided at the discretion of the physician. Thus, the dosages may be separated in time by a clinically acceptable time frame, for example, from about 2 days to about 10 days, or from about 3 or 4 days to about 7 or 8 days.
In some embodiments, the route of administration of the "pharmaceutical composition" or "pharmaceutical formulation" includes, but is not limited to: intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, intrarectal, and the like. When administered orally, a coating may be formulated that protects the active ingredient in the pharmaceutical composition from degradation in the stomach. In addition, the active ingredient may be administered by any device capable of being transferred to the target tissue. In particular embodiments, the pharmaceutical compositions provided herein can be formulated into various dosage forms according to actual needs, and the dosage beneficial to the patient can be determined by the clinician based on the type, age, weight and general disease condition of the subject, mode of administration, and the like. The mode of administration may be, for example, injection or any other suitable mode of administration known to those skilled in the art.
As used herein, the term "pharmaceutically acceptable carrier and/or adjuvant" refers to a substance suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers and/or excipients useful in the present invention are conventional, remington's Pharmaceutical Sciences, written by e.w. martin, mack Publishing co., easton, PA, 15 th edition (1975) describe compositions and formulations suitable for drug delivery of one or more therapeutic compounds, molecules or agents.
Generally, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically comprise injectable fluids including pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like, as a carrier. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to the bio-neutral carrier, the pharmaceutical composition or pharmaceutical formulation to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
As used herein, the term "effective amount" is an amount that reduces the symptoms of a given disorder or pathology, preferably normalizes the physiological response of an individual suffering from the disorder or pathology. The reduction of symptoms or normalization of physiological responses may be determined using methods conventional in the art and may vary according to the given condition or pathology. In one aspect, a therapeutically effective amount of one or more GLP-2 analogues or a pharmaceutical composition containing one or more GLP-2 analogues is an amount of: which restores the detectable physiological parameter to a value substantially identical to (preferably within +30%, more preferably within +20%, and still more preferably within +10%) of the value in an individual not suffering from the disorder or condition.
In some embodiments, the "effective amount" will vary with the age, weight, and species of mammal being treated, the particular compound being used, the particular mode of administration, and the desired effect and therapeutic indication. Since the relationship of these factors to determining this amount is well known in the medical arts, it will be within the ability of the skilled artisan to determine the therapeutically effective dosage level, the amount needed to achieve the desired results for preventing and/or treating the intestinal and gastric related diseases described herein, and other medical indications disclosed herein.
In some embodiments, the effective therapeutic dose may vary as a function of different variables, particularly the mode of administration, the disease state, age and weight of the subject, the responsiveness of the subject to treatment, the type of concurrent therapy, the frequency of treatment, and/or the need for treatment or prophylaxis.
In some embodiments, when directed to the use for preventing a disease, the agonistic peptide or pharmaceutically acceptable salt, pharmaceutical composition and/or pharmaceutical formulation of the invention is administered in a dosage sufficient to prevent or delay the onset and/or establishment and/or recurrence of a pathological condition (e.g., a gastric and/or intestinal related disease), particularly in a subject at risk of developing a disease.
In some embodiments, when referring to the use of treating a disease, an effective therapeutic dose may be a dose that results in a visible improvement in clinical status as compared to the basal or intended status in which no treatment is being performed, such as an increase in small intestine mass, an increase in cell activity of small intestine epithelial cells, an increase in small intestine length, an increase in small intestine weight, an increase in small intestine villus number, an increase in crypt depth, an extension of remission, stabilization of disease status (i.e., no deterioration), a delay or slowing of disease progression or severity, an improvement or alleviation of disease status, an extension of survival, a better response to conventional treatment, an increase in quality of life, a reduction in mortality, and the like.
The invention is further illustrated below in conjunction with specific examples, which are provided solely to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.
The reagents and materials used in the present invention are readily available to those of ordinary skill in the art, and unless otherwise indicated, are commercially available, and the invention is not limited to specific conditions, and the invention may be practiced by routine or manufacturer-recommended procedures, and the person skilled in the art may prepare the agonist peptides of the invention and pharmaceutically acceptable salts thereof in a synthetic manner which is not limited to the examples of the invention. In particular, the following examples are only illustrative of the invention and should not be construed as limiting the scope of the invention in any way.
EXAMPLE 1 Synthesis of peptide Compounds
1. Experimental materials
The materials and reagents used in the invention are purchased from commercial products, and the protective amino acids used in the whole synthesis process are as follows ：Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Ala-O、Fmo-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Lys(Boc)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Phe-OH、Fmoc-Aib-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(iLEde)-OH、Fmoc-α-Me-Leu-OH、Fmoc-Ile-OH、Boc-Tyr(tBu)-OH、Eicosanedioic acid(mon-tBu)-γGlu(α-OtBu)-AEEA-OH.
2. Taking A1 as an example, the synthetic preparation method of the agonistic peptide of the invention is described (the preparation of other agonistic peptides of the invention only needs to replace the synthesis sequence of amino acid raw materials)
1) Pretreatment of resin: 26.30g Rink Amide AM Resin of resin was weighed into a reaction kettle, 200mL of DMF was added, and the mixture was stirred and swelled for 30min under nitrogen. When swollen, the solvent was removed by suction filtration and the resin was washed 3 times with 200mL of DMF each time;
2) Preparing an amino acid solution: fmoc-Ser (tBu) -OH 11.50g and HOBt 4.04g were weighed and dissolved in 150mL DMF, then 3.78g DIC was added and mixed well;
3) Deprotection: 200mL of 20% piperidine/DMF solvent was added to the reaction vessel, and the reaction was stirred with nitrogen for 35min. At the end of the reaction, the resin was washed 6 times with 200mL of DMF. After the washing is finished, detecting the resin by using ninhydrin reagent, wherein the resin is positive;
4) Coupling reaction: adding the prepared amino acid solution into a reaction kettle, controlling the temperature to be 25-35 ℃, introducing nitrogen and stirring for reaction. The progress of the reaction was monitored with ninhydrin reagent until the resin was negative indicating completion of the reaction. After the reaction was completed, the resin was washed 3 times with 200mL of DMF each time. Repeating the above steps, and sequentially coupling corresponding protected amino acids according to the sequence of the A1 peptide until the synthesis of the peptide main chain is completed;
5) And removing the Lys side chain protecting group iLEde: 200mL of 8% hydrazine hydrate/DMF solution was added to the reaction vessel, the reaction was stirred with nitrogen for 1 hour, the solvent was removed by suction filtration when the reaction was completed, the operation was repeated 1 time, and the resin was washed 12 times with DMF, 200mL each time. After the washing is finished, detecting the resin by using ninhydrin reagent, wherein the resin is positive;
6) Modification of Lys side chain: eicosanedioic acid (mon-tBu) -gamma Glu (alpha-OtBu) -AEEA-OH 14.58g and HOBt 2.70g are weighed and dissolved by 300mL of DMF, then 2.60g of DIC is added, and after uniform mixing, the mixture is added into a reaction kettle, the temperature is controlled to be 25-35 ℃, and nitrogen is introduced for stirring reaction. The progress of the reaction was monitored with ninhydrin reagent until the resin was negative indicating completion of the reaction. After the reaction was completed, the resin was washed 4 times with DMF, 2 times with DCM, 3 times with methyl tert-butyl ether, 200mL each time. Drying the resin for later use;
7) Peptide resin cleavage: preparing a cutting fluid according to TFA/TIS/DTT/H 2 O=180/5/5/10, adding the dried peptide resin into the cutting fluid, and stirring and reacting for 3H. Filtering when the reaction is carried out, adding the filtrate into 5 times volume of ice methyl tertiary butyl ether for precipitation, carrying out suction filtration, washing a filter cake with methyl tertiary butyl ether slurry for 3 times, and carrying out vacuum drying to obtain a crude product;
8) Purifying and refining: the crude product obtained was purified by refining with a C18 reversed phase preparative chromatography system to obtain an A1 purified product having a purity of not less than 90%, and the compound was analyzed by analytical HPLC and LC-MS.
3. Preparation results
The molecular weight measurement results of each agonistic peptide compound after completion of the preparation are shown in the following table 1, and the results indicate that the prepared compounds are correct.
Table 1 list of synthetic agonistic peptide compounds and molecular weights
EXAMPLE 2 EC 50 test of peptide Compounds
1. Experimental materials
The materials and reagents used in this test were purchased from commercial products.
2. Experimental method
The following examples of compounds A1-A6 were used to conduct the cell EC50 test, specifically as follows:
1) Construction of cell model
HEK293 cells highly expressing human GLP2R were constructed (by transfection of human GLP 2R).
The method comprises the following specific steps:
A. Constructing a plasmid which overexpresses a human GLP-2 receptor;
B. performing transfection by using a Lip3000 transfection reagent to obtain a target cell with high expression of a GLP-2 receptor;
C. cell verification is carried out, and whether transfection is successful or not is detected through an RT-qPCR experiment;
D. Extracting Total RNA of cells before and after transfection, carrying out RT-qPCR reaction, and analyzing the result;
E. After successful cell transfection, drug (peptide compound) tests were performed.
2) Peptide compound assay
A. Culture of HEK293 cells: HEK293 cells were cultured using a medium containing dmem+10% fbs+1% penicillin+1% streptomycin at 37 ℃,5% CO 2;
B. Removing the culture medium when the cells are in the logarithmic growth phase, and cleaning the cells with PBS (phosphate buffered saline) without phosphorus and magnesium;
C. protease treatment is carried out by Ackutase;
D. Washing and re-suspending the separated cells in a determination buffer to determine the cell density;
E. Inoculating the cell suspension into a 96-well plate according to the ratio of 4×10 5 cells/mL;
F. Adding a test peptide chemical;
G. Incubating for 30 minutes at room temperature;
H. Using cAMP Gs kit to make determination;
I. Adding a lysis buffer, diluting the HTRF reagent, and incubating for 1 hour;
J. measured at 665/620 nm;
K. standard curves were drawn by ELISA and then EC50 values for the polypeptides were calculated.
3. Experimental results
The experimental results are shown in the following table 2, and the EC50 values of A1-A6 are all better than A0 for the agonism effect of GLP2R targets.
Table 2 EC50 test of peptide compounds
EXAMPLE 3 functional Activity of peptide Compounds on mouse Mode-k cells
1. Experimental materials
The materials and reagents used in this example were purchased from commercial products. Basal medium for Mode-k cells (DMEM+10% FBS+0.38mg/mL G418+1% P/S).
2. Experimental method
The following test for cell activity was conducted using the compounds A1 to A6 as an example, and specifically as follows:
Taking Mode-k cells in logarithmic growth phase, inoculating the Mode-k cells into a 96-well plate according to the ratio of 5×10 4, continuously culturing for 12 hours, adding polypeptide, continuously incubating for 24 hours, and then detecting CCK-8 cytotoxicity. The polypeptide was used at 10. Mu. Mol/L.
3. Experimental results
The results are shown in FIG. 1, which shows that the A1-A6 molecules all had better cell activity than the A0 group.
Example 4 peptide Compounds for mice intestinal quality testing
1. Experimental materials
Female mice of wild type C57 at 12 weeks of age.
2. Experimental method
In vivo mouse intestinal mass tests were performed using compounds A1 and A0 as examples, specifically as follows:
C57 mice of 12 weeks of age were grouped into Vechicle, A1 (4 mg/kg) and A0 (4 mg/kg) groups and then subcutaneously injected with different peptide compounds 100 μl/time/week for two consecutive weeks. The mice were taken after 14 days for intestinal mass and functional activity detection. N=3.
3. Experimental results
The results are shown in FIG. 2, which shows that both group A1 (4 mg/kg) and group A0 (4 mg/kg) increase the plasma Cit content compared to group Vechicle. In vivo A1 has a stronger effect on the small intestine than A0.
Example 5 peptide Compounds for the test of the Small intestine Length of mice
1. Experimental materials
Female mice of wild type C57 at 12 weeks of age.
2. Experimental method
In vivo experiments were performed on the length of small intestine villus and depth of crypt of mice using compounds A1 and A0 as examples, specifically as follows:
C57 mice of 12 weeks of age were grouped into Vechicle, A1 (4 mg/kg) and A0 (4 mg/kg) groups and then subcutaneously injected with different peptide compounds 100 μl/time/week for two consecutive weeks. The whole section of the mouse small intestine was taken after 14 days. Fixation in 4% paraformaldehyde, paraffin embedding, sectioning and HE staining were performed to perform mouse intestinal crypt depth detection. N=3.
3. Experimental results
The results are shown in FIG. 3, which shows that the A1 group (4 mg/kg) and the A0 group (4 mg/kg) also changed the crypt depth compared to Vechicle groups. In vivo A1 has a stronger effect on the small intestine than A0.
EXAMPLE 6 peptide Compounds tested on small intestine villus Length and crypt depth
1. Experimental materials
Female mice of wild type C57 at 12 weeks of age.
2. Experimental method
In the following, the in vivo test of the small intestine length, small intestine villus length and crypt depth of the mice was performed using the compounds A1 and A0 as examples, specifically as follows:
C57 mice of 12 weeks of age were grouped into Vechicle, A1 (4 mg/kg) and A0 (4 mg/kg) groups and then subcutaneously injected with different peptide compounds 100 μl/time/week for two consecutive weeks. After 14 days the whole section of the small intestine of the mouse is taken, the length of the small intestine is measured and weighed. Fixed in 4% paraformaldehyde, paraffin embedded, sectioned and HE stained for small intestine length, small intestine weight, small intestine villus length and crypt depth detection in mice. N=3.
3. Experimental results
The results are shown in FIGS. 4 and 5, and show that both group A1 (4 mg/kg) and group A0 (4 mg/kg) increased the length and weight of the small intestine, and the amount of villi and depth of crypt were also changed, as compared with group Vechicle. In vivo A1 has a stronger effect on the small intestine than A0.
EXAMPLE 7 evaluation of mouse safety of peptide Compounds
1. Experimental materials
The materials and reagents used in this example were purchased from commercial products. Wild type C57 female mice of 12 weeks of age.
2. Experimental method
The safety evaluation of mice was carried out by taking the compounds A1 and A0 as examples, and specifically as follows:
Wild-type C57 female mice of 12 weeks of age were grouped into Vechicle, A1 (4 mg/kg) and A0 (4 mg/kg). Each group was subcutaneously injected every 3 days with 4mg/kg of peptide compound and after 21 days of injection, tail blood was taken for serum Creatine Kinase (CK) and glutamic pyruvic transaminase (ALT) detection, n=3.
3. Experimental results
The results are shown in FIG. 6, and the results show that the serum ALT/AST of the A0 group (4 mg/kg) and the serum ALT/AST of the A1 group (4 mg/kg) have no statistical difference compared with the serum ALT/AST of the Vechicle group (Control), so that the in vivo safety of the peptide compound prepared by the invention is good.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

1. An agonistic peptide of a GLP2R target or a pharmaceutically acceptable salt thereof, characterized in that said agonistic peptide or a pharmaceutically acceptable salt thereof comprises or consists of an amino acid sequence of general formula i:
general formula (VI) Ⅰ：X1-X2-DGSFSDE-Nle-DPhe-TILD-X16-LAARDFINWLIQT-X30-ITD-XX1-R1;
Wherein,
X 1 is H or Y;
X 2 is Aib or Mfe-Gly or other unnatural amino acid;
x 16 is N or L;
x 30 is ψ, ψ is Lys with side chains modified;
XX 1 is GGPSSGAPPPS、GGGPSSGAPPPS、GGPSSGAdPPPS、GGGPSSGAdPPPS、GGPSSGAPPPSKVSRA、GGGPSSGAPPPSKVSRA、GGPSSGAdPPPSKVSRA or GGGPSSGA d PPPSKVSRA;
R 1 is NH 2 or OH, or a pharmaceutically acceptable salt and/or ester thereof.
2. The agonistic peptide or pharmaceutically acceptable salt thereof according to claim 1, wherein ψ is Lys having a side chain modified by a structure having the following general formula ii;
General formula II: B-Z;
Wherein B is (AEEA or Glu) a - (AEEA or Glu) b - (AEEA or Glu) c, wherein a, B, c are each independently 0 or 1, and a, B, c are not simultaneously 0; the carboxyl end of the B is connected with epsilon-amino of a side chain of Lys, Z is-CO- (CH 2)m-R2), m is an integer between 6 and 24, and R 2 is selected from-COOH.
3. The agonistic peptide or pharmaceutically acceptable salt thereof according to claim 2, wherein said general formula ii is AEEA-yg1u—co (CH 2)18 COOH;
preferably, the agonistic peptide or a pharmaceutically acceptable salt thereof is chemically modified at least one site by a fatty acid side chain group.
4. The agonist peptide or pharmaceutically acceptable salt thereof according to claim 3, wherein the agonist peptide or pharmaceutically acceptable salt thereof is selected from any one of the following:
H-Aib-DGSFSDE-Nle-DPhe-TILD-L-LAARDFINWLIQT-K(AEEA-K-C18)-ITD-GGPSSGAPPPS-NH2；
H-Aib-DGSFSDE-Nle-DPhe-TILD-D-LAARDFINWLIQT-K(AEEA-K-C18)-ITD-GGGPSSGAPPPS-NH2；
H-Aib-DGSFSDE-Nle-DPhe-TILD-D-LAARDFINWLIQT-K(AEEA-K-C18)-ITD-GGPSSGAPPPS-NH2；
H-Aib-DGSFSDE-Nle-DPhe-TILD-L-LAARDFINWLIQT-K(AEEA-K-C20)-ITD-GGPSSGAPPPS-NH2；
H-Aib-DGSFSDE-Nle-DPhe-TILD-D-LAARDFINWLIQT-K(AEEA-K-C20)-ITD-GGPSSGAPPPS-NH2；
H-Aib-DGSFSDE-Nle-DPhe-TILD-D-LAARDFINWLIQT-K(AEEA-K-C20)-ITD-GGGPSSGAPPPS-NH2。
5. The agonistic peptide or pharmaceutically acceptable salt thereof according to claim 4, wherein said agonistic peptide or pharmaceutically acceptable salt thereof is capable of significantly improving the intestinal quality;
Preferably, the agonistic peptide or pharmaceutically acceptable salt thereof is capable of significantly enhancing the cellular activity of small intestine epithelial cells;
Preferably, the agonistic peptide or pharmaceutically acceptable salt thereof is capable of significantly increasing the small intestine length;
Preferably, the agonistic peptide or pharmaceutically acceptable salt thereof is capable of significantly increasing the weight of the small intestine;
preferably, the agonistic peptide or pharmaceutically acceptable salt thereof is capable of significantly increasing the amount of small intestinal villi;
Preferably, the agonistic peptide or a pharmaceutically acceptable salt thereof is capable of significantly increasing crypt depth.
6. A pharmaceutical composition comprising an agonistic peptide according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof;
preferably, the pharmaceutical composition may further comprise other drugs which can be used for the treatment and/or prevention of stomach and/or intestine related diseases;
More preferably, the stomach and/or intestine related diseases comprise short bowel syndrome, gastrointestinal acute graft versus host disease, malabsorption syndrome, cullet syndrome, inflammatory bowel disease, abdominal type site-prednisole diarrhea, tropical type site-prednisole diarrhea, low-propylene globulinemia type site-prednisole diarrhea, mucositis and diarrhea caused by chemotherapy and/or radiation therapy, ulcers, digestive disorders, enteritis, crohn's disease, small intestine injury, metabolic syndrome, obesity and/or diabetes.
7. A pharmaceutical formulation comprising the pharmaceutical composition of claim 6;
Preferably, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier and/or adjuvant;
More preferably, the pharmaceutically acceptable carrier and/or adjuvant comprises a buffer, a non-ionic tonicity modifier;
More preferably, the buffer comprises a histidine buffer, a mesylate buffer, an acetate buffer, a glycine buffer, a lysine buffer, a TRIS buffer, a Bis-TRIS buffer and/or a MOPS buffer;
More preferably, the nonionic tonicity modifier comprises mannitol, sucrose, glycerol, sorbitol and/or trehalose;
preferably, the dosage form of the pharmaceutical preparation comprises a liquid preparation, a solid preparation, a semisolid preparation and a gas preparation;
more preferably, the liquid formulation comprises injection, solution, suspension;
More preferably, the solid formulation comprises a tablet, capsule, powder, granule;
more preferably, the semi-solid formulation comprises a gel, cream, suppository;
more preferably, the gas formulation comprises an aerosol, a spray, a powder spray.
8. A product of any one of the following, characterized in that it comprises:
(1) A kit comprising the pharmaceutical composition of claim 6 or the pharmaceutical formulation of claim 7, and a container containing the pharmaceutical composition or pharmaceutical formulation;
(2) A delivery device comprising a liquid formulation of an agonistic peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1-5;
preferably, the delivery device comprises a prefilled syringe, a syringe device, an injection pen, a dispensable automatic syringe, a disposable automatic syringe, a wearable syringe, an infusion pump.
9. A method of preparing an agonistic peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, comprising the steps of:
(1) Pretreatment of resin: adding the resin into a reaction kettle, adding DMF, introducing nitrogen, stirring for swelling, filtering to remove the solvent, and washing;
(2) Preparing an amino acid solution: fmoc-Ser (tBu) -OH and HOBt are dissolved by DMF, DIC is added, and the mixture is uniformly mixed;
(3) Deprotection: adding 20% piperidine/DMF solvent into a reaction kettle, introducing nitrogen, stirring for reaction, and washing the obtained resin after the reaction is completed;
(4) Coupling reaction: adding the amino acid solution prepared in the step (2) into a reaction kettle, introducing nitrogen at a controlled temperature to stir for reaction, washing the obtained resin after the reaction is completed, and repeating the above operations, and sequentially coupling corresponding protective amino acids according to the amino acid sequence of the agonistic peptide or the pharmaceutically acceptable salt thereof in any one of claims 1-5 until the synthesis of the backbone of the agonistic peptide is completed;
(5) And removing the Lys side chain protecting group iLEde: adding 8% hydrazine hydrate/DMF solution into a reaction kettle, introducing nitrogen, stirring for reaction, filtering to remove solvent after the reaction is completed, and washing;
(6) Modification of Lys side chain: dissolving Eicosanedioic acid (mon-tBu) -gamma Glu (alpha-OtBu) -AEEA-OH and HOBt with DMF, adding DIC, mixing, adding into a reaction kettle, controlling temperature, introducing nitrogen, stirring for reaction, washing the obtained resin after the reaction is completed to obtain peptide resin containing the agonistic peptide or pharmaceutically acceptable salt thereof according to any one of claims 1-5, and drying;
preferably, the method further comprises the steps of:
(a) Peptide resin cleavage: preparing cutting fluid, adding the peptide resin obtained by drying in the step (6) into the cutting fluid, stirring for reaction, filtering after the reaction is completed, adding filtrate into ice methyl tertiary butyl ether for precipitation, carrying out suction filtration, washing a filter cake with methyl tertiary butyl ether slurry, and carrying out vacuum drying to obtain a crude product;
(b) Purifying and refining: refining and purifying the crude product by a C18 reversed phase preparation chromatographic system to obtain a purified product;
Preferably, the resin in step (1) is RINK AMIDE AM RESIN resin;
Preferably, the resin in step (1) is used in a dose of 26.30g;
preferably, the DMF used in step (1) is used in a dosage of 200mL;
preferably, the swelling time in step (1) is 30min;
Preferably, the conditions of the washing in step (1) are 3 washes with DMF, 200mL each time;
preferably, the Fmoc-Ser (tBu) -OH, HOBt, DMF, DIC in step (2) is used in a dose of 11.50g, 4.04g, 150mL, 3.78g, respectively;
Preferably, the 20% piperidine/DMF solvent of step (3) is used in a dosage of 200mL;
preferably, the time of the reaction in step (3) is 35min;
preferably, the conditions of the washing in step (3) are 6 washes with DMF 200mL each;
preferably, the step (3) further comprises detecting the washed resin with ninhydrin reagent, wherein the resin is positive;
Preferably, the temperature control condition in the step (4) is 25-35 ℃;
preferably, step (4) further comprises monitoring the progress of the reaction with ninhydrin reagent during the reaction until the resin is negative indicating that the reaction is complete;
preferably, the conditions of the washing in step (4) are 3 washes with DMF 200mL each;
preferably, the 8% hydrazine hydrate/DMF solution in step (5) is used in an amount of 200mL;
preferably, the time of the reaction in step (5) is 1h;
preferably, the conditions of the washing in step (5) are 12 washes with DMF, 200mL each time;
Preferably, the step (5) further comprises taking resin and detecting the resin by ninhydrin reagent after the washing is finished, wherein the resin is positive;
Preferably, the dosage of Eicosanedioic acid (mon-tBu) - γGlu (α -OtBu) -AEEA-OH, HOBt, DMF, DIC used in step (6) is 14.58g, 2.70g, 300mL, 2.60g, respectively;
preferably, the temperature control condition in the step (6) is 25-35 ℃;
Preferably, step (6) further comprises monitoring the progress of the reaction with ninhydrin reagent until the resin is negative indicating that the reaction is complete;
preferably, the conditions of the washing in step (6) are 4 times with DMF, 2 times with DCM, 3 times with methyl tert-butyl ether, 200mL each;
More preferably, the cutting fluid in step (a) is formulated according to TFA/TIS/DTT/H 2 o=180/5/5/10;
more preferably, the time of the reaction in step (a) is 3h;
more preferably, the ice methyl tertiary butyl ether in step (a) is 5 volumes of ice methyl tertiary butyl ether;
More preferably, the sizing conditions in step (a) are 3 times with methyl tert-butyl ether.
10. An application according to any one of the following, characterized in that the application comprises:
(1) Use of an agonistic peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 for the manufacture of a medicament for the treatment and/or prevention of gastric and/or intestinal related diseases;
(2) Use of an agonistic peptide according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof and/or a pharmaceutical composition according to claim 6 for the preparation of a pharmaceutical formulation for the treatment and/or prevention of gastric and/or intestinal related diseases;
(3) Use of an agonistic peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, a pharmaceutical composition according to claim 6 and/or a pharmaceutical formulation according to claim 7 for the preparation of a kit for the treatment and/or prevention of gastric and/or intestinal related diseases;
(4) Use of an agonistic peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, a pharmaceutical composition according to claim 6 and/or a pharmaceutical formulation according to claim 7 for the manufacture of a delivery device for the treatment and/or prevention of gastric and/or intestinal related diseases;
(5) Use of an agonistic peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, a pharmaceutical composition according to claim 6, a pharmaceutical formulation according to claim 7 and/or a delivery device according to claim 8 for the manufacture of an agent or medicament for increasing the mass of the small intestine, increasing the cellular activity of the epithelial cells of the small intestine, increasing the length of the small intestine, increasing the weight of the small intestine, increasing the number of villi of the small intestine and/or increasing the depth of the crypt;
Preferably, the stomach and/or intestine related diseases comprise short bowel syndrome, gastrointestinal acute graft versus host disease, malabsorption syndrome, cullet syndrome, inflammatory bowel disease, abdominal type site-prednisole diarrhea, tropical type site-prednisole diarrhea, low-propisochlainemia type site-prednisole diarrhea, mucositis and diarrhea caused by chemotherapy and/or radiation therapy, ulcers, digestive disorders, enteritis, crohn's disease, small intestine injury, metabolic syndrome, obesity and/or diabetes.
CN202410283735.3A 2024-03-13 2024-03-13 Preparation method and application of GLP-2 peptide analogue Pending CN118146350A (en)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102942626A (en) * 2009-03-05 2013-02-27 连云港恒邦医药科技有限公司 Glucagon-like peptide-2 analogue, its preparation method and application
CN102949731A (en) * 2011-09-16 2013-03-06 西藏海思科药业集团股份有限公司 Medicine fusion specifically bound with GLP-2 receptor
WO2018104558A1 (en) * 2016-12-09 2018-06-14 Zealand Pharma A/S Acylated glp-1/glp-2 dual agonists
CN109641946A (en) * 2016-03-23 2019-04-16 巴切姆股份公司 The preparation method of glucagon-like peptide
WO2021186169A1 (en) * 2020-03-16 2021-09-23 Heptares Therapeutics Limited Oral glp receptor agonists
CN113710692A (en) * 2019-02-11 2021-11-26 Opko生物科学有限公司 Long-acting GLP-2 analogs
CN115666622A (en) * 2020-03-16 2023-01-31 赫普泰雅治疗有限公司 GLP receptor agonists

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102942626A (en) * 2009-03-05 2013-02-27 连云港恒邦医药科技有限公司 Glucagon-like peptide-2 analogue, its preparation method and application
CN102949731A (en) * 2011-09-16 2013-03-06 西藏海思科药业集团股份有限公司 Medicine fusion specifically bound with GLP-2 receptor
CN109641946A (en) * 2016-03-23 2019-04-16 巴切姆股份公司 The preparation method of glucagon-like peptide
WO2018104558A1 (en) * 2016-12-09 2018-06-14 Zealand Pharma A/S Acylated glp-1/glp-2 dual agonists
CN113710692A (en) * 2019-02-11 2021-11-26 Opko生物科学有限公司 Long-acting GLP-2 analogs
WO2021186169A1 (en) * 2020-03-16 2021-09-23 Heptares Therapeutics Limited Oral glp receptor agonists
CN115666622A (en) * 2020-03-16 2023-01-31 赫普泰雅治疗有限公司 GLP receptor agonists

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