CN100503637C - Glucagon-like peptide analogs, compositions and methods of use thereof - Google Patents

Abstract

The present invention provides a kind of glucagon-like peptide analog and its composition, which are used in up regulating insulin expression in mammal body to treat diabetes. These peptide derivatives may be used in treating diabetes and other insulinotropic hormone peptide related diseases as well as glucagon level related gastrointestinal function activity diseases.

Description

Glucagon-like peptide analogs, compositions and methods of use thereof

technical field

The present invention relates to glucagon-like peptide analogs and compositions thereof, which are used to up-regulate insulin expression in mammals, thereby treating diabetes. In particular, these peptide derivatives can be used for the long-term treatment of diabetes and other diseases associated with insulinotropic peptides, as well as functionally active diseases of the gastrointestinal tract related to glucagon levels.

Background technique

Islet endocrine control is governed by a complex set of mechanisms not only influenced by metabolites such as glucose, amino acids and catecholamines, but also by local paracrine influences. The major islet hormones, glucagon, insulin, and somatostatin bind to specific pancreatic cells (A cells, B cells, and D cells, respectively) to regulate secretory responses. Although insulin secretion is primarily determined by blood insulin levels, human growth hormone releases inhibin to inhibit glucose-regulated insulin secretion. In addition to the insular paracrine regulation of insulin secretion, the existence of intestinal insulinotropic factors has been demonstrated.

The endocrine concept arose from the observation that energy derived from food intake or intestinal glucose absorption elicits a greater stimulus to insulin release than the same amount of energy (glucose) injected intravenously ( Elrick, H. et al., J. Clin. Endocrinol. Metab., 24 , 1076-1082, 1964; McIntyre, N. et al., J. Clin. Endocrinol. Metab., 25 , 1317-1324, 1965). Thus, it was hypothesized that gut-derived signals elicited by oral nutrient intake represent potent insulin secretagogues that drive increased insulin release when energy is taken up through the gastrointestinal tract rather than parenterally (Dupre, J. et al., Diabetes, 15 , 555-559, 1966).

Although some neurotransmitters and gut hormones also have endocrine-like activities, there is substantial evidence from immune, antagonist, and knockout studies that glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide (GLP)-1 represents the dominant peptide responsible for most nutrient-stimulated insulin secretion. The observation that meal-stimulated insulin secretion release in patients with type 2 diabetes exhibits a markedly reduced magnitude has aroused interest in investigating whether incomplete endocrine or endocrine resistance is associated with β-cell dysfunction in diabetics related to the pathophysiology of.

In 1987, glucagon-like peptide-1 (GLP-1) was discovered for the first time in humans, and it was considered to be an endocrine hormone, a peptide secreted by the intestine after food intake. GLP-1 is secreted by intestinal L cells following a proteolytic process of a 160 amino acid precursor protein (ie, preproglucagon). After the preproglucagon (preproglucagon) is cleaved, a peptide GLP-1 composed of 37 amino acids is firstly generated, that is, GLP-1(1-37)OH, and the activity of this peptide is very weak. Subsequent cleavage at position 7 yields biologically active GLP-1(7-37)OH. Approximately 80% of the synthesized GLP-1(7-37)OH is amidated at the terminal C-position after removal of the terminal glycine residue in L-cells. The biological effects and metabolic turnover of the free acid GLP-1(7-37)OH and the amide compound GLP-1(7-37) _NH2 were indistinguishable.

It is well known that GLP-1 stimulates insulin secretion to cause an increase in glucose concentration in cells, thereby reducing blood sugar levels (Moisov, S. et al., J.Clin.Invest., 79 , 616-619, 1987; Kreymann, B. et al., Lancet ii, 1300-1304, 1987; Orskov, C. et al., Endocrinology, 123 , 2009-2013, 1988). Acute intraventricular injection of GLP-1 or GLP-1 receptor constriction produces a transient reduction in food intake (Turton MD et al., Nature, 379 , 60-72, 1996), however some studies have shown that more chronic intraventricular Administration of injectable or parenteral GLP-1 receptor agonists has been associated with weight loss (Meeran, K. et al., Endocrinology, 140 , 244-250, 1999; Davies, HRJr., Obes. Res., 6 , 147-156, 1998; Szayna, M. et al., Endocrinology, 141 , 1936-1941, 2000; Larsen, PJ et al., Diabetes, 50 , 2530-2539, 2001). A large number of GLP-1 analogs with insulinotropic effects are known in the art. For example, these analogs include GLP-1(7-36), Gln ⁹ -GLP-1(7-37), D-Gln9-GLP-1(7-37), Acetyl-Lys9-GLP-1(7 -37), Thr16-Lys18-GLP-1(7-37) and Lys18-GLP-1(7-37); derivatives of GLP-1 include acid addition salts, carboxylates, lower alkyl esters and amides compounds (WO91/11457; EP0733,644; US Patent 5,512,549).

Most of the effects of GLP-1 delineated in preclinical experiments have also been confirmed in human studies. Under fast glucose intake and food intake state, inject GLP-1 (7-36) NH in normal human body _Can stimulate insulin secretion, obviously reduce blood sugar (Orskov, people such as C., Diabetes, 42 , 658-661 , 1993; Qualmann, C. et al., Acta. Diabetol., 32 , 13-16, 1995).

For diabetic patients who fail to be treated with sulfonylureas, GLP-1-based peptides will definitely have a good therapeutic effect (Nauck, MA et al., Diabetes Care, 21 , 1925-1931, 1998). GLP-1 stimulates insulin secretion only in the context of hyperglycemia. This property of GLP-1 makes it safer than insulin, and it has been observed that the amount of insulin secreted is directly proportional to the degree of hyperglycemia. In addition, GLP-1 treatment will cause insulin to be released from the pancreas and act on the first pass of insulin in the liver. This results in lower circulating levels of peripheral insulin compared with subcutaneous insulin injections. GLP-1 slows the emptying of the gastrointestinal tract, which allows nutrients to be absorbed over a longer period of time, thereby reducing the postprandial glucose peak. Some reports suggest that GLP-1 can increase insulin sensitivity in peripheral tissues such as muscle, liver and fat. Finally, GLP-1 also exhibits a potential appetite-regulating function.

The therapeutic potential of GLP-1 and its analogs will be even more prominent if applied to patients with type 1 diabetes. Numerous studies have demonstrated the effectiveness of natural GLP-1 in the treatment of insulin-dependent diabetes mellitus (IDDM). Similar to patients with non-insulin-dependent diabetes mellitus (NIDDM), GLP-1 can effectively alleviate starvation hyperglycemia through its glucagonostatic properties. Additional studies suggest that GLP-1 also reduces postprandial glycemic excursions in IDDM patients, likely by prolonging gastric emptying. These observations suggest that GLP-1 may be useful in the treatment of IDDM and NIDDM.

However, the biological half-life of the native GLP-1 molecule is rather short, influenced by the activity of dipeptidase IV (DPP IV). For example, the biological half-life of GLP-1(7-37)OH is only 3-5 minutes (US Patent 5118666). Sustained lowering of blood glucose concentrations can only be achieved by continuous infusion, as demonstrated in studies where 24-hour intravenous infusions can control GLP-1 concentrations (Larsen, J. et al., DiabetesCare, 24 , 1416- 1421, 2001). The enzyme DPP IV is a serine protease that preferentially hydrolyzes peptides at the position after the penultimate NH2-terminal proline (Xaa-Pro-) or alanine (Xaa-Ala-) (Mentlein, R., Regul. Pept., 85 , 9-25, 1999), this enzyme has been shown to rapidly metabolize GLP-1 in vitro. Therefore, long-acting GLP-1-based peptides resistant to DPP IV have great therapeutic potential in treating diabetic patients.

Contents of the invention

The present invention provides analogues of GLP-1 which are more persistent than native GLP-1 and are completely resistant to hydrolysis by the enzyme DPP IV.

The invention includes a compound of the following general formula I or a pharmaceutically acceptable salt thereof:

R ¹ -XR ²

Formula I

In the formula,

^R1 is selected from: L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl - histidine, alpha-methylhistidine;

X is a linker unit selected from the following group:

R ² is a peptide moiety or fragment selected from the group consisting of:

-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val -Lys-Gly-Arg-R ⁴

-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val -Lys-Gly-Arg-Gly-R ⁴

-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val -Lys-Gly-Arg-Lys-R ⁴

-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-

-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-

-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val -Lys-Gly-Arg-R ⁴

-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val -Lys-Gly-Arg-Gly- ^R4 ;

Y and Z are independently selected from: Glu, Gln, Ala, Thr, Ser, and Gly;

R ³ is selected from C _1-6 alkyl;

R ⁴ is selected from NH ₂ and OH, which represent the free acid or amide compound form of the terminal amino acid;

R ⁵ is selected from C _6-10 unbranched acyl groups.

The present invention also provides a compound of the following formula or a pharmaceutically acceptable salt thereof:

R ¹ -XR ⁶

in:

^R1 is selected from: L-histidine, D-histidine, deaminohistidine, 2-aminohistidine, β-hydroxyhistidine, homohistidine, α-fluoromethylhistidine and α-methylhistidine;

X is as defined above;

^R6 are peptide fragments which contain one or more additional amino acids at their carboxyl terminus in addition to the fragments substantially homologous to naturally occurring GLP-1 peptides.

The present invention also provides a compound of the following formula or a pharmaceutically acceptable salt thereof:

R ¹ -XR ⁷

in:

R ¹ is tyrosine;

X is as defined above;

R ⁷ is a peptide fragment, these fragments contain the same peptide as the naturally occurring GIP(3-42):

- EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ ⁴² substantially homologous sequences.

The present invention also provides chemically reactive derivatives of the above compounds, said derivatives can react with available functional groups on cell carriers to form covalent bonds, wherein said cell carriers include mobile blood proteins.

The present invention also provides a method of treating diabetes, insulin resistance and nervous system disorders, characterized in that it comprises administering a non-toxic, therapeutically effective amount of the compound of the present invention to a patient in need of treatment. Preferably, the compound is administered orally, intravenously, subcutaneously or by aerosol once a day, twice a day or three times a day.

The present invention also provides a pharmaceutical composition, which contains the compound described in the present invention and a pharmaceutically acceptable carrier.

The present invention also provides the use of the compound, which is used to prepare medicines for treating diabetes, insulin resistance and nervous system disorders.

Detailed ways

The key of the present invention is to replace the amide bond in the Ala ⁸ of the amino terminal of GLP-1 with a linking unit containing CF ₃ , which is the recognition site of DPP-IV. Recently, it has been reported that in the main peptide backbone of a class of metalloprotease inhibitors, the new linker [-CH(CF ₃ )NH-] may be used as a mimic of the conventional linker (NHCO) (Organic Letters, 2, 1827- 1830, 2000; Tetrahedron Letters, 42 , 3143-3144, 2001). Based on the same principle, [-CH(CF ₃ )NH-]-modified GLP-1 analogues retain the same biological activity as insulinotropic substances and are long-acting.

Peptide parts (peptide fragments) selected from the determined amino acid sequence of human GLP-1 constitute the starting point for the development of the present invention. The interchangeable terms "peptide fragment" and "peptide portion" are meant to include both synthetic and natural amino acid sequences derived from naturally occurring amino acid sequences. Some researchers have reported the amino acid sequence of GLP (Lopez, people such as LC, Acad.Sci., USA 80,5485-5489,1983 ; Bell, people such as GI, Nature 302 :716-718 (1983); Heinrich, G. et al., Endocrinol, 115 :2176-2181 (1984)). The structure of preproglucagon mRNA and its corresponding amino acid sequence is well known. The conversion of the precursor gene product (ie, proglucagon) to glucagon and the proteolysis of the two insulinotropic peptides have also been identified. As used herein, the notation GLP-1(1-37) represents a GLP-1 polypeptide having all amino acids from position 1 (N-terminus) to position 37 (C-terminus). Similarly, GLP-1(7-37) represents a GLP-1 polypeptide having all amino acids from position 7 (N-terminus) to position 37 (C-terminus). Likewise, a polypeptide represented by GLP-1(7-36) is a GLP-1 polypeptide having all amino acids from position 7 (N-terminal) to position 36 (C-terminal).

The same practice can be applied to the glucose-dependent insulinotropic polypeptide (GIP), a 42 amino acid polypeptide derived from a larger 153 amino acid precursor (Takeda, J. et al., Proc. Natl. Acad. .Sci., USA 84 , 7005-7008, 1987). Like GLP-1, GIP is cleaved at the alanine at position 2 by the ubiquitously expressed aminopeptidase DPP IV, thereby generating the truncated inactive peptide GIP(3-42). A GIP analog (4 diastereomers) of the formula is resistant to DPP IV and exerts long-acting GIP effects.

The present invention also provides pharmaceutical compositions comprising a compound of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients.

The principles of solid phase synthesis of polypeptides are well known in the art and may also be found in general works in this field, for example: Dugas, H. and Penney, C., Bioorganic Chemistry (1981) Springer-Verlag, New York, pages 54-92; Merrifield, JM, Chem. Soc., 85 , 2149, 1962, and Stewart and Young, Solid Phase Peptide Synthesis, pages 24-66, Freeman (San Francisco, 1969).

For example, the polypeptide fragments of the present invention can be synthesized by a solid-phase method using an Applied Biosystems 430 peptide synthesizer (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA94404) and a synthetic cycle device provided by Applied Biosystems. Boc-protected amino acids and other reagents are commercially available from Applied Biosystems and other chemical suppliers. Sequential BOC chemistry using a double coupling protocol can be applied to p-methylbenzhydrylamine resins to generate C-terminal carboxamides. For the production of C-terminal acids, the corresponding PAM resins can be used. Aspartic acid, glutamine, and arginine can be coupled using preformed hydroxybenzotriazole esters.

The desired fragment containing CF ₃ can be synthesized according to the following reaction procedure. Protected glutamic acid 1 can react with methyl hemiacetal in CF ₃ CHO to generate 2. 2 reacts with acetate anion to generate 3, which is deprotected to generate carboxylic acid 4. Treatment of 4 with (PhO) ₂ PON ₃ in the presence of Et ₃ N in THF with tert-butanol gave 5. Catalytic hydrogenation over palladium-on-carbon Pd/C catalyst led to the formation of 5 as a diastereomeric mixture of 6. 6 was separated by HPLC to obtain all pure diastereoisomers A, B, C, D.

Use Applied Biosystems 430 peptide synthesizer to link A, B, C, and D to the peptide fragments bound to the polymer by solid-phase synthesis, then deprotect and add R ¹ , R ¹ is selected from: L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine and α-methylhistidine acid. Deprotection and cleavage from the polymeric solid support yielded a new class of GLP analogs that are resistant to DPP IV hydrolysis and have long-lasting insulinotropic effects.

The present invention also includes salt forms of GLP-1 (glucagon-like peptide-1) analogs. GLP-1 analogs can be strongly acidic or basic, and can react with many inorganic bases and inorganic acids to form salts. The inorganic acids used to synthesize acid addition salts are usually hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and the organic acids are p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, Carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of salts include: sulfates, sulfates, bisulfates, sulfites, bisulfites, phosphates, hydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, Iodide, propionate, caprate, caprylate, acrylate, formate, isobutyrate, heptanoate, propiolate, oxalate, malonate, succinate, Suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dimethyl, benzoate, chlorobenzoate , benzoate, dinitrobenzoate, hydroxybenzoate, hydroxybenzoate, phthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropanoid salt, phenylbutyrate, citrate, lactate, 3-hydroxyphenylbutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, 1-sulfonic acid naphthalene salt, 2-Naphthalene sulfonate, mandelate, etc. Preferred acid addition salts are those formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, especially hydrochloric acid.

Base addition salts can be formed from inorganic bases, such as ammonium, alkali or alkaline earth metal hydroxides, carbonates, bicarbonates and the like. The following basic substances are commonly used to form the salt forms of this product, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate and the like. Salt forms of the GLP-1 analogs are particularly preferred. Of course, if the compounds of the present invention are to be used in therapy, these compounds may also be salts, but they must be pharmaceutically acceptable salts.

The improved GLP-1 analogs of the present invention can have multiple uses, they can be used to treat diabetes, can be used as a sedative, can treat nervous system dysfunction, can act on the central nervous system to produce anxiolytic effect, and can stimulate the central nervous system system, which can be used as postoperative therapy and to treat insulin resistance.

A. Diabetes treatment

Modified GLP-1 analogs of the invention generally normalize hyperglycemia through a glucose-dependent mechanism. Therefore, the modified GLP-1 analog can be used as a leading drug for treating type II diabetes, and can also be used as an adjuvant drug for treating type I diabetes.

Treatment of diabetes with effective doses of modified GLP-1 analogs is more effective than unmodified GLP-1. Because the modified GLP-1 analogs are more stable in vivo, effective therapeutic effects can be achieved with a smaller dosage. The present invention is especially suitable for the treatment of diabetic patients, regardless of type I or type II, because the activity of the polypeptide depends on the concentration of glucose in the blood, so compared with the currently used treatment method, the risk of hypoglycemia side effect is greatly reduced in the new method.

The present invention also provides a method for treating diabetes in humans, wherein the method comprises the steps of: providing a modified GLP-1 analogue in an amount sufficient to treat diabetes; wherein the composition comprises the modified GLP-1 analogue.

B. Treatment of nervous system disorders

The modified GLP-1 analogs of the invention may also be used as sedatives. On the one hand, for some mammals whose activity of the central or peripheral nervous system is enhanced due to some abnormality, an attractive or Anxiolytic effect, thereby calming it. Such modified GLP-1 analogs can be injected intracerebroventricularly, orally, subcutaneously, intramuscularly or intravenously. This approach is effective in treating or improving neurological symptoms such as anxiety, movement disturbances, aggressive behavior, confusion, madness, panic attacks, hysteria, and insomnia.

In another related aspect, the invention also includes a method of increasing the mobility of a mammal by administering to a subject a modified GLP-1 analog in an amount sufficient to cause it to act in the mammal and to activate efficacy in the subject. This method is more suitable for mammals with reduced central or peripheral nervous system activity. It is especially suitable for treating or improving a series of symptoms such as depression, affective cutting disorder, sleep apnea syndrome, attention-distraction disorder, amnesia, amnesia, and narcolepsy. In these conditions, it may be beneficial to awaken the central nervous system.

In the treatment or improvement of depression, affective cutting disorder, sleep apnea syndrome, distraction disorder, amnesia, amnesia, narcolepsy and other diseases, the modified GLP-1 analog of the present invention can be used Wake up the patient. The efficacy of GLP-1 analogs can be assessed by counseling patients to assess their disorders, by psychological or neurological testing, or by improvement in symptoms associated with these disorders. For example, the curative effect on narcolepsy can be evaluated from the degree of control over the onset of narcolepsy. As another example, any of a variety of different diagnostic tests known to those skilled in the art can be used to assess the effect of a modified GLP-1 analog on concentration or memory in a patient.

C. Postoperative treatment

The modified GLP-1 analogs of the invention can be used as post-operative therapy. Patients may require such modified GLP-1 analogues either 1-16 hours before surgery, during surgery, or up to 5 days after surgery.

Patients are administered the modified GLP-1 analogue from 16 hours to 1 hour before the start of surgery. The duration of administration of the compounds of the invention to patients to reduce catabolic effects and insulin resistance depends on many factors. These factors are well known to physicians of ordinary skill and include: Most importantly, whether the patient is fasting or receiving glucose infusions or drinking beverages or other forms of food in preparation for surgery. Other important factors include the patient's sex, weight, age, severity of glycemic dysregulation, potential triggers of any inability to regulate blood glucose, severity of anticipated trauma from surgery, route of administration and bioavailability, individual tolerance, formulation, and efficacy of the drug. The preferred time period for administering the modified GLP-1 analogs of the invention varies from one hour to ten hours before surgery. The optimal time period to start dosing is two hours to eight hours before surgery.

Insulin resistance after a particular type of surgery, such as elective abdominal surgery, is worst on the first postoperative day, lasts at least five days, and may take up to three weeks to return to normal. Therefore, after undergoing surgical trauma, the patient needs to administer the modified GLP-1 analogue of the present invention for a period of time after the operation, and the length of time for drug administration depends on whether the patient fasted after the operation or performed glucose infusion or drank beverages or other forms of food. At the same time, the patient's gender, weight, age, severity of blood sugar imbalance, potential cause of loss of blood sugar regulation function, actual severity of surgical trauma, route of administration, bioavailability, individual endurance, formulation, and drug efficacy are also factors. are all influencing factors. The preferred time of administration of the drugs administered in the present invention is no more than five days post-surgery.

D. Treatment of insulin resistance

Treatment of Insulin Resistance Unlike its use in postoperative therapy, modified GLP-1 analogs can be used to treat insulin resistance. Insulin resistance may be caused by a decrease in the amount of insulin bound to receptors on the cell surface or by changes in intracellular metabolism. The first type of insulin resistance is characterized by decreased insulin sensitivity and is typically treated by increasing insulin doses. The second is characterized by reduced insulin responsiveness, which is ineffective for high-dose insulin therapy. The damage caused by insulin resistance can be treated with insulin doses proportional to the degree of insulin resistance, so it is clear that this damage is caused by reduced insulin sensitivity.

The effective dose of the modified GLP-1 analog to normalize the patient's blood sugar level is affected by the following factors: the dose of the GLP-1 analog, the presence or absence of contraindications, the sex, weight and age of the patient, the severity of the loss of blood glucose regulation, Potential etiology that can cause loss of blood sugar regulation function, whether to give glucose or other sugars at the same time, route of administration and bioavailability, patient tolerance, formulation and drug efficacy.

To test the ability of GLP-1 analogs to stimulate insulin secretion, GLP-1 analogs can be injected into artificially cultivated animal cells, such as RIN-38 mouse islet β-cell tumor cells, and then the immune cells released into the culture medium can be monitored. Amount of responsive insulin (IRI). Another approach is to inject GLP-1 analogs into animals and then monitor the plasma concentration of immunoreactive insulin (IRI).

Immunoreactive insulin (IRI) was detected by radioimmunoassay against insulin. Any radioimmunoassay capable of detecting IRI can be used; one such method is a modification of the method developed by Albano, JDM, et al., Acta Endocrinol. 70:487-509 (1972). Phosphate/albumin buffer at pH 7.4 was used in this modification. Inject 500 μl of phosphate buffered saline, 50 μl of perfusate sample or perfusate containing standard mouse insulin, 100 μl of anti-insulin antiserum (Wellcome Laboratories: diluted 1:40,000), and 100 μl of [ ¹²⁵ I] insulin into the culture vessel , with a total capacity of 750 μl, placed in a 10×75mm disposable glass test tube. Incubate at 4°C for 2-3 days, and use charcoal to separate free insulin from antibody-bound insulin. The assay sensitivity is 1-2 uU/ml. Proinsulin can be radiolabeled in order to measure the amount of IRI released into the growth medium of cells in tissue culture. Although any radioactive label capable of labeling a polypeptide can be used, preferably ³ H leucine is used to obtain labeled proinsulin.

Pancreatic perfusion experiments can also be used to determine whether GLP-1 analogs have the function of promoting insulin. The mouse pancreas in situ perfusion assay is a modification of the method of Penhos, J, C. et al. (Diabetes, 18:733-738 (1969)). Fasted male Charles River rats weighing 350-600 g were anesthetized by intraperitoneal injection of amobarbital (Eli Lilly and Co, 160 ng/kg). Ligate the blood vessels of the kidneys, adrenal glands, stomach and lower colon. The entire bowel was nonfunctional except for about 4 cm of the duodenum and descending colon and rectum. Only a small portion of the intestine is thus perfused, minimizing the effects of an immune reaction between the glucagon-like peptide and intestinal material. The perfusate was a modified Kreba-Ringer bicarbonate buffer containing 4% T70 dextran and 0.2% bovine serum albumin (fragment V), bubbled with 95% _O2 and 5% _CO2 . An extracorporeal circulation machine (Buchler polystatic, Buchler Instruments Division, Nuclear-Chicago Corp) with a 4-channel roller-bearing pump was used, with a three-way valve controlling the direction of perfusate flow. The perfusion method was performed, monitored and analyzed according to the method of Weir, GC et al. (J. Clin., Investigat. 54:1403-1412 (1974), which is incorporated herein by reference).

The present invention also provides a pharmaceutical composition, which contains the GLP-1 analog of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition can be processed by traditional pharmaceutical techniques, and can be taken alone or mixed with other therapeutic agents, especially through parenteral administration. The most suitable routes of administration include intramuscular and subcutaneous injections.

Administration by parenteral route is once a day, and the dosage is between 1 pg/kg and 1000 μg/kg, although the dosage can be higher or lower than this standard. The specific dosage depends on the severity of the patient's condition and the patient's height, weight, sex, age and medical history.

In manufacturing the compositions of the present invention, the active ingredient comprising at least one protein usually needs to be mixed with or diluted with an excipient. If the excipient is used as a diluent, it can be in solid, semi-solid or liquid form, thereby serving as a carrier or matrix for the active ingredient.

When preparing a formulation, if the active protein is completely insoluble, it may be necessary to first grind the active mixture to a particle size suitable for incorporation with the other ingredients. Usually ground to below 200 mesh. If the active mixture is completely water soluble, the particles will usually be adjusted to a roughly uniform size, such as about 40 mesh.

Useful excipients include lactose, glucose, sucrose, trehalose, sorbitol, mannitol, starch, acacia, calcium silicate, microcrystalline cellulose, water, syrup, and methylcellulose. Lubricants (such as talc, magnesium, and mineral oil), wetting agents, emulsifying and suspending agents, preservatives (such as methylhydroxybenzoate and propylhydroxybenzoate), sweeteners may also be added to the formulation. or flavor enhancers. The compositions of the present invention can be formulated so that the active ingredient has a rapid onset of action after administration by routes conventional in the art, and can also be formulated so that the active ingredient has a sustained or slow release onset of action.

The compositions are conveniently presented in unit dosage form, each unit dosage generally comprising from about 50 [mu]g to 100 mg, or more usually from about 1 mg to 10 mg, of the active ingredient. "Unit dose" refers to a single dosage unit suitable for humans and other mammals, each unit comprising a predetermined amount of active substance, mixed with an appropriate amount of pharmaceutically acceptable excipients, capable of producing the desired therapeutic effect.

For parenteral administration, the protein-containing composition of the present invention is preferably mixed with distilled water and adjusted to a pH of about 6.0 to 9.0.

Other pharmaceutical methods can also be used to control the duration of the response. Controlled release formulations can be prepared through the use of polymers to complex or absorb the compositions of the invention. Selection of the appropriate polymer (e.g., polyester, polyamic acid, polyvinylpyrrolidone, vinyl acetate, methylcellulose, carboxymethylcellulose, and protamine), its concentration, and method of incorporation to control drug delivery, as well Can be used for controlled drug release.

Another possible method of controlling duration of action is to incorporate the proteins of the invention into polymeric particles (eg, polyesters, polyamines, hydrogels, polyethylene (lactic acid), or vinyl acetonitrile copolymers).

In addition to incorporating compounds into polymer particles, the mixture can also be encapsulated in microcapsules, such as hydroxymethylcellulose or gel microcapsules, or in colloidal drug delivery systems using agglomeration or interfacial polymerization techniques , such as forming liposomes, albumin microspheres, microemulsion particles, microparticles, micromicelles, or incorporation into macroemulsions (macroemulation), as disclosed in Remington's Pharmaceutical Sciences (1980).

Likewise, the present invention provides a method for treating diabetes or hyperglycemia in a mammal (especially a human) in need thereof, comprising the step of administering to the mammal an effective amount of a GLP-1 analogue or composition of the present invention.

By way of illustration, the following examples are provided to help describe embodiments for making and practicing the invention. These examples are not to be construed in any way as limiting the scope of the invention.

Example

review

By solid-phase peptide chemistry, on an Applied Biosystems (ABI) 460A peptide synthesizer, using MBHA resin (Applied Biosystems Inc., lot#A1A023, 0.77mmol/g), generated conjugated to methylbenzhydrylamine (MBHA) Resin intermediate peptide fragment. All amino acids are protected at their α-amino groups by tert-tert-butoxycarbonyl (t-Boc). Amino acids with active side chains are protected as follows: arginine (Tos); lysine (Cl-Z); tryptophan (CHO); glutamic acid (CHex); tyrosine (Br-Z); Bzl); aspartic acid (OBzl); threonine (Bzl).

The protected amino acid was activated with dichloromethane (DCM) and half an equivalent of dicyclohexylcarbodiimide (DCC) per equivalent of amino acid to form the symmetrical amino acid anhydride. However, arginine, glutamine, and glycine residues are activated by forming their respective 1-hydroxybenzotriazole (HOBt) esters (amino acids, HOBt, and DCC are dissolved in dimethyl in formamide).

Through a series of coupling and deprotection cyclic reactions, the reactive residues gradually move from the C-terminus to the N-terminus. Coupling refers to the nucleophilic substitution reaction between the activated amino acid and the first free amine in the pre-coupled amino acid. Deprotection refers to the removal of the N-terminal protecting group Boc with anhydrous trifluoroacetic acid (TFA). After neutralization with diisopropylethylamine (DIEA), a free amine group is generated.

The synthesis scale was 0.5 mmol. The concentration of the MBHA resin functional area was 0.77mmol/g, and a total of 649mg of resin was used. In addition, it is necessary to prepare symmetrical anhydrides (dehydrating agents) twice the weight of the resin for all amino acids. The C-terminal arginine was coupled to MBHA resin via standard synthesis. All residues are double coupled. This means that each residue is coupled to the resin twice to ensure complete reaction of the _NH2 groups on the resin. In the second coupling, no Boc deprotection was performed before re-adding the amino acid, which allowed the full reaction of all free amine groups in the resin. The tryptophan residues were coupled four times. After the second coupling in each double coupling cycle reaction, the terminal Boc protecting group was removed with anhydrous trifluoroacetic acid (TFA) and neutralized with diisopropylethylamine (DIEA).

The formyl side chain protecting group on the tryptophan residue was reacted with piperidine in dimethylformamide (DMF) prior to separation of the peptide from the resin. After the peptide-resin conjugate was transferred to a 50 ml glass flask, several washes were performed with dichloromethane (DCM) and dimethylformamide (DMF). Then 3-5 ml of a 50/50 piperidine/DMF solution was added until the peptide-resin was completely covered. After five minutes the piperidine/DMF was removed with a vacuum filter and 3-5 ml of piperidine/DMF was added. After ten minutes the piperidine/DMF was removed again with a vacuum filter and 15-20 ml of piperidine/DMF was added. After fifteen minutes the piperidine/DMF was removed and the peptide-resin was washed several times with DMF and then with DCM. The peptide-resin is then placed in an incubator (without heating) to remove the solvent.

Alternatively, polymers incorporating peptide fragments can also be protected with fluorenylmethoxycarbonyl (Fmoc). Will Rink

Amide MBHA resin, Fmoc-amino acid, O-benzotriazole-N,N,N′,N′-tetramethyluronium tetrafluoroborate (HBTU) were dissolved in dimethylformamide (DMF) and washed with Activation with N-methylmorpholine and deprotection of the Fmoc group with piperidine (step 1). If necessary, the lysine (Aloc) group can be selectively artificially deprotected, and the resin is placed in _three equal parts of 5ml CHCl _{3 :} NMM:HOAc(18:1 :0.5) in solution for two hours (step 2). The resin was then washed with _CHCl3 (6x5ml), 20% HOAc in DCM (6x5ml), DCM (6x5ml) and DMF (6x5ml). Occasionally, the synthesis becomes spontaneous again (step 3) due to the addition of hydroxyethylethylenediamine (AEEA) groups, acetic acid, or 3-maleimide propionic acid (MPA). Resin removal and product isolation were performed using 85% trifluoroacetic acid (TFA)/5% TIS/5% thioanisole and 5% phenol, followed by immediate treatment with dry ice-frozen _Et2O (step 4). Use Varian (Rainin) preparative binary HPLC system, the product is purified by preparative reverse-phase HPLC: use the 10 μ phenyl-hexyl that Phenomenex Luna company produces, 21mm * 25cm post and wavelength are the ultraviolet detector of 214, 254nm ( Varian Dynamax UVD II), at a flow rate of 9.5 ml per minute, washed with 30-55% B (0.045% TFA in water (A) and 0.045% TFA in CH ₃ CN (B)) gradient washing for 180 minutes. The degree of purification was 95% as determined by RP-HPLC mass spectrometry using a Hewlett Packard LCMS-1100 series mass spectrometer equipped with a diode detector and electrospray ionization.

Protective groups are chemical groups that prevent amino acids from reacting spontaneously. These protecting groups include acetyl, 9-fluorenylmethoxycarbonyl (FMOC), tert-butoxycarbonyl (Boc), benzyloxycarbonyl (CBZ) and the like. Specific amino acids with protective groups are shown in Table 1.

Table 1

Example 1

Synthesis of the following substances:

step 1

Fmoc-Rink Amide MBHA Resin

The solid-phase peptide synthesis of the described analogs on a 100 μmol scale was carried out as follows: using artificial solid-phase synthesis and a Symphony peptide synthesizer, Fmoc-protected Rink Amide MBHA resin, Fmoc-protected amino acids, O-benzotriazole -1-yl-N,N,N',N'-tetramethyl-uronium tetrafluoroborate (HBTU) was dissolved in dimethylformamide (DMF), activated with N-methylmorpholine, and Piperidine deprotects the Fmoc group (step 1). The Boc group was removed from the product of step 2 before coupling with Fmoc-His(Trt)-OH. Resin removal and product isolation were performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation with dry ice frozen _Et2O (step 2). Use Varian (Rainin) preparative binary HPLC system, the product is purified by preparative reverse phase HPLC: use the 10 μm phenyl-hexyl that Phenomenex Luna company produces, 21mm * 25cm column and the ultraviolet detector (VarianDynamax UVD of wavelength 214nm and 254nm II), with a flow rate of 9.5ml per minute, gradient elution with 30-55% B (0.045% TFA aqueous solution (A) and 0.045% TFA in CH ₃ CN solution (B)) for 180 minutes, determined by RP-HPLC, The desired peptide was obtained in >95% purity.

Example 2

Synthesis of the following substances:

step 1

The synthesis steps and conditions of GLP-1 analogs are as described in Example 1.

The desired peptide was obtained with >96% purity as determined by RP-HPLC.

Example 3

Synthesis of the following substances:

step 1

The synthesis steps and conditions of GLP-1 analogs are as described in Example 1.

The desired peptide was obtained with >95% purity as determined by RP-HPLC.

Example 4

Synthesis of the following substances:

step 1

The synthesis steps and conditions of GLP-1 analogs are as described in Example 1.

The desired peptide was obtained with >95% purity as determined by RP-HPLC.

test case

Insulin-stimulating ability of GLP-1 analogs

The GLP-1 analogs prepared in Examples 1-4 were respectively injected into cultured RIN-38 mouse islet β-cell tumor cells, and then the amount of immunoreactive insulin (IRI) released into the medium was monitored. Methods The improved method of Albano, JDM et al. (Acta Endocrinol.70: 487-509 (1972)) was used: 500 μl of phosphate buffer solution, 50 μl of perfusate sample or perfusate containing standard amount of mouse insulin were sequentially injected into the culture vessel , 100 μl anti-insulin antiserum (Wellcome Laboratories: diluted 1:40,000), and 100 μl [ ¹²⁵ I] insulin, with a total volume of 750 μl, were placed in a 10×75 mm disposable glass test tube. Incubate at 4°C for 2-3 days, and use charcoal to separate free insulin from antibody-bound insulin.

As a result, the amount of radioactivity detected is proportional to the amount of test substance added. This shows that the GLP-1 analogs prepared in Examples 1-4 have the ability to stimulate insulin secretion.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (3)

1. A compound of formula I:

R¹-X-R²

formula I

Wherein,

R¹selected from: l-histidine, D-histidine, deaminated histidine, 2-aminohistidine, beta-hydroxyhistidine, alpha-fluoromethylhistidine and alpha-methylhistidine;

x is a linking unit selected from the group consisting of:

R²is a peptide portion or fragment selected from the group consisting of:

-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R⁴；

R³is selected from methyl;

R⁴is selected from NH₂。

2. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.

3. Use of a compound according to claim 1 for the preparation of a medicament for the treatment of diabetes, insulin resistance and neurological disorders.