CN100503637C - Glucagon-like peptide analog, its composition and its using method - 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 thereof, and methods of use thereof

Technical Field

The present invention relates to glucagon-like peptide analogs and compositions thereof, which are useful for up-regulating insulin expression in a mammal, thereby treating diabetes. In particular, these peptide derivatives are useful for the long-term treatment of diabetes and other diseases associated with insulinotropic peptides, as well as diseases of functional activity of the gastrointestinal tract associated with glucagon levels.

Background

The endocrine function of the islets is governed by a complex set of mechanisms that are influenced not only by metabolites such as glucose, amino acids and catecholamines, but also by local paracrine secretion. The major insulins, glucagon, insulin and somatostatin bind to specific pancreatic cells (a cells, B cells and D cells, respectively) to regulate the secretory response. Although insulin secretion is mainly determined by blood insulin levels, human growth hormone releases inhibin to inhibit the secretion of glucose-regulated insulin. In addition to the juxtacral secretion regulation of insulin secretion, the presence of the incretin factor is indeed demonstrated.

The concept of endocrine secretion stems from the observation of the following phenomena: energy from food intake or intestinal glucose absorption can stimulate greater stimulation to promote insulin release than the same amount of energy (glucose) injected intravenously (Elrick, h. et al, j.clin.endocrinol.meta,241076-1082, 1964; McIntyre, n, et al, j.clin.endocrinol.meta,25,1317-1324, 1965). It is therefore hypothesized that the gut-derived signals triggered by the uptake of oral nutrients represent a potent insulin secretagogue that promotes increased insulin release when energy is taken up through the gastrointestinal tract rather than through the parenteral route (Dupre, j. et al, Diabetes,15，555-559，1966)。

although some neurotransmitters and gut hormones also have endocrine-like activity, a great deal of evidence from immune, antagonist and knock-out studies suggests that glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide (GLP) -1 represent the peptides that play a dominant role in insulin secretion caused by stimulation with most nutrients. The observation that type 2 diabetic patients show a marked reduction in the number of meal-stimulated insulin secretory releases has led to interest in the following studies: whether incomplete endocrine or endocrine resistance is associated with the pathophysiology of beta-cell dysfunction in diabetic patients.

In 1987, glucagon-like peptide-1 (GLP-1) was first discovered by humans and is considered to be an endocrine hormone, a peptide secreted by the intestinal tract after food intake. GLP-1 is secreted by intestinal L cells after proteolytic processing by a 160 amino acid precursor protein (i.e., preproglucagon).The cleavage of preproglucagon firstly produces a peptide GLP-1 consisting of 37 amino acids, i.e. GLP-1(1-37) OH, which is very weak in activity. Subsequent cleavage at position 7 produces the biologically active GLP-1(7-37) OH. When the terminal glycine residue is to be removed in L-cells, about 80% of the synthetic GLP-1(7-37) OH is amidated at the terminal C-position. Free acid GLP-1(7-37) OH and amide compound GLP-1(7-37) NH₂Is indistinguishable from metabolic turnover.

It is well known that stimulation of insulin secretion by GLP-1 causes an increase in glucose concentration in cells, thereby lowering blood glucose levels (Moisov, s. et al, j.clin.invest.,79616-619, 1987; kreymann, B. et al, Lancet ii, 1300-; orskov, C. et al, Endocrinology,123,2009-2013, 1988). Acute intraventricular injections of GLP-1 or contraction of the GLP-1 receptor produce a transient decrease in food intake (turtle m.d. et al, Nature,37960-72, 1996), however, several studies have shown that more long-term intraventricular injections or administration of parenteral GLP-1 receptor agonists are associated with weight loss (Meeran, k. et al, Endocrinology,140，244-250，1999；Davies，H.R.Jr.，Obes.Res.，6147-; szayna, m, et al, Endocrinology,1411936-1941, 2000; larsen, p.j. et al, Diabetes,50,2530-2539, 2001). A large number of GLP-1 analogs having insulinotropic action are known in the art. For example, such analogs include GLP-1(7-36), Gln⁹-GLP-1(7-37), D-Gln9-GLP-1(7-37), acetyl-Lys 9-GLP-1(7-37), Thr16-Lys18-GLP-1(7-37) and Lys18-GLP-1 (7-37); derivatives of GLP-1 include acid addition salts, carboxylic acid salts, lower alkyl esters and amide compounds (WO 91/11457; EP0733, 644; U.S. Pat. No. 3, 5,512,549).

Most of the GLP-1 effects described in preclinical trials have also been demonstrated in human studies. Injecting GLP-1(7-36) NH into normal human body under rapid glucose intake and food intake state₂Can stimulate insulin secretion and significantly reduce blood glucose (Orskov, c. et al, Diabetes,42658 & 661, 1993; qualmann, C. et al, acta, Diabetol.,32，13-16，1995)。

the choice of GLP-1 based peptides for treatment of diabetic patients who have failed sulfonylureas (Nauck, m.a. et al, Diabetes Care,21,1925-1931, 1998). GLP-1 stimulates insulin secretion only in case of hyperglycemia. This property of GLP-1 makes it safer than insulin, and it has been observed that the amount of insulin secreted is proportional to the degree of hyperglycemia. In addition, treatment with GLP-1 will result in insulin release from the pancreas and play a role in the first pass of insulin in the liver. This can result in low circulating levels of peripheral insulin compared to subcutaneous insulin injections. GLP-1 slows emptying of the gastrointestinal tract, which is more beneficial for allowing nutrients to be absorbed over a longer period of time, thereby reducing the post-prandial glucose peak. Several reports suggest that GLP-1 may increase insulin sensitivity in peripheral tissues such as muscle, liver and fat. Finally, GLP-1 also exhibits potential appetite regulating functions.

The therapeutic potential of GLP-1 and its analogs is even more pronounced if it is administered to type 1 diabetics. Numerous studies have demonstrated the effectiveness of native GLP-1 in treating insulin-dependent diabetes mellitus (IDDM). Similar to non-insulin dependent diabetes mellitus (NIDDM) patients, GLP-1 is effective in relieving hunger hyperglycemia through its glucagon-stabilizing (glucostatic) property. Additional studies have shown that GLP-1 also reduces postprandial blood glucose excursions in IDDM patients, most likely by prolonging gastric emptying. These observations suggest that GLP-1 can be used to treat IDDM and NIDDM.

However, the biological half-life of the native GLP-1 molecule is influenced by dipeptidase iv (dpp iv) activity and is rather short lived. For example, GLP-1(7-37) OH has a biological half-life of only 3-5 minutes (U.S. Pat. No. 5118666). The blood glucose concentration was continuously reduced only by continuous infusion, as demonstrated in the study, 24 hour intravenous infusion controlled GLP-1 concentration (Larsen, j. et al,DiabetesCare，24,1416-1421, 2001). The enzyme DPP IV is a serine protease which preferentially hydrolyzes peptides at a position after the penultimate NH 2-terminal proline (Xaa-Pro-) or alanine (Xaa-Ala-) (Mentlein, R., Regul. pept.),859-25, 1999), this enzyme has been shown to rapidly produce metabolic changes in GLP-1 in vitro. Thus, long-acting GLP-1 based peptides that are resistant to DPP IV have great therapeutic potential in treating diabetic patients.

Disclosure of Invention

The present invention provides GLP-1 analogs that exert a longer lasting effect than native GLP-1 and are completely resistant to hydrolysis by the enzyme DPP IV.

The invention includes compounds having the following general formula I:

R¹-X-R²

formula I

In the formula,

R¹selected from: l-histidine, D-histidine, deaminated-histidine, 2-amino-histidine, β -hydroxy-histidine, homohistidine, α -fluoromethyl-histidine, α -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

-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-R⁴

-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-R⁴

-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-R⁴

-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;

R³is selected from C_1-6An alkyl group;

R⁴is selected from NH₂And OH, which represent free acid or amide forms of the terminal amino acid;

R⁵is selected from C_6-10An unbranched acyl group.

The present invention also provides a compound of the formula:

R¹-X-R⁶

wherein:

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

x is as defined above;

R⁶are peptide fragments that contain one or more additional amino acids at their carboxy terminus in addition to a fragment that is substantially homologous to the naturally occurring GLP-1 peptide.

The present invention also provides a compound of the formula:

R¹-X-R⁷

wherein:

R¹is tyrosine;

x is as defined above;

R⁷are peptide fragments that contain a peptide that is homologous to a naturally occurring GIP (3-42) peptide:

-EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ⁴²a substantially homologous sequence.

The invention also provides chemically reactive derivative compounds of the above compounds that react with available functional groups on a cell carrier to form covalent bonds, wherein the cell carrier comprises mobile blood proteins.

The invention also provides a method of treating diabetes, insulin resistance and nervous system disorders which comprises administering to a patient in need thereof a non-toxic, therapeutically effective amount of a compound of the invention. Preferably, the compound is administered once a day, twice a day or three times a day, in the form of an oral, intravenous, subcutaneous or aerosol formulation.

The invention also provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier.

The invention also provides the use of said compounds for the preparation of a medicament for the treatment of diabetes, insulin resistance and neurological disorders.

Detailed Description

The key point of the invention is that the catalyst contains CF₃The linking unit of (a) replaces Ala at the amino terminus of GLP-1⁸The position is a recognition site of DPP-IV. Recently, it has been reported that a novel linker unit [ -CH (CF) is present in the main peptide backbone of a class of metalloprotease inhibitors₃)NH-]May be used as mimetics of the conventional linker unit (NHCO) (organic Letters, 2, 1827-,42,3143-3144, 2001). Based on the same principle, via [ -CH (CF)₃)NH-]The modified GLP-1 analogs retain the same biological activity as the insulinotropic substance and are long acting.

Peptide moieties (peptide fragments) selected from defined human GLP-1 amino acid sequences 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. Several researchers have reported the amino acid sequence of GLP (Lopez, l.c. et al, acad.sci., USA)805485-5489, 1983; bell, G.I. et al, Nature302: 716-718 (1983); heinrich, G. et al, Endocrinol,115: 2176-2181(1984)). The structure of the mRNA of preproglucagon (preproglucagon) and its corresponding amino acid sequence is well known. The proteolytic process of conversion of the precursor gene product (i.e., preproglucagon) to glucagon and of the two insulinotropic peptides has also been established. As used herein, the notation of GLP-1(1-37) denotes 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). Similarly, GLP-1(7-36) represents a polypeptide which is a GLP-1 polypeptide having all amino acids from position 7 (N-terminus) to position 36 (C-terminus).

The same practice can be applied to glucose-dependent insulinotropic polypeptide (GIP), which is a 42 amino acid polypeptide from one of the larger 153 amino acid precursors (Takeda, j. et al, proc. natl. acad. sci., USA)84,7005-7008, 1987). Like GLP-1, GIP is cleaved at the alanine in position 2 by the broadly expressed aminopeptidase DPP IV, thereby generating a truncated inactive peptide GIP (3-42). A GIP analog having the formula (4 diastereomer) is resistant to DPP IV and exerts a long-lasting GIP effect.

The invention also provides pharmaceutical compositions comprising a compound of the 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 common works in this field, for example: dugas, H.and Penney, C., Bioorganic Chemistry (1981) Springer-Verlag, New York, page 54-92; merrifield, j.m., chem.soc.,85，2149，1962，and Stewart and Young，Solid Phase Peptide Synthesis，page 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 Centre Drive, Foster City, Calif. 94404) and a synthesis cycle apparatus provided by Applied Biosystems. Boc protected amino acids and other reagents are available from Applied Biosystems and other chemical suppliers. Para-methylbenzhydrylamine resin can be applied using sequential BOC chemistry in a double coupling scheme to produce a C-terminal carboxamide (carboxamide). For the production of the C-terminal acid, the corresponding PAM resin can be used. Aspartic acid, glutamine and arginine can be coupled using preformed hydroxybenzotriazole esters.

Desired to contain CF₃The fragment of (a) can be synthesized according to the following reaction procedure. Protected glutamic acid 1 may be reacted with CF₃The methyl hemiacetal in CHO reacts to form 2. 2 reacts with acetate anion to form 3, 3 is deprotected to form carboxylic acid 4. In Et₃In the presence of N (in tetrahydrofuran with tert-butanol) with (PhO)₂PON₃Process 4, yield 5. The 5 is catalyzed and hydrogenated by palladium carbon Pd/C catalyst to generate 6 diastereoisomer mixture. 6 all pure diastereoisomers A, B, C, D can be obtained by HPLC separation.

Attachment of A, B, C, D to polymer-bound peptide fragments by solid phase synthesis using an Applied Biosystems 430 peptide synthesizer, deprotection and addition of R¹，R¹Selected from: l-histidine, D-histidine, deaminated-histidine, 2-amino-histidine, β -hydroxy-histidine, homohistidine, α -fluoromethyl-histidine and α -methylhistidine. Deprotection and cleavage from a polymeric solid support provides a novel class of GLP analogs that are resistant to hydrolysis by DPP IV and have long-term insulinotropic activity.

The invention also includes salt forms of GLP-1 (glucagon-like peptide-1) analogs. The GLP-1 analogue can be strong acid or strong alkali and can react with a plurality of inorganic bases and inorganic acids to generate a salt. The inorganic acids used to synthesize the acid addition salts are typically hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and the organic acids are p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of salts include: sulfate, bisulfate, sulfite, bisulfite, phosphate, hydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, 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, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, 3-hydroxybenzutyrate, glycolate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, propionate, caprylate, acrylate, formate, isobutyrate, heptanoate, propiolate, oxalate, malonate, fumarate, benzoate, phenylbutyrate, citrate, lactate, 3-hydroxybenzutyrate, glycolate, or the like, Tartrate, methanesulfonate, propanesulfonate, naphthalenesulfonate 1-sulfonate, naphthalenesulfonate 2-sulfonate, mandelate and the like. Preferred acid addition salts are those formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, and especially hydrochloric acid.

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

The improved GLP-1 analogs of the invention have a variety of uses, they can be used to treat diabetes, they can be used as sedatives, they can treat neurological disorders, they can act on the central nervous system to produce anxiolytic effects, they can stimulate the central nervous system, they can be used as post-operative treatments and they can be used to treat insulin resistance.

A. Treatment of diabetes

The modified GLP-1 analogs of the invention are generally able to normalize hyperglycemia by a glucose-dependent mechanism. Thus, the modified GLP-1 analogs can be used as leading drugs for treating type II diabetes, and can also be used as adjuvant drugs for treating type I diabetes.

The modified GLP-1 analogs are used at effective doses to treat diabetes more effectively than unmodified GLP-1. Because the modified GLP-1 analogs are more stable in vivo, effective therapeutic effects can be achieved with smaller doses. The invention is particularly suitable for the treatment of diabetic patients, both type I and type II, because the activity of the polypeptide is dependent on the blood glucose level, and the risk of hypoglycemia as a side effect in the new method is greatly reduced compared to currently used treatments.

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

B. Treatment of neurological disorders

The modified GLP-1 analogs of the invention can also be used as sedatives. In one aspect, the modified GLP-1 analog is administered in an amount sufficient to induce an attractive or anxiolytic effect in mammals whose central or peripheral nervous system activity has been enhanced by an abnormality, thereby sedating the mammal. Such modified GLP-1 analogs can be administered intracerebroventricularly, orally, subcutaneously, intramuscularly, or intravenously. The method is effective in treating or ameliorating nervous system symptoms such as anxiety, motor confusion, aggressive behavior, delirium, mania, panic attacks, hysterisis, and insomnia.

In another related aspect, the invention also includes a method of increasing mobility in a mammal comprising: administering to the subject a modified GLP-1 analog in an amount sufficient to produce an effect in the mammal and to activate the efficacy of the subject. The method is more applicable to 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 disorder, sleep apnea syndrome, distraction disorder, amnesia, lethargy, etc. In these conditions, it may be advantageous to wake the central nervous system.

The modified GLP-1 analogues of the present invention may be used to wake a patient in the treatment or amelioration of depression, schizoaffective disorder, sleep apnea syndrome, distraction, amnesia, lethargy and other conditions. The efficacy of GLP-1 analogs can be assessed by consulting patients to assess their condition, by psychological or neurological tests, or by amelioration of symptoms associated with such conditions. For example, the efficacy for hypersomnia can be evaluated from the degree of control over the onset of hypersomnia. As another example, the effect of a modified GLP-1 analog on focusing attention or improving memory in a patient can be assessed using any of a variety of different diagnostic tests well known to those skilled in the art.

C. Post-operative treatment

The modified GLP-1 analogs of the invention can be used as a post-operative treatment. Such modified GLP-1 analogs can be required by the patient, whether 1-16 hours prior to surgery, during surgery, or within 5 days after surgery.

The modified GLP-1 analogue is administered to the patient from 16 hours prior to the start of surgery to the first 1 hour. The compounds of the present invention are administered to a patient in order to reduce catabolic effects and insulin resistance, and the length of time of administration depends on a number of factors. These factors are well known to practitioners of ordinary skill and include: most important is whether the patient is fasted or glucose infused or drinks beverages or other forms of food during the surgical preparation phase. Other important factors include the sex, weight, age of the patient, the severity of the blood glucose imbalance, any potential causes of inability to regulate blood glucose, the severity of the trauma anticipated by surgery, the route of administration and bioavailability, the endurance, formulation and efficacy of the individual. Preferred time periods for administration of the modified GLP-1 analogs of the invention range from one hour to ten hours prior to surgery. The optimal time period for starting the administration is two hours before the operation to eight hours before the operation.

Insulin resistance after a particular type of surgery, such as selective abdominal surgery, is most severe the first day after surgery, lasting at least five days, and possibly three weeks before returning to normal. Thus, after undergoing surgical trauma, the patient needs to be administered the modified GLP-1 analogue of the invention for a period of time after surgery, depending on whether the patient is fasting or has had a glucose infusion or has consumed a drink or other form of food after surgery. Also, sex, body weight, age, severity of blood glucose imbalance of the patient, potential causes of loss of blood glucose regulation function, actual severity of surgical trauma, route of administration and bioavailability, individual endurance, formulation and efficacy of the drug are all factors. The preferred time of use of the drug administered in the present invention is no more than five days post-operatively.

D. Treatment of insulin resistance

Treatment of insulin resistance, unlike its use in post-operative treatment, 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 cell surface receptors or by a change in intracellular metabolism. The first type of insulin resistance is characterized by decreased insulin sensitivity, and a typical treatment is increased insulin dosage. The second is characterized by reduced insulin responsiveness and ineffective high dose insulin therapy. Insulin resistance-induced damage can be treated with doses of insulin proportional to the degree of insulin resistance, and it is therefore apparent that such damage is caused by decreased insulin sensitivity.

An effective dose of a modified GLP-1 analog that normalizes blood glucose levels in a patient is affected by: dosage of GLP-1 analogue, presence or absence of contraindication, sex, weight and age of patient, severity of loss of blood glucose regulation function, underlying cause of loss of blood glucose regulation function, simultaneous administration of glucose or other saccharide, administration route and bioavailability, patient tolerance, formulation and drug effect.

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

Immunoreactive insulin (IRI) is detected using radioimmunoassays directed to insulin. Any radioimmunoassay method that can detect IRI can be used; one of them is a method of improvement of the method invented by Albano, j.d.m., et al, Acta endocrinol.70: 487-509(1972). In this modification a phosphate/albumin buffer at pH 7.4 was used. Into the incubator were sequentially injected 500. mu.l of a phosphate buffer solution, 50. mu.l of a sample of a perfusate or a perfusate containing a standard amount of mouse insulin, 100. mu.l of an insulin-resistant antiserum (Wellcome Laboratories: diluted 1:40,000), and 100. mu.l of¹²⁵I]Insulin, 750. mu.l in total volume, was placed in disposable glass tubes of 10X 75 mm. The incubation was carried out at 4 ℃ for 2-3 days, and the free insulin was separated from the antibody-bound insulin by activated charcoal. The assay sensitivity was 1-2 uU/ml. To determine the amount of IRI released into the cell growth medium in tissue culture, proinsulin can be radiolabeled. Although any radiolabel capable of labelling a polypeptide may be used, it is preferred that any radiolabel be used³H leucine to obtain a labelled proinsulin.

The pancreatic islet-promoting function of a GLP-1 analog can also be determined by a pancreatic perfusion assay. The mouse pancreas in situ perfusion assay is an improvement over the method of Penhos, J, C, et al (Diabetes, 18: 733-738 (1969)). Rats of fasted male Charles River species varying in body weight 350-. Blood vessels of the kidney, adrenal gland, stomach and lower colon were ligated. The entire intestine is not functional except for the duodenum and descending colon, which are about 4 cm, and the rectum. Thus only a small segment of the intestine is perfused, minimizing the effects of the immune response between the glucagon-like peptide and intestinal material. The perfusate was a modified Kreba-Ringer bicarbonate buffer containing 4% T70 dextroseGlycosides and 0.2% bovine serum albumin (fragment V), with 95% O₂And 5% CO₂And (4) blowing bubbles. An extracorporeal circulation machine (Buchler polystatic, Buchler Instruments Division, Nuclear-chicago corp) with a 4-channel roller bearing pump was used, together with a three-way valve to control the flow of the perfusion fluid. The perfusion method was performed, monitored and analyzed according to the method of Weir, G.C. et al (J.Clin., Investigat.54: 1403-1412(1974), which is incorporated herein by reference).

The invention also provides a pharmaceutical composition comprising a GLP-1 analog of the invention and a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical compositions may be formulated by conventional pharmaceutical techniques and may be administered alone or in admixture with other therapeutic agents, especially by parenteral administration. The most suitable routes of administration include intramuscular and subcutaneous injection.

Administration by parenteral route is once a day in an amount of between 1pg/kg and 1000 μ g/kg, although the amount may be above or below this standard. The specific amount depends on the severity of the patient's condition and the patient's height, weight, sex, age and medical history.

In making the compositions of the present invention, the active ingredient comprising at least one protein will generally need to be mixed or diluted with an excipient. If the excipient is used as a diluent, it may be in the form of a solid, semi-solid or liquid which acts as a carrier or matrix for the active ingredient.

In preparing the formulation, if the active protein is not soluble at all, it may be necessary to first grind the active mixture to particles of suitable size for combination with the other ingredients. Usually, the resulting product is ground to 200 mesh or less. If the active mixture is completely water soluble, the particles are often adjusted to a generally uniform size, such as about 40 mesh.

Useful excipients include lactose, glucose, sucrose, trehalose, sorbitol, mannitol, starch, gum acacia, calcium silicate, microcrystalline cellulose, water, syrup, and methyl cellulose. The preparation may also contain lubricant (such as pulvis Talci, magnesium and mineral oil), humectant, emulsifying and suspending agent, antiseptic (such as methyl hydroxybenzoate and propyl hydroxybenzoate), sweetener or flavoring agent. The compositions of the present invention may be formulated so that the active ingredient is rapidly effective upon administration by any route conventional in the art, or so that it is effective for sustained or sustained release.

The compositions are suitably presented in unit dosage form, typically comprising from about 50 μ g to 100mg, or more typically from about 1mg to 10mg, of the active ingredient per unit dose. "Unit dose" means a single dosage unit suitable for administration to humans and other mammals, each unit containing a predetermined quantity of active material in admixture with suitable pharmaceutically acceptable excipients, to produce the desired therapeutic effect.

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

Other pharmaceutical methods may also be used to control the duration of the reaction. Controlled release formulations can be prepared by using polymers to complex or absorb the compositions of the present invention. The selection of suitable polymers (e.g., polyesters, polyurethanes, polyvinylpyrrolidone, vinyl acetate, methylcellulose, carboxymethylcellulose, and protamine), their concentrations, and methods of incorporation to control drug delivery can also be used to control drug release.

Another possible method of controlling duration of action is to incorporate the proteins of the invention into polymer particles (e.g., polyesters, polyamino acids, hydrogels, polyethylene (lactic acid), or vinyl acetonitrile interpolymers).

In addition to incorporating the compound into the polymeric particles, the mixture may be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules, or in a colloidal drug delivery system, such as to form liposomes, albumin microspheres, microemulsion particles, very fine micelles, or incorporated into macroemulsions (macroemulsification), as disclosed in Remington's pharmaceutical Sciences (1980).

Also, the present invention provides a method of treating diabetes or hyperglycemia in a mammal (particularly a human) in need of such treatment, comprising the step of administering to the mammal an effective amount of a GLP-1 analog or composition of the present invention.

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

Examples

Overview

Intermediate peptide fragments bound to Methylbenzhydrylamine (MBHA) resin were generated by solid phase peptide chemistry on an Applied Biosystems (ABI)460A peptide synthesizer using MBHA resin (Applied Biosystems Inc., lot # A1A023, 0.77 mmol/g). All amino acids have their alpha-amino group protected by t-butyloxycarbonyl (t-Boc). Amino acids with reactive side chains are protected as follows: arginine (Tos); lysine (Cl-Z); tryptophan (CHO); glutamic acid (CHex); tyrosine (Br-Z); serine (Bzl); aspartic acid (OBzl); threonine (Bzl).

The protected amino acid was activated with Dichloromethane (DCM) and half an equivalent of Dicyclohexylcarbodiimide (DCC) was used per equivalent of amino acid to form a symmetrical amino acid anhydride. But the arginine, glutamine and glycine residues are activated by forming the respective 1-hydroxybenzotriazole (HOBt) esters (amino acids, HOBt and DCC are dissolved in dimethylformamide at 1:1: 1).

Through a series of coupling and deprotection cycles, the reactive residues are gradually moved from the C-terminus to the N-terminus. Coupling refers to the nucleophilic substitution reaction of the activated amino acid with the first free amine in the previously coupled amino acid. Deprotection refers to the removal of the protecting group Boc at the N-terminus with anhydrous trifluoroacetic acid (TFA). After neutralization with Diisopropylethylamine (DIEA), a free amine group is formed.

The combinationThe scale of formation was 0.5 mmol. The functional domain concentration of MBHA resin was 0.77mmol/g, and 649mg of resin was used in total. A symmetrical acid anhydride (dehydrating agent) having twice the weight of the resin was prepared for all the amino acids. The C-terminal arginine was coupled to MBHA resin by standard synthesis. All residues were double coupled. That is to say that each residue is coupled to the resin twice to ensure NH on the resin₂The groups are fully reacted. In the second coupling, no Boc deprotection was performed before the amino acid was reapplied, which allowed all free amine groups in the resin to react well. Four couplings of tryptophan residues were performed. After the second coupling in each double coupling cycle, 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 separating the peptide from the resin. The peptide-resin conjugate was transferred to a 50ml glass flask and washed several times with Dichloromethane (DCM) and Dimethylformamide (DMF). Then 3-5ml of 50/50 piperidine/DMF solution was added until the peptide-resin was completely covered. After five minutes the piperidine/DMF was removed by vacuum filtration and 3-5ml piperidine/DMF was added. Ten minutes later the piperidine/DMF was again removed by vacuum filtration and 15-20ml piperidine/DMF was added. Fifteen minutes later to remove piperidine/DMF, DMF washing peptide-resin several times, then DCM washing. The peptide-resin was then placed in an incubator (without heating) to remove the solvent.

In addition, the polymer to which the peptide fragment is bonded may also be protected with fluorenylmethoxycarbonyl (Fmoc). Sink will be

Amide MBHA resin, Fmoc-amino acid, O-benzotriazole-N, N' -tetramethyluronium tetrafluoroborate (HBTU) was dissolved in Dimethylformamide (DMF), activated with N-methylmorpholine, and the Fmoc group was deprotected with piperidine (step 1). Optionally, lysine (Aloc) groups were optionally deprotected manually, and the resin was placed in triplicate of Pd (PPh) in solution₃)₄5ml of CHCl₃Namely, NMM, HOAc (18:1:0.5) solution for two hours (step 2). Then using CHCl₃(6x 5ml), 20% HOAc in DCM (6X 5ml), DCM (6X 5ml) and DMF (6X 5ml) wash the resin. Sometimes, the synthesis becomes spontaneous again due to the addition of hydroxyethylethylenediamine (AEEA) groups, acetic acid or 3-maleimidopropionic acid (MPA) (step 3). Resin stripping and product isolation using 85% trifluoroacetic acid (TFA)/5% TIS/5% thioanisole and 5% phenol, immediately followed by Et frozen on dry ice₂And (4) performing O treatment. The product was purified by preparative reverse phase HPLC using a varian (rainin) preparative binary HPLC system: using a 10. mu.phenyl-hexyl column from Phenomenex Luna, a 21mm X25 cm column and an ultraviolet detector (Varian Dynamax UVD II) having a wavelength of 214 and 254nm, 30-55% B (0.045% aqueous TFA (A) and 0.045% TFA in CH at a flow rate of 9.5 ml/min₃CN solution (B)) for 180 minutes. The degree of purification was 95% by RP-HPLC mass spectrometry using a Hewlett Packard LCMS-1100 series mass spectrometer equipped with a diode detector and electrospray ionization.

A protecting group is a chemical group that prevents the spontaneous reaction of an amino acid. These protective groups include acetyl, 9-Fluorenylmethoxycarbonyl (FMOC), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (CBZ), and the like. Specific protected amino acids are shown in Table 1.

TABLE 1

Example 1

Synthesis of the following materials:

step 1

Fmoc-Rink Amide MBHA resin

Solid phase peptide synthesis of the analogs at 100 μmol scale was performed as follows: using an artificial solid phase synthesis method and a Symphony peptide synthesizer, Fmoc-protected Rink Amide MBHA resin, Fmoc-protected amino acid, O-benzotriazol-1-yl-N, N' -tetramethyl-uronium tetrafluoroborate (HBTU) were dissolved in Dimethylformamide (DMF), activated with N-methylmorpholine, and the Fmoc group was deprotected with piperidine (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 using 85% TFA/5% TIS/5% thioanisole and 5% phenol followed by dry ice frozen Et₂O precipitates (step 2). The product was purified by preparative reverse phase HPLC using a varian (rainin) preparative binary HPLC system: using a 10 μm phenyl-hexyl column from Phenomenex Luna, a 21mm X25 cm column and an ultraviolet detector (VarianDynamax UVD II) having a wavelength of 214nm and 254nm, 30-55% B (0.045% aqueous TFA (A) and 0.045% TFA in CH at a flow rate of 9.5 ml/min₃CN solution (B)) was gradient eluted for 180 minutes to give the desired purity as determined by RP-HPLC>95% of peptides.

Example 2

Synthesis of the following materials:

step 1

The synthetic procedures and conditions for GLP-1 analogs are described in example 1.

RP-HPLC analysis gave the desired peptide with a purity of > 96%.

Example 3

Synthesis of the following materials:

step 1

The synthetic procedures and conditions for GLP-1 analogs are described in example 1.

RP-HPLC analysis gave the desired peptide with a purity of > 95%.

Example 4

Synthesis of the following materials:

step 1

The synthetic procedures and conditions for GLP-1 analogs are described in example 1.

RP-HPLC analysis gave the desired peptide with a purity of > 95%.

Test example

Insulin secretion stimulating ability of GLP-1 analogs

GLP-1 analogs prepared in examples 1-4 were injected into cultured RIN-38 mouse islet beta cell tumor cells, respectively, and the release of the immunization into the medium was monitoredAmount of reactive insulin (IRI). The method adopts an improved method of Albano, J.D.M. et al (Acta Endocrinol.70: 487-509 (1972)): into the incubator were sequentially injected 500. mu.l of a phosphate buffer solution, 50. mu.l of a sample of a perfusate or a perfusate containing a standard amount of mouse insulin, 100. mu.l of an insulin-resistant antiserum (Wellcome Laboratories: diluted 1:40,000), and 100. mu.l of¹²⁵I]Insulin, 750. mu.l in total volume, was placed in disposable glass tubes of 10X 75 mm. The incubation was carried out at 4 ℃ for 2-3 days, and the free insulin was separated from the antibody-bound insulin by activated charcoal.

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

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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.