WO2016090628A1

WO2016090628A1 - Oxyntomodulin (oxm) analogs, synthesis and use thereof

Info

Publication number: WO2016090628A1
Application number: PCT/CN2014/093681
Authority: WO
Inventors: 马涛; 许树森; 张爱红; 邱红娟; 车美英
Original assignee: 北京韩美药品有限公司
Priority date: 2014-12-12
Filing date: 2014-12-12
Publication date: 2016-06-16

Abstract

Provided are oxyntomodulin (OXM) analogs having GLP-1 receptor antagonist activity and GCG receptor antagonist activity. Also provided are pharmaceutical compositions comprising the OXM analogs, and a use thereof.

Description

Gastric acid regulating peptide (OXM) analogue, its synthesis and application

Technical field

The present invention relates to oxyntomodulin (OXM) analogs, their synthesis and their use. In particular, the present invention relates to oxyntomodulin analogs and pharmaceutical compositions thereof, and to methods and uses thereof as effective drug candidates for clinical treatment of metabolic disorders such as obesity diseases.

Background technique

Overweight and obesity have become a worldwide health problem. According to estimates by the World Health Organization, 1.6 billion people in the world are overweight (BMI between 25-30) and 400 million people are obese (BMI over 30), accounting for 24% and 6% of the total population, respectively. Studies have shown that obesity can significantly increase the risk of cardiovascular disease, stroke, type 2 diabetes, osteoporosis and cancer (Whitlock, et al. Lancet, 2009, 373, 1083–96; Kopelman, et al. Nature , 2000, 404, 635–43). For every BMI above 24.9, it is possible for women to increase their risk of heart disease by 5%, while men increase the risk of 7% (Kenchaiah, et al. The New England Journal of Medicine, 2002, 347, 305). The rapid increase in obesity and the consequences of obesity place a heavy burden on the health systems of countries, and this burden is particularly acute in developing countries.

Current methods of treating and controlling obesity are very limited. It can produce certain effects by controlling diet and increasing exercise, but this method requires long-term persistence and is therefore difficult for individuals who are overweight or obese. Another method is to reduce or control body weight by medication. There are few clinically licensed obesity treatments. One example that is more widely used is Orlistat, a lipase inhibitor that reduces the digestion and absorption of fat and thereby reduces the amount of heat absorbed by the body (Guerciolini. International Journal of Obesity and Related Metabolic Disorders, 1997, Jun.21Supplement 3, S12–23). At present, the clinically applied drug treatment effect is not obvious, and it needs to be taken for a long time with obvious side effects (Padwal, et al. Lancet, 2007, 369, 71-7). Therefore, it is necessary to develop new therapeutic drugs to meet the growing Clinical needs.

For severely obese patients at risk of life, the only effective method is to perform weight loss surgery (Adams, et al, The New England Journal of Medicine, 2007, 357, 753–61), but this treatment is very high. The risk is only applicable to a few serious patients. In one of the procedures, Roux-en-Y gastric bypass (RYGB) produced better weight loss than other procedures. Researchers believe that at least part of the RYGB's weight loss effect is caused by changes in intestinal hormone secretion (Kellum, et al. Annals of Surgery, 1990, 211, 763–70; Le, et al. Annals of Surgery, 2006). , 243, 108–14). These intestinal hormones include Peptide YY, glucagon-like peptide-1 (GLP-1) and oxyntomodulin (OXM).

Among these hormones, GLP-1 has been the most studied as a new drug development target. GLP-1 is a 30 or 31 amino acid polypeptide secreted by small intestinal L cells. It binds to the GLP-1 receptor in the islets to produce an incretin effect, which promotes insulin release in a glucose-dependent manner (Kreymann, et al. Lancet, 1987, 2, 1300-4). GLP-1 also inhibits glucagon secretion, slows gastric emptying and reduces food intake Ingestion (Larsen. Diabetes, 2001, 50, 2530-9; Turton. Nature, 1996, 379, 69-72; Tang-Christensen, et al. American Journal of Physiology, 1996, 271, R848-56). Studies have shown that the satiety produced by GLP–1 may be the result of its direct interaction with the CNS, not just by slowing gastric emptying (Abbott, et al. Brain Research, 2005, 1044, 127–31). GLP-1's incretin action and satiety make it the preferred target for the development of anti-diabetic and anti-obesity drugs. Natural GLP-1 is rapidly degraded in vivo by the DPP-IV enzyme and therefore cannot be directly used as a drug in clinical applications (Deacon, et al. Journal of Clinical Endocrinology & Metabolism, 1995, 80, 952-957).

The GLP-1 analogue exenatide developed by Eli Lilly and the GLP-1 analogue liraglutide developed by Novo Nordisk have been marketed as anti-type 2 diabetes drugs. Their clinical trials have shown that these GLP-1 analogues are effective in reducing body weight in addition to controlling blood glucose, so they have potential as anti-obesity drugs (Ratner, et al. Diabetes, Obesity and Metabolism, 2006, 8, 419– 28; Nauck, et al. Diabetes Care, 2009, 32, 84–90).

Oxytoxin (OXM) is a 37 amino acid polypeptide whose sequence includes all 29 amino acids of glucagon and is called IP-1 (intervening peptide–1) at the C-terminus. Amino acid elongation (Bataille, et al. Peptides, 1981, 2, Supplement 2, 41-44). OXM is rapidly secreted after a meal, and the main physiological effects include reducing gastric acid secretion, reducing pancreatic exocrine and delaying gastric emptying (Schjoldager, et al. European Journal of Clinical Investigation, 1988, 18, 5, 499-503). In addition, OXM has the effect of reducing food intake and increasing energy consumption.

OXM has been shown to activate both the GLP-1 receptor and the glucagon receptor. Its inhibitory effect on food intake is likely to be achieved by binding to the GLP-1 receptor. OXM did not cause appetite suppression in GLP-1 receptor knockout mice, but was not affected in glucagon receptor knockout mice. However, there is also evidence that the physiological role of OXM may not be entirely dependent on the GLP-1 receptor. For example, OXM has a 50-fold lower affinity for GLP-1 receptor than native GLP-1, but the same molar amount of OXM and GLP-1 can produce similar food uptake inhibition (Darkin, et al. Endocrinology, 2001, 142, 10, 4244 - 4250).

Administration of OXM in the ventricles of rodents or direct injection of the polypeptide into the hypothalamus can reduce food intake in animals (Dakin, et al. American Journal of Physiology - Endocrinology and Metabolism, 2002, 283, 6, E1173 - E1177). Dakin et al. found that intraperitoneal injections and repeated intraventricular injections of OXM twice daily for seven consecutive days reduced body weight gain and reduced obesity. Animals administered OXM had more body weight loss than the blank group at the same food intake, and their basal body temperature and heart rate also increased. This suggests that OXM can increase energy expenditure (Dakin, et al. Endocrinology, 2004, 145, 6, 2687-2695; American Journal of Physiology - Endocrinology and Metabolism, 2002, 283, 6, E1173 - E1177).

Clinical trials in overweight and obese patients have shown that subcutaneous injection of OXM 30 minutes before a meal can reduce energy intake by 25%. More importantly, for 4 weeks of continuous administration, all patients who received OXM had a weight loss of about 2.3 kg while the control group had a decrease of only 0.5 kg (Wynne, et al. Diabetes, 54, 82390-2395). Wynne et al. concluded from another set of clinical trials that additional weight loss may be achieved by increasing energy expenditure (Wynne, et al. International Journal of Obesity (London), 2006, 30, 12, 1729 - 1736). Interestingly, OXM did not alter blood glucose levels in healthy subjects in clinical trials, suggesting that it has only a very weak incretin effect in the human body. Considering the weaker incretin effect of OXM, it is similar to GLP-1 The ability of foot sensation and the effect of weight loss in clinical trials, OXM is attracting more and more attention as an anti-obesity treatment for non-diabetic people.

The association of OXM activation of glucagon receptor with its weight loss activity has not been well studied. The main pharmacological activity of glucagon is to promote glycogenolysis and gluconeogenesis in the state of hypoglycemia (Exton. Advances in Enzyme Regulation, 1968, 6, 391–407), so its clinical application is limited to emergency treatment of insulin injection. Caused by hypoglycemia or used as an esophageal muscle relaxant. Glucagon also has the effect of increasing lipid breakdown, increasing satiety, increasing heat production and energy expenditure (Habegger, et al. Nature Reviews Endocrinology, 2010, 6, 689-697). Glucagon increases satiety and promotes energy expenditure, making it a potential target for obesity treatment, but its role in raising blood sugar and accelerating insulin resistance limits its application.

Two articles published by Day and Pocai et al. in 2009 showed that simultaneous activation of the glucagon receptor and the GLP-1 receptor have a good therapeutic effect on obesity and glucose intolerance caused by obesity (Day, et Al. Nature Chemical Biology, 2009, 5, 749-757; Pocai, et al. Diabetes, 2009, 58, 2258-2266). Day et al. administered a GLP-1/glucagon dual agonist and an equimolar concentration of a single GLP-1 receptor agonist in a DIO animal model and compared their pharmacological results. The researchers found that both dual agonists and selective GLP-1 receptor agonists significantly reduced food intake, body weight, and adipose tissue levels compared to placebo. At the same time, they can also reduce blood sugar and increase glucose tolerance. It is noteworthy that dual agonists with similar activation at the GLP-1 receptor and glucagon receptor are more effective than GLP-1 receptor agonists in controlling body weight, adipose tissue content, and glucose homeostasis. Have better activity. Dual agonists can also significantly increase energy expenditure and promote lipid metabolism. In particular, continuous use of this dual agonist did not result in an increase in blood glucose produced by glucagon. One reason for this may be that activation of the GLP-1 receptor counteracts the glycemic effect caused by the glucagon receptor (Day, et al. Nature Chemical Biology, 2009, 5, 749-757).

In summary, OXM and other GLP-1/glucagon receptor dual agonists offer a new direction in the development of anti-obesity and metabolic disease drugs. Novel peptide drugs can provide finer regulation of metabolism in the body, which may have better activity and fewer side effects.

Summary of the invention

A first aspect of the invention relates to an OXM analog derived from a native OXM sequence having enhanced GlP-1 receptor agonistic activity and GCG receptor agonistic activity and having the amino acid sequence of Formula I below:

His-A2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-A16-A17-A18-A19-A20-A21-Phe-A23-A24-Trp- Leu-A27-A28-A29-Y (Formula I)

among them,

A2 is selected from the group consisting of Ala, Gly, sarcosine, Aib, d-Ala, and d-Ser;

A16 is selected from the group consisting of Ser, Gln, Glu, Asp, Asn, and Lys;

A17 is selected from the group consisting of Arg, Asn, Asp, Lys, Lys-Z1, Glu, and Gln;

A18 is selected from the group consisting of Arg, Ala, Aib, and N-methyl Ala;

A19 is selected from the group consisting of Ala and Aib;

A20 is selected from the group consisting of Gln, Glu, Lys, and Lys-Z2;

A21 is selected from the group consisting of Glu and Asp;

A23 is selected from the group consisting of Val and Ile;

A24 is selected from the group consisting of Gln, Glu, Ala, Lys-Z3, and Cys-Z4;

A27 is selected from the group consisting of Met, Leu, and Lys-Z5;

A28 is selected from the group consisting of Asn, Ala, and Lys-Z6;

A29 is selected from the group consisting of Thr and Gly;

Y is selected from Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-A38, Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-A38 and Lys-Arg-Asn-Arg-Asn-Asn a group consisting of -Ile-Ala-A38;

A38 is selected from the group consisting of (Lys)n-Z7, Cys-Z8, (Glu) _m- Z9 and deletions;

Z1 to Z9 are independently selected from the group consisting of a bridge-PEG, a bridge-biotin, and a bridge-fatty acid;

n is an integer selected from 1 to 6,

ie

1, 2, 3, 4, 5 or 6;

m is an integer selected from 0 to 3,

ie

0, 1, 2 or 3;

A linker is a peptide having 0-5 amino acid residues,

ie

0, 1, 2, 3, 4 or 5 amino acid residues;

Forming a lactam ring between A16 and A20 or between A17 and A21;

Or a pharmaceutically acceptable salt thereof.

In some embodiments, in Formula I:

Y is Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-A38;

Z7 to Z9 are independently selected from the group consisting of -(Glu) _a -PEG, -(Glu) _b -biotin and -(Glu) _c -fatty acid and deletion;

n is an integer selected from 1 to 6,

ie

1, 2, 3, 4, 5 or 6;

m is an integer selected from 0 to 3,

ie

0, 1, 2 or 3;

a, b and c are independently selected from integers from 0 to 5,

ie

1, 2, 3, 4 or 5.

In some embodiments, in Formula I,

Y is Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-A38;

A38 is selected from the group consisting of -(Lys) _n -Z7 and -Cys-Z8 and a deletion;

Z7 or Z8 is independently selected from the group consisting of -(Glu) _a -PEG, -(Glu) _b -biotin, -(Glu) _c -fatty acid, and deletion;

n is an integer selected from 1 to 6,

ie

1, 2, 3, 4, 5 or 6;

m is an integer selected from 0 to 3,

ie

0, 1, 2 or 3;

a, b and c are independently selected from integers from 0 to 5,

ie

1, 2, 3, 4 or 5.

In some embodiments, in Formula I,

Y is Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala-A38;

A38 is selected from the group consisting of -(Lys) _n -Z7, -Cys-Z8 and deletions;

n is an integer selected from 1 to 6,

ie

1, 2, 3, 4, 5 or 6;

m is an integer selected from 0 to 3,

ie

0, 1, 2 or 3;

a, b and c are independently selected from integers from 0 to 5,

ie

1, 2, 3, 4 or 5.

In some embodiments, in Formula I,

A2 is Aib.

In some embodiments, in Formula I,

A24 is Cys-Z4;

Z4 is -(Glu) _a -PEG;

a is an integer selected from 0 to 3, that is, 0, 1, 2 or 3.

In some embodiments, in Formula I,

A38 is selected from (Lys) _n -Z7;

Z7 is selected from the group consisting of -(Glu) _a -PEG, -(Glu) _b -biotin, -(Glu) _c -fatty acid, and deletion;

n is an integer selected from 1 to 6,

ie

1, 2, 3, 4, 5 or 6;

m is an integer selected from 0 to 3,

ie

0, 1, 2 or 3;

a, b and c are independently selected from integers from 0 to 5,

ie

1, 2, 3, 4 or 5.

In some embodiments, in Formula I,

Z1 to Z9 are independently selected from the group consisting of (Glu) _a -PEG and (Glu) _c -fatty acid;

a or c is independently selected from an integer from 0 to 2,

ie

0, 1 or 2, for example Glu is γ-Glu.

In some embodiments, in Formula I,

Z1 to Z9 are (Glu) _a -PEG; a is an integer selected from 0 to 2, for example, Glu is γ-Glu.

In some embodiments, in Formula I,

Z1 to Z9 are -(Glu) _c -fatty acids;

c is an integer selected from 0 to 2, that is, 0, 1, or 2, for example, Glu is γ-Glu.

In some embodiments, in Formula I, the fatty acid can be selected from the group consisting of myristic acid, palmitic acid, stearic acid, and cholic acid.

In some embodiments, in Formula I, the molecular weight of the PEG can range from 5 kDa to 40 kDa, such as 20 kDa, 30 kDa, or 40 kDa.

In some embodiments, the OXM analog or a pharmaceutically acceptable salt thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (analog 034), SEQ ID NO: 2 (analog 044), SEQ ID NO: 3 (analog 045), SEQ ID NO: 4 (analog 046), SEQ ID NO: 5 (analog 051), SEQ ID NO: 6 (analog 052), SEQ ID NO: 7 (analog 053) SEQ ID NO: 8 (analog 054), SEQ ID NO: 9 (analog 058), SEQ ID NO: 10 (analog 060), SEQ ID NO: 11 (analog 067), SEQ ID NO: 12 (analog 068), SEQ ID NO: 13 (analog 069), SEQ ID NO: 14 (analog 070), SEQ ID NO: 15 (analog 072), SEQ ID NO: 16 (analog 073), SEQ ID NO: 17 (analog 074), SEQ ID NO: 18 (analog 075), SEQ ID NO: 19 (analog 082), SEQ ID NO: 20 (analog 083), SEQ ID NO: 21 ( Analog 084), SEQ ID NO: 22 (analog 085), SEQ ID NO: 23 (analog 100), SEQ ID NO: 24 (analog 101), SEQ ID NO: 25 (analog 055), SEQ ID NO: 26 (analog 056), SEQ ID NO: 27 (analog 057), SEQ ID NO: 28 (analog 061), SEQ ID NO: 29 (analog 071) , SEQ ID NO: 30 (analog 080), SEQ ID NO: 31 (analog 081). In a further embodiment, the OXM analog or a pharmaceutically acceptable salt thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (analog 034), SEQ ID NO: 2 (analog 044), SEQ ID NO : 4 (analog 046), SEQ ID NO: 5 (analog 051), SEQ ID NO: 6 (analog 052), SEQ ID NO: 7 (analog 053), SEQ ID NO: 9 (analog 058) ), SEQ ID NO: 11 (analog 067), SEQ ID NO: 13 (analog 069), SEQ ID NO: 15 (analog 072), SEQ ID NO: 17 (analog 074), SEQ ID NO: a group consisting of 19 (analog 082), SEQ ID NO: 21 (analog 084), or SEQ ID NO: 23 (analog 100). In a still further embodiment, the amino acid sequence encompassed by the OXM analog or a pharmaceutically acceptable salt thereof is SEQ ID NO: 1 (analog 034), SEQ ID NO: 5 (analog 051), SEQ ID NO : 9 (analog 058), SEQ ID NO: 13 (analog 069), SEQ ID NO: 21 (analog 084) or SEQ ID NO: 23 (analog 100).

Among them, the correspondence between the amino acid sequence and the analog is as follows:

Analog 034:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂ ,

Analog 044:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂ [lactam ring between 16 and 20],

Analog 045:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂ [lactam ring between 16 and 20],

Analog 046:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂ ,

Analog 051:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂ ,

Analog 052:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Ile-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂ ,

Analog 053:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Gln-G lu-Phe-Ile-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂ ,

Analog 054:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys(palmitoyl)-Arg-Ala-Gln-Glu-Phe-Ile- Gln-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂ ,

Analog 055:

Analog 056:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys-NH ₂ ,

Analog 057:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-NH ₂ ,

Analog 058:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys-NH ₂ ,

Analog 060:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys(PEG-40k)-NH ₂ ,

Analog 061:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys(PEG-40k)-NH ₂ ,

Analog 067:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-NH ₂ ,

Analog 068:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-N H ₂ ,

Analog 069:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂ ,

Analog 070:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂ ,

Analog 071:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-NH ₂ ,

Analog 072:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-A sp-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂ ,

Analog 073:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-A sp-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂ ,

Analog 074:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-A sp-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-NH ₂ ,

Analog 075:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-A sp-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-NH ₂ ,

Analog 080:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Ile-Ala-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys-NH ₂ ,

Analog 081:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Ile-Ala-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys(PEG-40k)-NH ₂ ,

Analog 082:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂ ,

Analog 083:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂ ,

Analog 084:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-NH ₂ ,

Analog 085:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-N H ₂ ,

Analog 100:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Aib-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂ [lactam ring between 17 and 21], or

Analog 101:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Aib-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂ [Lactam ring between position 17 and position 21].

A second aspect of the invention relates to a pharmaceutical composition comprising an effective amount of an OXM analog as described above, a pharmaceutically acceptable diluent, carrier or excipient, and optionally an anti-diabetic agent, The anti-diabetic agent is selected from the group consisting of insulins, biguanides, sulfonylureas, rosiglitazone or pioglitazone, alpha-glucosidase inhibitors, and aminodipeptidase IV inhibitors.

In some embodiments, the pharmaceutical composition is in the form of an injection or lyophilized powder.

In some embodiments, an OXM analog as described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, is used to treat a metabolic disease.

In a further embodiment, the OXM analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, for use in the treatment of a metabolic disease, wherein the metabolic disease is selected from the group consisting of diabetes, obesity, and bone A group consisting of looseness.

A third aspect of the invention relates to an OXM analog as described above, or a pharmaceutically acceptable salt thereof, or as described above Use of a pharmaceutical composition for the preparation of a medicament for the treatment of a metabolic disease.

In some embodiments, the metabolic disease is selected from the group consisting of diabetes, obesity, and osteoporosis.

A fourth aspect of the invention relates to a method of treating and/or preventing a metabolic disease, comprising administering to a subject in need thereof an effective amount of an OXM analog as described above or a pharmaceutically acceptable salt thereof or A pharmaceutical composition as described above.

A fifth aspect of the invention relates to a method for simultaneously activating GCG and GLP-1 receptors in vivo and/or in vitro comprising administering an OXM analog as described above or a pharmaceutically acceptable salt thereof or as described above Pharmaceutical composition.

DRAWINGS

Figure 1: shows an in vitro activity screening assay for analog 034 on CHO cells overexpressing the human GLP-1 receptor.

Figure 2: shows in vitro activity screening assay for analog 034 on CHO cells overexpressing human GCG receptor.

Figure 3: shows in vitro activity screening assay for analog 069 on CHO cells overexpressing the human GLP-1 receptor.

Figure 4: shows in vitro activity screening assay for analog 069 on CHO cells overexpressing the human GCG receptor.

detailed description

The OXM analogs referred to in the present invention are engineered based on the native OXM sequence (1 - 37, SEQ ID NO: 32 (analog 013)) and are characterized by the glucagon receptor and GLP - The agonistic activity of the 1 receptor was significantly enhanced compared to the native OXM. The invention also provides pharmaceutical methods for treating or preventing a metabolic disease or condition using an OXM analog, which is primarily referred to as obesity and diabetes.

Studies have shown that natural OXM (1-37) agonist activity of glucagon receptor is weak, only one-tenth of the native glucagon activity (EC ₅₀ = 0.7759nM, EC glucagon ₅₀ =0.0905 nM), but OXM is roughly equivalent to glucagon's agonistic activity at the GLP-1 receptor, which is only one percent of native GLP-1 (Riber, et al. WO 2008152403, 2008, June, 16). . Thus, a novel OXM analog can be constructed by modifying the OXM (1-37) sequence by a hybridization concept, ie, a specific site.

For example, in the second position of the sequence, the substitution of Ser with the non-natural amino acid Aib can achieve stability of DPP-IV metabolism; the amino acid residue Glu with a negative charge in the side chain is substituted at the 16th and 21st positions, respectively. The amino acid residue Lys with a positive charge in the chain is substituted at positions 17 and 20, respectively, to give analogs such as analogs 060, 067, 068, 069, 070, 082 and 083). Modification of the above sites results in a mid-segment of the above analog polypeptide sequence having two salt bridges (between 16 and 20 positions, and between positions 17 and 21, respectively) to immobilize the a-helix configuration. Fatty acids and hydrophilic macromolecules (eg, polyethylene glycol PEG) are linked to the polypeptide chain by covalent bonds. All of the above measures can significantly prolong the pharmacokinetic properties of the compound in vivo, such as the analogs 060, 068, 070, 083 and 085.

definition:

In the present invention, the following terms will be consistent with the following terms.

The term "similar" or "close" as used herein means 10% greater or less than the specified value or range of values, but does not specify any value or range of values only in this region. Each numerical value or range of values described by "similar" or "close" also includes the specified absolute or numerical range.

The term "pharmaceutically acceptable carrier" as used herein includes any standard pharmaceutical carrier, such as a physiological saline buffer of phosphate, water, an emulsion such as an oil/water or water/oil emulsion, and various humectants.

The term "pharmaceutically acceptable salt" as used herein, refers to salts of the compounds which retain the biological activity of the parent, and those which are not biologically active or otherwise different forms of the compound. Many of the compounds described herein are capable of forming salts of acids and/or bases via amino and/or carboxyl groups or other similar groups.

Pharmaceutically acceptable base salts are available from inorganic and organic bases. Salts derived from inorganic bases include only the examples, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines.

Pharmaceutically acceptable acid salts can be obtained from inorganic acids and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, hydroxysuccinic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid. , mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and other similar acids.

The term "treatment" as used herein includes the prevention of a particular disorder or condition, or the alleviation of a condition associated with a particular disorder or condition, and/or the prevention or elimination of said condition.

As used herein, "effective amount" and "therapeutically effective amount" of an OXM analog refer to an amount that is non-toxic but that achieves the desired therapeutic effect. For example, expected therapeutic effects include weight loss or prevention of weight gain, as measured by the level of weight loss. The "effective" dose will vary with the age and condition of the individual, the management mode, and the like. Therefore, it is unlikely that an accurate "effective dose" will be determined. However, a moderate "effective" dose for each person may need to be determined by routine experimentation.

The term "purified" and like terms, as used herein, refers to the separation of an impurity or a molecule or compound in its free form, which is usually associated with a natural molecule or compound. However, the term "purification" as used herein does not only include purity, but also relative meaning. As used herein, "purified polypeptide" refers to polypeptides isolated from other compounds, including, but not limited to, nucleic acid molecules, liposomes, and carbohydrates.

The term "isolated" refers to the isolation of the material from its original environment. For example, a natural polynucleotide represents a living organism but is not isolated, but the same polynucleotide, from some coexisting organisms. Being picked out is separation.

The term "peptide" as referred to herein refers to a sequence of three or more, but less than 50 amino acids, which may be natural or non-natural. A non-natural amino acid refers to an amino acid that is not found in vivo, but may be included in the sequences described herein.

The "OXM analog" referred to herein is the amino acid sequence including the analog 013 (SEQ ID NO: 32). Or any peptide of any of the analogous amino acid sequences of SEQ ID NOS: 1 to 31, which includes substitution, addition, deletion or modification of an amino acid (eg, methylation, acetylation, alkylation, panthromination, molecule Internal covalent bonds: such as formation of lactam bridges, polyethylene glycol modifications, etc.), wherein these analogs are capable of stimulating the glucagon receptor and/or the GLP-1 receptor, as described herein for cAMP in vitro activity screening test.

As used herein, "modification" of an amino acid refers to a substitution, addition or deletion of an amino acid, including substitution or addition of any of the 20 natural amino acids. In the present invention, any of the specific amino acid positions mentioned (e.g., position 24) refers to an amino acid at a specific position in the native OXM (analog 013) or any of the analogs.

The term "native GLP-1" as referred to herein refers to a polypeptide comprising human GLP-1 (7-36 or 7-37) sequences, and the term "native OXM" refers to a polypeptide of human OXM sequence (1-37). . The term "GLP-1" or "OXM" as used herein, unless otherwise explained, refers to native GLP-1 or native OXM, respectively.

As used herein, "substitution" of an amino acid means that one amino acid residue is replaced by another amino acid residue.

As used herein, "polyethylene glycol" or "PEG" refers to a polymer of ethylene oxide and water, in the form of a straight or branched chain, of the formula H(OCH ₂ CH ₂ ) _n OH,n The minimum is equal to 9. Unless otherwise stated, the term refers to compounds having an average total molecular weight of polyethylene glycol of between 5,000 and 40,000 Daltons. "Polyethylene glycol" or "PEG" is used with a numerical suffix to indicate the average molecular weight. For example, PEG-5000 means that the average molecular weight of polyethylene glycol is 5000 Daltons.

The term "polyethylene glycol modification" or like terms, as used herein, refers to the modification of the original site of a polypeptide by attachment of polyethylene glycol. A "peptide modified by polyethylene glycol" refers to a polypeptide that is covalently bonded to polyethylene glycol.

A peptide as referred to herein refers to a polypeptide modified at the N-terminus and the C-terminus. For example, a natural amino acid chain is in the form of a carboxyl group at the C-terminus, and the modified peptide may be in the form of an amide.

As used herein, "linker" refers to a group that links a bond, a molecule, or a link to two separate entities. A bridge can optimize the spatial structure of two entities or provide a variable bond that separates two entities. Variable binding bonds include photolytic groups, acid labile groups, base labile groups, and enzymatic groups.

As used herein, "dimer" refers to a complex composed of two units covalently bonded by a linkage. Dimers include homodimers and hybrid dimers. A homodimer includes two identical structural units, and a hybrid dimer includes two different structural units, although the two units are very similar.

As used herein, "charged amino acid" refers to an amino acid having a negative charge (ie, deprotonated) or a positive charge (ie, protonated) in the side chain within the pH range of the physiological solution. For example, negatively charged amino acids include aspartic acid, glutamic acid, cysteine, homocysteine, homologous glutamic acid, while positively charged amino acids include arginine, lysine, and Histidine. Charged amino acids include the charged amino acids of the 20 natural amino acids and the unnatural amino acids.

As used herein, "acidic amino acid" refers to an amino acid containing a second acidic group, including, for example, a carboxylic acid group or a sulfate group.

The peptides referred to herein, "GLP-1 activity" refers to native GLP-1 in the EC ₅₀ of GLP-1 receptor with a ratio of ₅₀ EC of this polypeptide on GLP-1 receptor.

"Glucagon activity" peptides referred to herein is the ratio of EC native glucagon at the glucagon receptor over the EC _{50 of} this polypeptide in the glucagon receptor _50.

The term "alkyl" as used herein refers to a straight or branched chain hydrocarbon containing a certain number of carbon atoms. For example, alkyl groups include methyl, ethyl and n-propyl groups.

The term "heteroalkyl" as used herein refers to a straight or branched chain hydrocarbon containing a number of carbon atoms, wherein the structure contains at least one heteroatom in the backbone. Heteroatoms suitable for use in the present invention include, but are not limited to, N, S, O.

The term "cycloalkyl" as used herein refers to a cyclic hydrocarbon containing a certain number of carbon atoms, for example, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl.

The term "heterocycloalkyl" as used herein refers to a cyclic hydrocarbon containing a certain number of carbon atoms and one to three heteroatoms, wherein the heteroatoms are selected from the group consisting of O, N, S. The heterocyclic ring is not limited to the following groups: piperidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, thiophene and the like.

The term "aryl" as used herein refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group containing a specified number of carbon atoms, such as benzene or naphthalene. Unless otherwise specified, an aryl group can be unsubstituted or substituted.

The term "Aib" as referred to herein means alpha-aminoisobutyric acid.

The term "sarcosine" as referred to herein means an analog of glycine whose amino group is modified by a methyl group.

Example

The OXM analogs of the invention can be obtained by standard polypeptide solid phase synthesis methods, recombinant DNA techniques, or any other method of preparing polypeptides and fusion proteins. The OXM analogs containing non-peptide moieties of the present invention can be synthesized by standard organic synthesis reactions in addition to standard peptide synthesis methods.

Example 1 General Method for Linear Polypeptide Synthesis

Polypeptides were prepared by standard solid phase peptide synthesis methods using an N-terminal Fmoc-protection strategy. The assembly of the peptide chain was performed manually according to the standard Fmoc method. Fmoc Rink-Amide resin (1% cross-linking degree, 100-200 mesh, degree of substitution 0.34-0.44 mmol/g) commercially available from Tianjin Nankai Synthetic Technology Co., Ltd. was used as a solid phase carrier.

The side chain protected amino acids used below are provided by Shanghai Jill Biochemical Co., Ltd.: Fmoc-Ala-OH,

Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH,

Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OAll)-OH, Fmoc-Gly-OH,

Fmoc-Gly(Allyl)-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,

Fmoc-Lys(Boc)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Met-OH, Fmoc-Nle-OH, Fmoc-Phe-OH,

Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH,

Fmoc-Tyr(tBu)-OH, Boc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Aib-OH. All chemicals (from different suppliers including Sigma-Aldrich, Holling, Shanghai Jill Biochemical) are synthetic grade.

Upon completion of the synthesis, the polypeptide is cleaved from the solid phase polymer support and all side chains are deprotected. This can be accomplished by treatment with trifluoroacetic acid (TFA) for 2 h, while 2.5% water and 2.5% 1,2-didecylethane (EDT) are added to the trifluoroacetic acid as a scavenger for the side chain protecting group. Peptide-TFA mixture from resin After filtering out, most of the TFA was removed, and cold diethyl ether was added to precipitate the polypeptide. It was centrifuged, washed with diethyl ether, and then dissolved in acetonitrile buffer.

After excision from the resin, the crude polypeptide was analyzed by a reverse phase HPLC analytical column. The chromatographic conditions were: Waters Xterra@MS system, C18 column (50 x 2.1 mm), detection wavelength 214 nm, flow rate 1 mL/min, elution with a mobile phase gradient containing 0.1% TFA (A buffer = 0.1% TFA / H ₂ O, B buffer = 0.1% TFA / 90% acetonitrile), gradient program: 0-15 min, 10% - 90%. After analysis, the crude polypeptide was purified by semi-preparative chromatography using a Vydac C4 or C8 column (2.2 x 25 cm) using a mobile phase containing 0.1% TFA. The pure product was characterized by LC-MS and then lyophilized to obtain the target polypeptide.

Example 2 Synthesis of Analog 013 (Anym containing Cys Monosubstituted)

0.05 mmol of Fmoc Rink-Amide resin was placed in a 10 mL reaction vessel, and a standard Fmoc-chemical solid phase polypeptide synthesis process was carried out according to the fitted sequence, wherein DIC/HOBt was used as a condensation reagent.

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp- Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala

When all the synthesis cycles were completed, the polypeptide-bound resin was treated with 20% piperidine in DMF to remove the N-terminal Fmoc group, followed by the excision reagent (95% TFA, 2.5% H ₂ O, 2.5% EDT). ), reaction 2h. The solid resin was filtered off, and the obtained filtrate was concentrated with nitrogen. The polypeptide was precipitated with cold ether and centrifuged to give a crude material. The crude peptide was dissolved in acetonitrile buffer and loaded onto a semi-preparative reversed phase column. Gradient elution was carried out using an HPLC system containing acetonitrile flowing relative to Waters. The appropriate fractions were characterized by LC-MS and pooled together for lyophilization. HPLC analysis showed that the purity of the resulting product was greater than 90%, and ESI-MS showed the ion signal of the target polypeptide.

The analogs 058, 082, 084, etc. were prepared in the same manner.

Synthesis of Example 3 Analog 100 (polypeptide analog containing lactam bridge)

0.05 mmol of Fmoc Rink-Amide resin was placed in a 10 mL reaction vessel, and a standard Fmoc-chemical solid phase polypeptide synthesis process was carried out in sequence, wherein DIC/HOBt was used as a condensation reagent.

After all the synthesis cycles were completed, the polypeptide-attached resin was treated with Pd(PPh ₃ ) ₄ for 1 h under nitrogen to remove the Alloc/OAll protecting group, and a lactam bond was formed under the action of the coupling reagent PyBOP/DIEA (17). Between bit and 21). It was then treated with a solution containing 20% piperidine in DMF access polypeptide resin to remove the N- terminal Fmoc group removal agent was added (95% TFA, 2.5% H 2 O, 2.5% EDT), the reaction 2h. The solid resin was filtered off, and the obtained filtrate was concentrated with nitrogen. The polypeptide was precipitated with cold ether and centrifuged to give a crude material. The crude peptide was dissolved in acetonitrile buffer and loaded onto a semi-preparative reversed phase column. Gradient elution was carried out using an HPLC system containing acetonitrile flowing relative to Waters. The appropriate fractions were characterized by LC-MS and pooled together for lyophilization. HPLC analysis showed that the purity of the resulting product was greater than 90%, and ESI-MS showed the ion signal of the target polypeptide.

Example 4 General Method for PEGylation of Polypeptides (Cys-Maleimide)

The analog containing 058 containing Cys was dissolved in phosphate buffer (~10 mg/mL), an equivalent of maleimide-activated methoxy reagent containing methoxy reagent was added, stirred at room temperature, and analyzed by analytical HPLC. monitor. After 10-24 h of reaction, the reaction mixture was acidified, loaded onto a semi-preparative chromatograph, and purified by gradient eluting with acetonitrile-purified HPLC system. The appropriate fractions are combined and lyophilized to give the desired PEGylated polypeptide.

The analogs 060, 061, 068, and 070 were all prepared in the same manner.

Example 5 Synthesis of Analog 034 and Polypeptide Analogs Linked with Fatty Acids

After all the synthesis cycles were completed, the polypeptide-attached resin was treated with Pd(PPh ₃ ) ₄ for 1 h under nitrogen to remove the Alloc protecting group, and the fatty acid was added to condense with the ε-amino group of Lys at position 17. It was then treated with a solution containing 20% piperidine in DMF access polypeptide resin to remove the N- terminal Fmoc group removal agent was added (95% TFA, 2.5% H 2 O, 2.5% EDT), the reaction 2h. The solid resin was filtered off, and the obtained filtrate was concentrated with nitrogen. The polypeptide was precipitated with cold ether and centrifuged to give a crude material. The crude peptide was dissolved in acetonitrile buffer and loaded onto a semi-preparative reversed phase column. Gradient elution was carried out using an HPLC system containing acetonitrile flowing relative to Waters. The appropriate fractions were characterized by LC-MS and pooled together for lyophilization. HPLC analysis showed that the purity of the resulting product was greater than 90%, and ESI-MS showed the ion signal of the target polypeptide.

The analogs 051, 52, 53 and the like were all prepared in the same manner.

The synthesized OXM analogs were all analyzed by HPLC and MS.

Example 6 Screening experiment of hGLP-1R and hGCGR target active compounds based on cAMP detection

material

Cell lines: CHO/hGLP-1R and CHO/hGCGR;

Cell culture medium: αMEM (Gibco, 12561-056), plus 10% FBS (Gibco, 10099), 1 mg/mL G418 (Invitrogen, 10031035), 10 nM MTX (Sigma, M4010);

96-well cell culture plate: Falcon, 353072;

cAMP assay kit: "cAMP Fluorescent Assay kit", Molecular Devices, R8089;

10× trypsin: Gibco, 15400 (diluted 10 times after use in DPBS);

Ultra-clean workbench: ESCO, SVE-4A1;

Cell counter: Invitrogen, CountessTM;

Cell culture incubator: Thermo, 3111;

KRBG: Sigma, M4892 (add 15 mM NaHCO ₃ when configured);

IBMX: Sigma, I5879;

Multichannel pipette: Eppendorf;

Microplate oscillator: TAITEC, M.BR-022UP;

Microplate reader: Teacon, infinite F200.

Fundamental:

This experiment uses a genetically engineered, highly expressed human GLP-1 receptor (hGLP-1R) or human GCG receptor (hGCGR) CHO cell line. When hGLP-1R/hGCGR is bound and activated by a specific ligand, an increase in intracytoplasmic cAMP levels can be induced. Therefore, the ability of the compound to activate hGLP-1R/hGCGR can be reflected by detecting the intracellular cAMP concentration, thereby achieving the purpose of activity screening.

Experimental steps:

Cells in logarithmic growth phase were harvested, trypsinized, and plated (5000-20000/well, 96-well cell culture plate). Incubate at 37 ° C, saturated humidity, 5% CO _{2 for} 18-20 hours, stand by;

Take the prepared cells, discard the culture solution, add 100 μL of KRBG per well to wash the cells and discard the cells. Then, 100 μL of KRBG containing 0.75 mM IBMX was added to each well, placed in a cell culture incubator for 10 minutes, and then 50 μL of KRBG (containing various concentrations of compound: 1000 nM---0, 10-fold gradient dilution) was added, and mixed. Placed in a cell culture incubator;

After 30 minutes, the cell culture plate was taken out, and 50 μL of cell lysate (Molecular Devices-R7097, supplied with the kit) was added to each well, followed by shaking into a microplate shaker for 10 minutes to fully lyse the cells;

40 μL of cell lysis supernatant was taken per well, and the cAMP content was detected using a kit (Molecular Devices-R8089) (method reference kit instructions);

Finally, the fluorescence value of each well (Ex: 490 nm, Em: 530 nm) was detected and obtained, and the cAMP standard curve was converted into cAMP concentration, and the relationship between the drug concentration and the cAMP concentration was used as a four-parameter logistic fit (Origin 8.0). The EC ₅₀ value of each compound was determined.

The synthesized OXM analogs were screened by the above in vitro activity, and the results of the higher activity are summarized in the following table and in Figures 1-4.

table

The polypeptide sequences of the OXM analogs of the present invention are listed below.

Analog 013:

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-As p-Phe-Val-Gln-Trp -Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala-NH ₂

Analog 034:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂

Analog 044:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂ [Lactam ring between 16 and 20 positions]

Analog 045:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂ [Lactam ring between 16 and 20 positions]

Analog 046:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂

Analog 051:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂

Analog 052:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Ile-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂

Analog 053:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Gln-G lu-Phe-Ile-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂

Analog 054:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys(palmitoyl)-Arg-Ala-Gln-Glu-Phe-Ile- Gln-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂

Analog 055:

Analog 056:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys-NH ₂

Analog 057:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-NH ₂

Analog 058:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys-NH ₂

Analog 060:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys(PEG-40k)-NH ₂

Analog 061:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys(PEG-40k)-NH ₂

Analog 067:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-NH ₂

Analog 068:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-N H ₂

Analog 069:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂

Analog 070:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH ₂

Analog 071:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-NH ₂

Analog 072:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-A sp-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂

Analog 073:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-A sp-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂

Analog 074:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-A sp-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-NH ₂

Analog 075:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-A sp-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-NH ₂

Analog 080:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Ile-Ala-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys-NH ₂

Analog 081:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Ile-Ala-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys(PEG-40k)-NH ₂

Analog 082:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂

Analog 083:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂

Analog 084:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-NH ₂

Analog 085:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-N H ₂

Analog 100:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Aib-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂ [Lactam ring between 17 and 21]

Analog 101:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Aib-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH ₂ [Lactam ring between 17 and 21]

Claims

An OXM analog derived from a native OXM sequence having enhanced GlP-1 receptor agonistic activity and GCG receptor agonistic activity and having the amino acid sequence of Formula I below:

His-A2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-A16-A17-A18-A19-A20-A21-Phe-A23-A24-Trp- Leu-A27-A28-A29-Y (Formula I)

among them,

A2 is selected from the group consisting of Ala, Gly, sarcosine, Aib, d-Ala, and d-Ser;

A16 is selected from the group consisting of Ser, Gln, Glu, Asp, Asn, and Lys;

A17 is selected from the group consisting of Arg, Asn, Asp, Lys, Lys-Z1, Glu, and Gln;

A18 is selected from the group consisting of Arg, Ala, Aib, and N-methyl Ala;

A19 is selected from the group consisting of Ala, Val and Aib;

A20 is selected from the group consisting of Aib, Gln, Glu, Lys, and Lys-Z2;

A21 is selected from the group consisting of Glu and Asp;

A23 is selected from the group consisting of Val and Ile;

A24 is selected from the group consisting of Gln, Glu, Ala, Lys-Z3, and Cys-Z4;

A27 is selected from the group consisting of Met, Leu, Val and Lys-Z5;

A28 is selected from the group consisting of Asn, Ala, and Lys-Z6;

A29 is selected from the group consisting of Thr and Gly;

Y is selected from Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-A38, Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-A38 and Lys-Arg-Asn-Arg-Asn-Asn a group consisting of -Ile-Ala-A38;

A38 is selected from the group consisting of (Lys)n-Z7, Cys-Z8, (Glu) m- Z9 and deletions;

Z1 to Z9 are independently selected from the group consisting of a bridge-PEG, a bridge-biotin, and a bridge-fatty acid;

n is an integer selected from 1 to 6;

m is an integer selected from 0 to 3;

A junction bridge is a peptide having 0-5 amino acid residues;

Forming a lactam ring between A16 and A20 or between A17 and A21;

Or a pharmaceutically acceptable salt thereof.
The OXM analogue of claim 1 wherein in Formula I:

Y is Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-A38;

A38 is selected from the group consisting of (Lys)n-Z7, Cys-Z8, (Glu) m- Z9 and deletions;

Z7 to Z9 are independently selected from the group consisting of -(Glu) a -PEG, -(Glu) b -biotin and -(Glu) c -fatty acid and deletion;

n is an integer selected from 1 to 6;

m is an integer selected from 0 to 3;

a, b and c are independently selected from integers from 0 to 5.
The OXM analog according to claim 1, wherein in Formula I,

Y is Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-A38;

A38 is selected from the group consisting of -(Lys) n -Z7 and -Cys-Z8 and a deletion;

Z7 or Z8 is independently selected from the group consisting of -(Glu) a -PEG, -(Glu) b -biotin, -(Glu) c -fatty acid, and deletion;

n is an integer selected from 1 to 6;

m is an integer selected from 0 to 3;

a, b and c are independently selected from integers from 0 to 5.
The OXM analog according to claim 1, wherein in Formula I,

Y is Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala-A38;

A38 is selected from the group consisting of -(Lys) n -Z7, -Cys-Z8 and deletions;

Z7 or Z8 is independently selected from the group consisting of -(Glu) a -PEG, -(Glu) b -biotin, -(Glu) c -fatty acid, and deletion;

n is an integer selected from 1 to 6;

m is an integer selected from 0 to 3;

a, b and c are independently selected from integers from 0 to 5.
The OXM analog according to any one of claims 1 to 5, wherein in the formula I,

A2 is Aib.
The OXM analog according to any one of claims 1 to 5, wherein in the formula I,

A24 is Cys-Z4;

Z4 is -(Glu) a -PEG;

a is an integer selected from 0 to 3.
The OXM analog according to any one of claims 1 to 6, wherein in Formula I,

A38 is selected from (Lys) n -Z7;

Z7 is selected from the group consisting of -(Glu) a -PEG, -(Glu) b -biotin, -(Glu) c -fatty acid, and deletion;

n is an integer selected from 1 to 6;

m is an integer selected from 0 to 3;

a, b and c are independently selected from integers from 0 to 5.
The OXM analog according to any one of claims 1 to 7, wherein in Formula I,

Z1 to Z9 are independently selected from the group consisting of (Glu) a -PEG and (Glu) c -fatty acid;

a or c is independently selected from an integer from 0 to 2, for example Glu is γ-Glu.
The OXM analog according to any one of claims 1 to 8, wherein in Formula I,

Z1 to Z9 are (Glu) a -PEG; a is an integer selected from 0 to 2, for example, Glu is γ-Glu.
The OXM analog according to any one of claims 1 to 9, wherein in Formula I,

Z1 to Z9 are -(Glu) c -fatty acids;

c is an integer selected from 0 to 2, for example, Glu is γ-Glu.
The OXM analog according to any one of claims 1 to 10, wherein in the formula I, the fatty acid may be selected from the group consisting of myristic acid, palmitic acid, stearic acid and cholic acid.
The OXM analog according to any one of claims 1 to 11, wherein in the formula I, the molecular weight of the PEG may be from 5 kDa to 40 kDa, such as 20 kDa, 30 kDa, or 40 kDa.
The OXM analog according to any one of claims 1 to 12, wherein the OXM analog or a pharmaceutically acceptable salt thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 31.
The OXM analog of claim 1 selected from the group consisting of:

Analog 034:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH 2 ,

Analog 044:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH 2 [lactam ring between 16 and 20],

Analog 045:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH 2 [lactam ring between 16 and 20],

Analog 046:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH 2 ,

Analog 051:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH 2 ,

Analog 052:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Ile-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH 2 ,

Analog 053:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Gln-G lu-Phe-Ile-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH 2 ,

Analog 054:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys(palmitoyl)-Arg-Ala-Gln-Glu-Phe-Ile- Gln-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH 2 ,

Analog 055:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH 2 ,

Analog 056:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys-NH 2 ,

Analog 057:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-NH 2 ,

Analog 058:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys-NH 2 ,

Analog 060:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys(PEG-40k)-NH 2 ,

Analog 061:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-A sp-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys(PEG-40k)-NH 2 ,

Analog 067:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-NH 2 ,

Analog 068:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-N H 2 ,

Analog 069:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH 2 ,

Analog 070:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys(palmitoyl)-NH 2 ,

Analog 071:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Gln-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-NH 2 ,

Analog 072:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-A sp-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH 2 ,

Analog 073:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-A sp-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH 2 ,

Analog 074:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-A sp-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-NH 2 ,

Analog 075:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-A sp-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Pro-Pro-Pro-Ser-NH 2 ,

Analog 080:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Ile-Ala-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys-NH 2 ,

Analog 081:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Ile-Ala-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Cys(PEG-40k)-NH 2 ,

Analog 082:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH 2 ,

Analog 083:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Lys-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH 2 ,

Analog 084:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-NH 2 ,

Analog 085:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-Lys-N H 2 ,

Analog 100:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Aib-G lu-Phe-Val-Cys-Trp -Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH 2 [lactam ring between 17 and 21], or

Analog 101:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Arg-Ala-Aib-G lu-Phe-Val-Cys (PEG -40k)-Trp-Leu-Met-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Ser-NH 2 [Lactam ring between position 17 and position 21].
The OXM analog according to claim 14, which is selected from the group consisting of analog 034, analog 044, analog 046, analog 051, analog 052, analog 053, analog 058, analog 067, analog 069, Analog 072, analog 074, analog 08, analog 084 or analog 100, preferably, is selected from analog 034, analog 051, analog 058, analog 069, analog 084 or analog 100.
A pharmaceutical composition comprising an effective amount of the OXM analog of any of claims 1-15, a pharmaceutically acceptable diluent, carrier or excipient, and optionally an anti-diabetic agent, The anti-diabetic agent is selected from the group consisting of insulins, biguanides, sulfonylureas, rosiglitazone or pioglitazone, alpha-glucosidase inhibitors, and aminodipeptidase IV inhibitors.
The pharmaceutical composition according to claim 16, wherein the pharmaceutical composition is in the form of an injection or a lyophilized powder.
The OXM analog according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 16 or 17, for use in the treatment of a metabolic disease.
The OXM analog according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 16 or 17, for use in the treatment of a metabolic disease, wherein the metabolic disease is selected A group consisting of free diabetes, obesity, and osteoporosis.
Use of an OXM analog according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 16 or 17, for the preparation of a medicament for the treatment of a metabolic disease.
The use according to claim 20, wherein the metabolic disease is selected from the group consisting of diabetes, obesity and osteoporosis.
A method of treating and/or preventing a metabolic disease, comprising administering to a subject in need thereof an effective amount of the OXM analog of any of claims 1-15, or a pharmaceutically acceptable salt thereof, or The pharmaceutical composition according to claim 16 or 17.
A method of simultaneously activating GCG and GLP-1 receptors in vivo and/or in vitro, which comprises administering an OXM analog according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, or according to claim 16. Or the pharmaceutical composition of 17.