LONG-ACTING GLP-1 AND GLUCAGON RECEPTOR DUAL AGONIST

Abstract

A novel long-acting acylated oxyntomodulin peptide analogue having dual agonism on GLP-1 and glucagon receptors (dual GLP-1R/GlucagonR agonism) and a pharmaceutical composition including the same are disclosed. The novel long-acting acylated oxyntomodulin peptide analogue and the composition are useful for the prevention and treatment of obesity and overweight, or non-insulin-dependent diabetes accompanied by obesity and overweight. The acylated oxyntomodulin peptide analog has dual agonism for GLP-1/glucagon receptors and has an excellent increased in vivo half-life, and the pharmaceutical composition containing the same is useful for the treatment of metabolic diseases such as obesity and diabetes.

Claims

1. An acylated oxyntomodulin peptide analog of the following Chemical Formula I: His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-X.sub.1-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Glu-Tyr-Glu-X.sub.2-Glu-Tyr-Glu (SEQ ID NO: 14) wherein: X.sub.1 is functionalized Lys with [(2-(2-(2-aminoethoxy)ethoxy)acetoyl)2]-[gammaglutamyl]-[octadecanoyl] conjugated to its side chain; X.sub.2 is functionalized Lys with a lipophilic lipid or spacer-lipophilic lipid or polymeric moiety-spacer-lipophilic lipid or spacer-polymeric moiety-spacer-lipophilic lipid conjugated to its side chain; the lipophilic lipid is of the following Structural Formula (1) or (2), ##STR00014## the polymeric moiety is 1-3 (one or two or three) of 2-(2-(2-aminoethoxy)ethoxy)acetoyl; and the spacer is r-Glu or Lys.

2. The acylated oxyntomodulin peptide analog according to claim 1, wherein the spacer is Lys, and the amine group of the amino acid residue of Lys spacer is covalently bonded to the lipophilic lipid or polymeric moiety.

3. The acylated oxyntomodulin peptide analog according to claim 1, wherein the spacer is r-Glu, and the α-amino group of the r-Glu spacer is covalently bonded to the lipophilic lipid or the polymeric moiety.

4. The acylated oxyntomodulin peptide analog of claim 1, wherein the acylated oxyntomodulin peptide analog is Compound 1 (SEQ ID NO: 2), Compound 2 (SEQ ID NO: 3), Compound 3 (SEQ ID NO: 4), Compound 4 (SEQ ID NO: 5), Compound 5 (SEQ ID NO: 6), Compound 6 (SEQ ID NO: 7) or Compound 7 (SEQ ID NO: 8).

5. A pharmaceutical composition comprising the oxyntomodulin peptide analog of Chemical Formula I according to claim 1 as an active ingredient, and comprising a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, wherein the composition is an injection formulation.

7. A pharmaceutical composition for the treatment or prevention of non-insulin-dependent diabetes mellitus accompanied by obesity or overweight, comprising the oxyntomodulin peptide analog of Chemical Formula I according to claim 1 as an active ingredient, and comprising a pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, wherein the composition is an injection formulation.

9. A method for preventing or treating a condition characterized by or caused by obesity or overweight, comprising administering an effective amount of a composition comprising the oxyntomodulin peptide analog of Chemical Formula I according to claim 1, as an active ingredient, and a pharmaceutically acceptable excipient to a subject in need thereof.

10. A method for preventing or treating non-insulin-dependent diabetes mellitus accompanied by obesity or overweight, administering an effective amount of a composition comprising the oxyntomodulin peptide analog of Chemical Formula I according to claim 1, as an active ingredient, and a pharmaceutically acceptable excipient to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 is a graph showing the results of body weight loss efficacy evaluation in mice after one week of repeated injection of Compound 1, Compound 2, or Compound 7 respectively, which are acylated oxyntomodulin peptide analogs according to the present invention. FIG. 1a shows the weight loss result, and FIG. 1b is a graph showing the result of cumulative feed intake.

[0046] FIG. 2 is a graph showing the results of body weight loss drug efficacy evaluation in mice after one week of repeated injection of Compound 3, which is an acylated oxyntomodulin peptide analog according to the present invention. FIG. 2a shows the weight loss result, and FIG. 2b is a graph showing the result of cumulative feed intake.

[0047] FIG. 3 is a graph showing the results of body weight loss efficacy evaluation in mice after one week of repeated injection of Compound 4, Compound 5, or Compound 6 respectively, which are acylated oxyntomodulin peptide analogs according to the present invention. FIG. 3a shows the weight loss result, and FIG. 3b is a graph showing the result of cumulative feed intake.

[0048] FIG. 4 is a graph showing the pharmacokinetic results after a single-dose administration in mice of the acylated oxyntomodulin peptide analog according to the present invention.

[0049] FIG. 5 is a graph showing the pharmacokinetic results after a single-dose administration in monkeys of the acylated oxyntomodulin peptide analog according to the present invention.

[0050] FIG. 6 is a graph showing the results of the glucose tolerance improvement assay in mice of the acylated oxyntomodulin peptide analog according to the present invention.

DESCRIPTION OF EMBODIMENTS

[0051] The present invention is further described in detail by reference to the following examples and experimental examples. These examples are provided for purposes of illustration only, to help a person skilled in the art understand the invention, and should not in any way be construed as limiting the scope of the present invention.

<Example 1> Synthesis of Acylated Oxyntomodulin Peptide Analogs According to the Present Invention

[0052] The peptides and canonical peptide sequences comprising some amino acids of the present invention can be synthesized or purchased from commercial peptide manufacturers such as American Peptide Company or Bachem in the United States, or Anygen in Korea.

[0053] The acylated oxyntomodulin peptide analog in the present invention were synthesized using the Symphony X (synthesis scale: 0.1 mmol) model of Protein Technologies, Inc. The structures of Compound 1 (SEQ ID NO: 2) through Compound 7 (SEQ ID NO: 8), which are acylated oxyntomodulin peptide analogs synthesized according to the present invention, are shown in Tables 1 and 2. The specific synthesis process is as follows:

[0054] A mixture of Fmoc-AA-OH (1 mmol), HBTU (1 mmol), NMM (n-methylmorpholine) (2 mmol) and DMF (7 ml) was placed in resin from which Fmoc has been removed and stirred at room temperature for 1 hour. The reaction solution drained and washed twice with 7 ml of DMF (N,N-Dimethylmethanamide). Fmoc cleavage reaction was performed twice for 5 minutes at room temperature with 20% piperidine DMF solution (5 ml), and the solution was washed 6 times with DMF (7 ml). This process was repeated using an automated synthesizer to couple the amino acids.

[0055] For the Lys side synthesis, the coupling performed using Fmoc-K(dde)-OH or Fmoc-K(Alloc)-OH, and the last His coupled using Boc-His(trt)-OH. The synthesis of functionalized Lys side chains carried out using 2% hydralazine or tetrakis(triphenylphosphine)palladium(0) to remove the protected dde or Alloc, and then the desired side chain moieties (PEG2, rE, K, C14-18 di-acid, etc.) are coupled.

[0056] To 0.1 mmol of the peptide-resin obtained above, 8 ml of a solution of Reagent K (trifluoroacetic acid, water, thioanisole, 1,2-ethandiol (87.5, 5.0, 5.0, 2.5)) was added after cooling to 5-10° C. combined filtrate 100 ml of diethyl ether for crystallization. The solid filtered he crude peptide was purified by preparative HPLC to obtain the target compound.

[0057] ShimadzuAxima Assurance MALDI-TOF was used for molecular weight analysis, with α-Cyano-4-hydroxycinnamic acid (CHCA) used as a matrix.

TABLE-US-00002 TABLE 1 Structure of acylated oxyntomodulin peptide analogs of the present invention Compound Structure Compound 1 [00002] Compound 2 [00003] Compound 3 [00004] Compound 4 [00005] Compound 5 [00006] Compound 6 [00007] Compound 7 [00008]

TABLE-US-00003 TABLE 2 Sequence and structure of acylated oxyntomodulin peptide analogs of the present invention SEQ ID NO: Name Sequence 1 OXM H-HSQGTFTSDKSKYLDSRRAQDFVQWLMNTKRNR (origi- NNIA-OH nal) 2 Com-  H-HAibQGTFTSDK([(2-(2-(2-amino- pound 1 ethoxy)ethoxy)acetoyl).sub.2]-[gamma- glutamyl]-[octadecanoyl])SKYLDAibR RAQDFVQWLMNTKEYEK(13-carboxy-1- oxotridecyl)EYE-OH 3 Com-  H-HAibQGTFTSDK([(2-(2-(2-amino- pound 2 ethoxy)ethoxy)acetoyl).sub.2]-[gamma- glutamyl]-[octadecanoyl])SKYLDAibR RAQDFVQWLMNTKEYEK(15-carboxy-1- oxopentadecyl)EYE-OH 4 Com-  H-HAibQGTFTSDK([(2-(2-(2-amino- pound 3 [ethoxy)ethoxy)acetoyl).sub.2]-gamma- glutamyl]-[octadecanoyl])SKYLDAibR RAQDFVQWLMNTKEYEK(17-carboxy-1- oxoheptadecyl)EYE-OH 5 Com-  H-HAibQGTFTSDK([(2-(2-(2-amino- pound 4 ethoxy)ethoxy)acetoyl).sub.2]-[gamma- glutamyl]-[octadecanoyl])SKYLDAibR RAQDFVQWLMNTKEYEK([lysyl]-[17- carboxy-1-oxoheptadecyl])EYE-OH 6 Com-  H-HAibQGTFTSDK([(2-(2-(2-amino- pound 5 ethoxy)ethoxy)acetoyl).sub.2]-[gamma- glutamyl]-[octadecanoyl])SKYLDAibR RAQDFVQWLMNTKEYEK([(2-(2-(2-amino- ethoxy)ethoxy)acetoyl).sub.3]-[gamma- glutamyl]-[17-carboxy-1- oxoheptadecyl])EYE-OH 7 Com-  H-HAibQGTFTSDK([(2-(2-(2-amino- pound 6 ethoxy)ethoxy)acetoyl).sub.2]-[gamma- glutamyl]-[octadecanoyl])SKYLDAibR RAQDFVQWLMNTKEYEK([lysyl]-[(2-(2- (2-aminoethoxy)ethoxy)acetoyl).sub.3]- [gammaglutamyl]-[17-carboxy-1- oxoheptadecyl])EYE-OH 8 Com-  H-HAibQGTFTSDK([(2-(2-(2-amino- pound 7 ethoxy)ethoxy)acetoyl).sub.2]-[gamma- glutamyl]-[octadecanoyl])SKYLDAibR RAQDFVQWLMNTKEYEK(17-aminocarboxy-1- oxoheptadecyl)EYE-OH

<Comparative Example 1> Synthesis of Acylated Oxyntomodulin Peptide Analogs

[0058] For comparison with the present invention, an acylated oxyntomodulin peptide analog having structural similarity was synthesized by the method of Example 1. The following Comparative Example Compound 8 is described in Korean Patent Application No. 2018-0095717 as Compound 3, and it is a peptide analog that does not have the 7 amino acids (EYEX2EYE) at the C-terminal. Comparative Example Compound 9 is a peptide analog that is Comparative Example Compound 8 with a ligand mentioned in the literature (ACS Chem Biol. 2016; 11:324-328) added. Comparative Example Compound 10 and Comparative Example Compound 11 are peptide analogs having an aminocarboxyl group at the carbon chain end of C14 and C16, respectively, as the lipophilic lipid in X.sub.2. Comparative Example Compound 12 is a peptide analog in which the C16 is linked with a polymeric moiety as the lipophilic lipid in X.sub.2. The structures of Comparative Example Compound 8 to Comparative Example Compound 12 are shown in Tables 3 and 4.

TABLE-US-00004 TABLE 3 Structure of acylated oxyntomodulin peptide analogs Compound Structure Compound 8* [00009] Compound 9 [00010] Compound 10 [00011] Compound 11 [00012] Compound 12 [00013]

TABLE-US-00005 TABLE 4 Sequence and structure of acylated  oxyntomodulin peptide analogs SEQ ID NO: Name Sequence  9 Com- H-HAibQGTFTSDK([(2-(2-(2-amino- pound ethoxy)ethoxy)acetoyl)2]-[gamma- 8 glutamyl]-[octadecanoyl])SKYLD AibRRAQDFVQWLMNTK-OH 10 Com- H-HAibQGTFTSDK([(2-(2-(2-amino- pound ethoxy)ethoxy)acetoyl)2]-[gam- 9 maglutamyl]-[octadecanoyl]) SKYLDAibRRAQDFVQWLMNTKEYEK (hexadecanoyl)EYE-OH 11 Com- H-HAibQGTFTSDK([(2-(2-(2-amino- pound ethoxy)ethoxy)acetoyl)2]-[gam- 10 maglutamyl]-[octadecanoyl]) SKYLDAibRRAQDFVQWLMNTKEYEK (13-aminocarboxy-1-oxotridecyl) EYE-OH 12 Com- H-HAibQGTFTSDK([(2-(2-(2-amino- pound ethoxy)ethoxy)acetoyl)2]-[gam- 11 maglutamyl]-[octadecanoyl]) SKYLDAibRRAQDFVQWLMNTKEYEK (15-aminocarboxy-1- oxopentadecyl)EYE-OH 13 Com- H-HAibQGTFTSDK([(2-(2-(2-amino- pound ethoxy)ethoxy)acetoyl)2]-[gam- 12 maglutamyl]-[octadecanoyl]) SKYLDAibRRAQDFVQWLMNTKEYEK ([(2-(2-(2-aminoethoxy)ethoxy) acetoyl)3]-[hexadecanoyl])EYE-OH

<Experimental Example 1> GLP-1 and Glucagon Receptor Activation Assay

[0059] Human GLP-1 or glucagon receptors were transiently overexpressed in cells, so that the acylated oxyntomodulin peptide analog of the present invention could activate the receptors resulting in a rise in cyclic adenosine monophosphate (cAMP), which successively activates cyclic adenosine monophosphate response elements (CRE). Then, the resulting increased luciferase activity was evaluated as a measurement of the effect on each receptor activation.

[0060] For positive control, endogenous ligand GLP-1 or glucagon was used for respective evaluation. The above-mentioned acylated oxyntomodulin peptide analogs of Comparative Example 8-12 were synthesized and used as comparative examples.

[0061] Human GLP-1 or glucagon expression vector (“OriGene”) was transiently transfected into Chinese hamster ovary cells (CHO-K1), with plasmid DNAs that can induce expression of firefly luciferase or Renilla luciferase, using Lipofectamine Plus Reagent (Invitrogen). After 3 hours of transfection, medium was exchanged to Alpha Minimal Essential Medium (α-MEM) comprising 10% fetal bovine serum (FBS). Next day, the medium was exchanged to α-MEM comprising the acylated oxyntomodulin peptide analog of the present invention and 0.1% bovine serum albumin (BSA). After 6 hours, dual luciferase assay reagent (Promega) was added in the same amount as the medium in which the cells were submerged, and firefly luciferase and Renilla luciferase activities were successively measured. Firefly luciferase activity values were corrected against Renilla luciferase activity to yield transfection efficiency.

[0062] To measure the receptor activation efficacy, multi-concentration test was performed on the acylated oxyntomodulin peptide analog of the present invention to obtain the relative activation (%) of the maximum effect of the analog on either GLP-1 or glucagon, and the concentration indicating 50% activation (EC50) was calculated using non-linear regression analysis. The resulting values are shown in Table 5.

TABLE-US-00006 TABLE 5 Human GLP-1/glucagon receptor activation ability of acylated oxyntomodulin peptide analogs Receptor activation GLP-1 receptor GCG receptor Maximum Maximum activation activation Compound EC50* (% vs GLP-1) EC50* (% vs GCG) Example Compound 1 A 101.7% A 107.0% Compound 2 B 103.9% B 100.1% Compound 3 B 98.3% B 98.3% Compound 4 B 87.9% C 95.4% Compound 5 B 93.6% B 99.4% Compound 6 B 83.8% B 89.1% Compound 7 A 90.8% A 100.1% Comparative Compound 8 A 99.3% A 102.8% Example Compound 9 B 70.4% B 73.3% Compound 10 A 105.3% A 110.5% Compound 11 A 92.8% A 98.5% Compound 12 A 101.9% A 89.6% *A: EC.sub.50 < 100 pM, B: 100 pM ≤ EC.sub.50 < 1000 pM, C: EC.sub.50 ≥ 1000 pM

[0063] Experimental results show that the compounds of the acylated oxyntomodulin peptide analogs according to the present invention exhibit dual agonism against GLP-1 and glucagon receptors like wild type oxyntomodulin, and exhibit sufficient activity comparable to the endogenous hormones GLP-1 and glucagon in terms of maximum efficacy.

<Experimental Example 2> Body Weight Loss Efficacy Evaluation by 1-Week Repeated Injection of the Oxyntomodulin Peptide Analog of the Present Invention

[0064] This experiment was conducted to confirm the weight loss efficacy of the acylated oxyntomodulin peptide analog according to the present invention. Male laboratory mice (C57BL/6 mouse) were provided with diet containing high fat. The mice with high-fat-diet-induced obesity were separated into groups by body weight before the experiment began. Compound 1, Compound 2, and Compound 7, which are examples according to the present invention, were each prepared in sterile distilled water for injection containing 0.1% Cremophor EL to a dosage of 30 nmol/kg. They were injected subcutaneously once a day for a total of 7 days as described in Table 6. Body weight and food intake was measured once a day, at the same time each day, to evaluate the body weight loss efficacy of the acylated oxyntomodulin analogs compared to the initial body weight. The results are shown in FIGS. 1a and 1b.

TABLE-US-00007 TABLE 6 Body weight loss efficacy evaluation by 1-week repeated injection of acylated oxyntomodulin peptide of present invention Method of Group Drug and dose administered administration Control 0.1% Cremophor EL, vehicle S.C. once a Experimental Compound 1, 30 nmol/kg/QD day × 7 group Compound 2, 30 nmol/kg/QD Compound 7, 30 nmol/kg/QD

[0065] From the experiment results, significant weight changes of −44.9%, −26.9% and −6.8% from the initial body weight were observed in Compound 1, Compound 2 and Compound 7, respectively, which are acylated oxyntomodulin peptide analogs of the present invention; and cumulative feed consumption was reduced by 75%, 46% and 10%, respectively, compared to the vehicle control group.

<Experimental Example 3> Body Weight Loss Efficacy Evaluation by 1-Week Repeated Injection of the Oxyntomodulin Peptide Analog of the Present Invention

[0066] This experiment was conducted to confirm the weight loss efficacy of the acylated oxyntomodulin peptide analog according to the present invention. Male laboratory mice (C57BL/6 mouse) were provided with diet containing high fat. The mice with high-fat-diet-induced obesity were separated into groups by body weight before the experiment began. Compound 3, which is an acylated oxyntomodulin peptide analog according to an embodiment of the present invention, was prepared in sterile distilled water for injection containing 0.1% Cremophor EL to a dose of 30 nmol/kg. It was injected subcutaneously once a day for a total of 7 days as described in Table 9. Body weight and feed intake were measured once daily at the same time each day, to evaluate the body weight loss efficacy over time compared to the initial body weight, and the results are shown in FIGS. 2a and 2b.

TABLE-US-00008 TABLE 7 Body weight loss efficacy evaluation by 1-week repeated injection of acylated oxyntomodulin peptide of present invention Method of Group Drug and dose administered administration Control 0.1% Cremophor EL, vehicle S.C. once a Experimental Compound 3, 30 nmol/kg/QD day × 7 group

[0067] From the experiment results, a significant change was observed in the body weight of the group injected with Compound 3, which is an acylated oxyntomodulin peptide analog according to the present invention, by −16.1% compared to the initial value, and the cumulative feed consumption was reduced by 40% compared to the vehicle control group.

<Experimental Example 4> Body Weight Loss Efficacy Evaluation by 1-Week Repeated Injection of the Oxyntomodulin Peptide Analog of the Present Invention

[0068] This experiment was conducted to confirm the weight loss efficacy of the acylated oxyntomodulin peptide analog according to the present invention. Male laboratory mice (C57BL/6 mouse) were provided with diet containing high fat. The mice with high-fat-diet-induced obesity were separated into groups by body weight before the experiment began. Compound 4, Compound 5, or Compound 6, which are acylated oxyntomodulin peptide analogs according to an embodiment of the present invention, was prepared in sterile distilled water for injection containing 0.1% Cremophor EL to a dose of 30 nmol/kg. It was injected subcutaneously once a day for a total of 7 days as described in Table 8. Body weight and feed intake were measured once daily at the same time each day, to evaluate the body weight loss efficacy of the acylated oxyntomodulin peptide analogs over time compared to the initial body weight, and the results are shown in FIGS. 3a and 3b.

TABLE-US-00009 TABLE 8 Body weight loss efficacy evaluation by 1-week repeated injection of acylated oxyntomodulin peptide of present invention Method of Group Drug and dose administered administration Control 0.1% Cremophor EL, vehicle S.C. once a Experimental Compound 4, 30 nmol/kg/QD day × 7 group Compound 5, 30 nmol/kg/QD Compound 6, 30 nmol/kg/QD

[0069] From the experiment results, significant weight changes of −24.3%, −30.3% and −26.9% of the initial body weight were observed with Compound 4, Compound 5 and Compound 6, respectively, which are acylated oxyntomodulin peptide analogs of the present invention, and cumulative feed consumption was reduced by 67%, 83%, and 75%, respectively, compared to the vehicle control group.

<Experimental Example 5> Assessment of Single-Dose Pharmacokinetic Profile in Mice

[0070] ICR mice were used to evaluate the pharmacokinetics in mice of the acylated oxyntomodulin peptide analogs according to the present invention. Compound 1, Compound 2, Compound 3, or Compound 7, which is an acylated oxyntomodulin peptide analog prepared by the present invention, was prepared in sterile distilled water for injection containing 0.1% Cremophore EL to a dose of 30 nmmol/kg, and injected to the ICR mice subcutaneously; then, blood was collected at a designated time after administration, and pharmacokinetic indicators were calculated through plasma drug concentration analysis, and the results are shown in Table 9 and FIG. 4.

TABLE-US-00010 TABLE 9 Evaluation of pharmacokinetics in mice of acylated oxyntomodulin peptide analogs Mouse PK Compound (S.C. half-life)* Example Compound 1 B Compound 2 B Compound 3 B Compound 7 B Comparative Compound 8 A Example Semaglutide B MEDI0382 A *A: Less than 6 hours, B: 6 hours or more

[0071] As seen in Table 9 and FIG. 4, the experimental results showed that the four acylated oxyntomodulin peptide analogs (Compound 1, Compound 2, Compound 3, Compound 7) according to the examples of the present invention exhibited a longer half-life of more than 6 hours compared to Comparative Example Compound 8 (Korea Patent Application No. 2018-0095717, Compound 3) and Comparative Example MEDI0382. It can be seen that the improved chemical stability due to the introduction of a novel albumin ligand leads to the significantly improved half-life compared to a single acylated oxyntomodulin peptide analog Comparative Example Compound 8.

[0072] Moreover, the half-life of Comparative Example Compound 8 was similar to the half-life of Comparative Example MEDI0382 under development as a once-a-day injection formulation, indicating that Comparative Example Compound 8 also can be developed only as a once-a-day injection formulation. On the other hand, the four acylated oxyntomodulin peptide analogs according to embodiments of the present invention showed improved PK half-life than Comparative Example Compound 8, and showed a half-life similar to Semaglutide developed as a once-weekly injection, indicating that the acylated oxyntomodulin peptide analog of the present invention can be developed as a long-acting drug in a once-weekly formulation.

<Experimental Example 6> Assessment of Single-Dose Pharmacokinetic Profile in Monkeys

[0073] Cynomolgus monkeys were used to evaluate the pharmacokinetics in monkeys of the acylated oxyntomodulin peptide analogs according to the present invention. Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, or Compound 7, which is an acylated oxyntomodulin peptide analog prepared by the present invention, was prepared in sterile distilled water for injection containing 0.1% Cremophore EL to a dose of 100 nmmol/kg, and injected to the Cynomolgus monkeys subcutaneously once; then, blood was collected at a designated time after administration, and pharmacokinetic indicators were calculated through plasma drug concentration analysis. The results are shown in Table 10 and FIG. 5.

TABLE-US-00011 TABLE 10 Pharmacokinetic evaluation in monkeys of acylated oxyntomodulin peptide analogs Monkey PK Compound (S.C. half-life)* Example Compound 1 C Compound 2 C Compound 3 E Compound 4 D Compound 5 D Compound 6 C Compound 7 E Comparative Compound 9 B Example Compound 10 A Compound 11 B Compound 12 B Semaglutide E MEDI0382 A *A: Less than 6 hours, B: 6 hours to less than 12 hours; C: 12 hours to less than 18 hours; D: 18 hours to less than 24 hours; E: 24 hours or more

[0074] As seen in Table 10 and FIG. 5, the experimental results show that the seven acylated oxyntomodulin peptide analogs (Compounds 1 to 7) according to examples of the present invention exhibit a longer half-life of 12 hours or more compared to the four Comparative Example Compounds and Comparative Example MEDI0382 under development as an once-daily injection, indicating that the half-life was significantly improved through chemical stability improvement from the introduction of a new albumin ligand. In particular, Compound 3 and Compound 7 show a half-life similar to Semaglutide, which was developed as a once-weekly injection, with a monkey PK half-life of more than 24 hours, indicating that they can be developed as a long-acting drug in a once-weekly formulation.

[0075] Comparative Example Compound 9 is Comparative Example Compound 8 (Korea Patent Application No. 2018-0095717, Compound 3) except with an albumin ligand referenced in the literature (Nat. Commun. 2017; 8:16092), and had a half-life of less than 12 hours, suggesting that long-acting peptide analogs cannot be developed only with the introduction of the albumin ligand of the literature.

[0076] Comparative Example Compound 10, Comparative Example Compound 11, and Compound 7, having a lipophilic lipid in the end form of an aminocarboxyl group on the Lys side chain at position 34 of the peptide analog backbone, each have a carbon chain length of C14, C16, and C18, respectively. Compound 7 had a half-life of 24 hours or longer, whereas Comparative Example Compound 10 and Comparative Example Compound 11 showed less than 12 hours, indicating that when the lipophilic lipid ends with an aminocarboxyl group, it has a long half-life only when the carbon chain length is C18.

[0077] Compound 1, Compound 2, and Compound 3, each having a lipophilic lipid in the end form of a carboxyl group on the Lys side chain at position 34 of the peptide analog backbone, each have a carbon chain length of C14, C16, and C18, respectively. All of these substances showed a half-life of more than 12 hours, and in particular, Compound 3 showed a half-life of more than 24 hours. In order to have a long half-life, it is preferable to have a carboxyl group substituent at the end of the lipophilic lipid, and the longer the carbon chain, the better the effect.

[0078] Compounds 4, 5, and 6, in which a lipophilic lipid is bound to the Lys side chain at position 34 of the peptide analog backbone by a combination of a spacer and a polymeric moiety, have a longer half-life of 12 hours to less than 24 hours compared to the Comparative Examples. On the other hand, Comparative Example 12 showed a shorter half-life of less than 12 hours despite the lipophilic lipid linked to the polymeric moiety, suggesting that the effect of the end of the lipophilic lipid is greater than the effect of the polymeric moiety on the half-life.

<Experimental Example 7> Testing the Glucose Tolerance Improvement Effect in Mice

[0079] In this experiment, glucose tolerance improvement effect in male laboratory mice (ICR mice) of acylated oxyntomodulin peptide analogs according to the present invention was evaluated as improvement of postprandial glycemic control. Laboratory mice were fasted the day before the experiment. Then, Compound 2 or 3 or 7 according to the present invention was prepared in sterile distilled water for injection containing 0.1% Cremophor EL and injected subcutaneously 6 hours before glucose loading. Glucose solution was orally administered 6 hours after the injection of oxyntomodulin peptide analog. Whole blood glucose was measured via tail vein immediately before administering the drug and glucose, and for 2 hours after glucose loading at designated times. From the results, the area under the curve (AUC) of the blood glucose curve over time was produced to calculate the ratio of blood glucose AUC of the acylated oxyntomodulin peptide analog against the vehicle control as percentages. The results are shown in FIG. 6.

[0080] As seen in FIG. 6, Compound 2, Compound 3, or Compound 7, which is an acylated oxyntomodulin peptide analog according to the present invention, showed significant reduction of blood glucose AUC of 42.1%, 26.7%, and 33.9%, respectively, compared to the glucose control group, at 30 nmol/kg. In addition to the half-life improvement due to chemical stability, the acylated oxyntomodulin peptide analogs of the present invention are effective at improving blood glucose level and weight loss through activity on GLP-1 and glucagon receptors.