KR20180087864A - Novel exenatide variants-polymer complex - Google Patents

Abstract

The present invention relates to a novel exenatide variant-polymer complex and a production method thereof. The novel exenatide variant-polymer complex of the present invention has higher resistance to protease as compared to conventional exenatide monomers, allows users to overcome problems such as kidney excretion due to low molecular weight and induction of an immune response in the body, and an increase the efficacy of drugs by about 57 times, thereby ultimately reducing the dose and frequency of exenatide treatment for patients with type 2 diabetes, and effectively replacing conventional exenatide. Additionally, in the case of producing the complex through the production method of the present invention, the complex can be produced at a high yield without using an organic solvent, and the economic efficiency can be remarkably improved compared to conventional methods.

Description

NOVEL EXENATIDE VARIANTS-POLYMER COMPLEX}

The present invention relates to a novel structure exenatide variant-polymer complex and a method for producing the same.

Exenatide is a synthetic peptide of Exendin-4, a glucagon-like peptide 1 (GLP-1) analogue, used to lower insulin secretion in patients with type 2 diabetes and lower blood glucose levels. It has about 53% homology with native GLP-1 and is resistant to the proteolytic enzyme DPP-IV as an agonist for the GLP-1 receptor and has a half-life of 2-4 hours. The initial dose is 5 mcg subcutaneously twice a day and can be administered up to 10 mcg twice a day. However, its use in type 1 diabetes has not yet been recognized.

Several studies of exenatide have consistently demonstrated the effect of reducing fasting and postprandial glucose, glycosylated hemoglobin, and body weight, but efforts have been made to improve it with a shorter half-life. A method for forming a complex of exenatide-polymer (polyethylene glycol (PEG) is mainly used as a polymer) includes sharing an amine coupling reaction between a lysine residue in exenatide and PEG-aldehyde There is a technique of covalently bonding a PEG-MA (maleimide) molecule to a thiol group after generating an exenatide mutant having a cysteine residue attached at its C-terminus.

However, the covalent bonding technique using an amine coupling reaction is not site-specific because all two lysine residues are pegylated, and it is difficult to separate positional isomers bonded to each lysine residue . In addition, a method of preparing a linear structure complex by attaching a cysteine residue to the C-terminus can be carried out by reacting a dimethylaricolide (TEA) containing triethylamine (TEA) or trifluoroacetic acid (TFA) dimethylsulfoxide (DMSO). DMSO is difficult to remove because of its high boiling point, and TEA and TFA organic solvents have toxicity, which can induce the denaturation of exenatide peptides. The process must be added.

Therefore, there is a continuing need for research and development for the production of high efficiency exenatide-polymer complexes in order to reduce patient convenience and manufacturing cost, such as reduction in the number of administrations.

The present invention is intended to provide a method for preparing an improved complex, which is difficult to separate and purify, and which is not a conventional method for producing a complex, which deteriorates the life-span of exenatide by non-specific binding of the polymer. Accordingly, the present invention provides a novel structure of exenatide having a branched structure of T or Y form by bonding a polymer to the intermediate position of exenatide instead of the exenatide-polymer complex of the existing linear structure - < / RTI >

In this respect, peptides of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; And a biodegradable polymer.

The present invention also relates to a method for producing a biocompatible polymer, which comprises mixing a peptide of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 with a biocompatible polymer in a molar ratio of 1: 1 to 3; And a method for producing a peptide-polymer complex, which comprises separating and purifying a desired peptide-polymer complex using cation exchange chromatography from the reaction mixture.

The present invention also relates to an exenatide mutant comprising a mutation in which the 12th lysine is replaced with cysteine (K12C) or the 27th lysine is replaced with cysteine (K27C) in the exenatide peptide of the amino acid sequence of SEQ ID NO: 3 .

Since the method for producing a peptide-polymer complex according to the present invention induces a site-specific binding, the desired complex and separation and purification can be easily performed. Therefore, problems such as denaturation of a peptide without using an organic solvent in preparation and separation / And the step of removing the organic solvent can be omitted in the manufacturing process of the medicament, so that it is efficient. In addition, since non-specific binding between the peptide and the polymer does not occur, problems such as reduction of the bioactivity of the peptide in the complex do not occur, and the complex can be produced at a high yield, and the peptide- Can significantly improve the half-life and can be used as a bio-improved drug that can replace conventional exenatide formulations.

Figures 1a and 1b show the results of determining whether exenatide variants were pegylated: (1a): SDS-PAGE using Coomassie Blue staining. (B) PEG staining. (KD), # 2: exenatide variant (K12C), # 3: PEG-MA (10 kD), # 4: K12C-PEG, # 5: K27C-PEG in lanes 1 and 1b it means.
Figures 2a, 2b, 2c, and 2d illustrate separation using a cation exchange chromatography (HiTrap SP) of exenatide variant-polymer conjugates prepared according to one embodiment of the present invention: (2a) K12C-PEG, Peak # 1-unreacted PEG-MA, Peak # 2-K12C-PEG; (2b) 60% Buffer B, K27C-PEG, Peak # 3-K27C-PEG; (2c) Coomassie blue SDS-PAGE; (2d) PEG staining. Lane # 1: marker (kD) in Figures 2c and 2d; Lane # 2: PEG-MA (10 kD); Lane # 3: peak # 1; Lane # 4: K12C-PEG from PEGylation mixture (before chromatography); Lane # 5: Peak # 2 (K12C-PEG); Lane # 6: means K27C-PEG (before chromatography), lane # 7: peak # 3 (K27C-PEG) from a PEGylation mixture.
Figure 3 shows the trypsin stability test of an exenatide monomer in a trypsin solution prepared according to one embodiment of the present invention and a K12C-PEG complex and a K27C-PEG complex according to one embodiment of the present invention Show the results.
4 is a graph comparing the bioactivity of exenatide (Exn) monomer with cAMP production according to the concentration of K12C-PEG complex and K27C-PEG complex in CHO cells expressing GLP-1R according to an embodiment of the present invention This is a graph showing a result.
FIG. 5 is a graph showing the results of comparing the bioactivity of the peptide-PEG complex according to the molecular weight of PEG according to an embodiment of the present invention.
Figure 6 shows the results of cation exchange chromatography analysis of the homotypic complex K12C-PEG 10kD-K12C prepared according to one embodiment of the present invention.
Figures 7a and 7b are mass spectral results of the mass spectrometry of the separated product by cation exchange chromatography of the homogeneous complex K12C-PEG 10kD-K12C prepared according to one embodiment of the present invention.
FIG. 8 is a graph showing the results of comparing the bioactivity of the allogeneic complex K12C-PEG 10kD-K12C prepared according to an embodiment of the present invention and the monoclonal peptide-polymer complex K12C-PEG 10kD by cAMP production.
FIG. 9 is a fluorescence image showing in vivo half-life of a peptide and a peptide-polymer complex prepared according to an embodiment of the present invention.
FIG. 10 is a graph showing the in vivo half-life of a peptide and a peptide-polymer complex prepared according to an embodiment of the present invention.

The present invention relates to an exenatide variant comprising a mutation in which the 12th lysine in the amino acid sequence of SEQ ID NO: 3 is substituted with cysteine (K12C) or in which the 27th lysine is replaced with cysteine (K27C) .

The present invention relates to the above exenatide variant peptide; And a biodegradable polymer.

The complex may be a dimer-type complex comprising two peptides per biocompatible polymer. A peptide of SEQ ID NO: 1 - a polymer - a peptide of SEQ ID NO: 1; A peptide of SEQ ID NO: 1 - a polymer - a peptide of SEQ ID NO: 2; A peptide of SEQ ID NO: 2 - a polymer - a peptide of SEQ ID NO: 2; Or peptide-polymer of SEQ ID NO: 2 - peptide complex of SEQ ID NO: 1.

The present invention also relates in this aspect to a process for the preparation of peptide-polymer complexes. Mixing the peptide of the present invention and the biocompatible polymer in a molar ratio of 1: 1 to 3; And separating and purifying the desired peptide-polymer complex from the reaction mixture using cation exchange chromatography.

Hereinafter, the present invention will be described in more detail.

The exenatide variant of the present invention has a peptide sequence in which a part of the amino acid has been substituted in the sequence of the exenatide peptide (SEQ ID NO: 3), specifically, it may be a peptide of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The peptide of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 means the peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The peptide of the present invention is a variant of exenatide used as a therapeutic agent for diabetes mellitus in the present invention. It is preferable that the exenatide is an amino acid of SEQ ID NO: 1 or SEQ ID NO: 2 unless it affects the ability of the exenatide to bind to GLP- A homology of 60% or more with the sequence, or 70% or more, or 80% or more, or 90% or more of homology with the sequence may be included in the present invention.

Exenatide variants of the present invention are those wherein the 12th lysine in the exenatide sequence is replaced by cysteine (SEQ ID NO: 1, K12C); Or the 27th lysine is substituted with cysteine (SEQ ID NO: 2, K27C). When the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 forms a complex with a polymer, it can react with the polymer in a position-specific manner at the 12th cysteine or 27th cysteine, thereby improving the yield and efficacy of the peptide- So that it has a very advantageous effect for the production of a peptide-polymer complex. Particularly, the conventional method for preparing the exenatide-polymer complex is characterized in that a cysteine is bonded to an amino acid terminal to prepare a linear complex, or an amine coupling reaction between a lysine residue in exenatide and an PEG-aldehyde is used to bond with a polymer , But it was not efficient because the two lysine residues were able to participate in binding in the course of the preparation of the complex and did not cause a site specific reaction and it was very difficult to separate the position isomers bound to each lysine residue. However, when a complex with a polymer is formed using the exenatide mutant of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention, a binding reaction occurs only between the mutated cysteine residue and the polymer, And it is advantageous in that there is no difficulty in separation and purification of the resulting complex.

In the preparation of exenatide variant-polymer complex according to an embodiment of the present invention, the yield of site-specific covalent bond reaction between the peptide and the polymer was 95% or more (Example 2).

In this respect, the invention relates to a peptide-polymer complex, preferably an exenatide variant-polymer complex.

The peptide-polymer complex of the present invention refers to a molecule in which a peptide and a polymer are combined, and specifically refers to a molecule to which the exenatide variant of the present invention and the biocompatible polymer are bound. It is possible to increase the half-life of the exenatide therapeutic agent conventionally used for the exenatide variant-biocompatible polymer complex of the present invention, to improve potency and efficacy, and to reduce the number of administrations Effect.

The peptide-polymer complex of the present invention comprises a peptide of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; And a biodegradable polymer, and a peptide-polymer complex comprising the biocompatible polymer.

In addition, the complex may be a dimer-type complex comprising two peptides having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 for one biocompatible polymer. A peptide of SEQ ID NO: 1 - a polymer - a peptide of SEQ ID NO: 1; A peptide of SEQ ID NO: 1 - a polymer - a peptide of SEQ ID NO: 2; A peptide of SEQ ID NO: 2 - a polymer - a peptide of SEQ ID NO: 2; Or peptide-polymer of SEQ ID NO: 2 - peptide complex of SEQ ID NO: 1.

In the present invention, the potency means the minimum concentration at which the peptide mutant of the present invention or the complex containing the peptide can exhibit its effect. It is understood that the lower the potency value, the better the potency.

In addition, the efficacy of the present invention means the maximum effect of the drug at a certain concentration, and it is understood that the higher the efficacy value, the better the efficacy (excellent).

In the present invention, the biocompatible polymer is a copolymer of polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, a polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid (PLA, polylactic acid, polylactic-glycolic acid (PLGA), lipopolymer, chitin, hyaluronic acid, and combinations thereof. Also included in the present invention are derivatives already known in the art of the present invention or derivatives which can be easily prepared at the technical level of the present invention.

The biocompatible polymer increases the molecular weight of the peptide of the present invention to inhibit extinction by elongation and increases the resistance to the in vivo protease, thereby prolonging the half-life of the peptide.

The polymer may preferably be a polymer having a molecular weight of more than 5 kD, and more preferably a polymer having a molecular weight of more than 5 kD and 50 kD or less. When the polymer has a molecular weight of 5 kD or less, it may be complexed with the peptide of SEQ ID NO: 1 or 2, but its molecular weight may be small and it may be purified by elongation in the body. In addition, when a polymer having a molecular weight of more than 50 kD is used, the blocking of the peptide by the polymer may be too severe, and the biological potency of the peptide-polymer complex may be lowered. have. However, depending on the nature of the complex and the formulation of the drug, the weight of the polymer can be set differently.

The peptide of the present invention and the biocompatible polymer may be linked via any covalent bond, preferably linked by a covalent bond between the cysteine residue of the peptide and the biocompatible polymer. More preferably, the 12th cysteine in the amino acid sequence of SEQ ID NO: 1 or the 27th cysteine in the amino acid sequence of SEQ ID NO: 2 and the biocompatible polymer may be covalently linked. The peptide-polymer complex of the present invention can be represented as a peptide-polymer complex of a branched structure. The half-life of the peptide-polymer complex of the branched structure containing the exenatide variant of SEQ ID NO: 1 or 2 of the present invention is improved, and it has an excellent half-life in the body as compared with the linear peptide-polymer complex. In addition, the use of the exenatide variants of SEQ ID NO: 1 or 2 has the advantage that it is easy to prepare and isolate and purify the site specific complex.

In this respect, the biocompatible polymer of the present invention may have a reactor at one end or both ends. The reactor at the terminal is preferably selected from the group consisting of a maleimide group and a succinimide group. In the above, examples of the succinimide group may include succinimidyl propionate, succinimidyl butanoate, hydroxysuccinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate. The biocompatible polymer may have the same or different end-to-end reactors. When polyethylene glycol having a hydroxy group at both terminals is used, the hydroxy group may be activated by the known chemical reaction with the various reactors, or may be prepared using polyethylene glycol having a commercially available modified reactive group.

In particular, the biocompatible polymer of the present invention preferably has a maleimide group at one end or both ends thereof in order to cause a position-specific reaction with the 12th cysteine or the 27th cysteine in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In this case, it was confirmed in the examples of the present invention that the position non-specific reaction can be minimized and a complex can be produced with a reaction yield of 90% or more.

The exenatide variants of the present invention also make it possible to prepare peptide-polymer complexes very easily, to produce position-specific peptide-polymer complexes, to facilitate separation and purification of complexes, and to produce peptide- And has superiority in that the yield and efficiency can be remarkably improved. In addition, the complex of the present invention has improved resistance to an in vivo proteolytic enzyme, and has an excellent half-life in the body.

In this respect, the invention also relates to a process for the preparation of peptide-polymer complexes.

The exenatide variant and the biocompatible polymer of the present invention and the complex containing the same may be applied as they are to the production method, but the substrate is omitted in order to avoid redundant description.

In the method of the present invention, the reaction molar ratio of the peptide of SEQ ID NO: 1 or 2 of the present invention and the biocompatible polymer may be 1: 1 to 3, preferably 1: 1 to 2, do. The reaction of the peptide with the polymer can be performed in a buffer containing EDTA, and the buffer can have a pH of 6.5 to 8.0. When the reaction is carried out while satisfying the above range, the production yield of the peptide-polymer complex can be increased.

The preparation method of the present invention comprises separating and purifying the desired peptide-polymer complex from the reaction mixture using cation exchange chromatography. The process for producing a peptide-polymer complex of the present invention can be carried out by using a peptide of SEQ ID NO: 1 or SEQ ID NO: 2 having a mutation of K12C or K27C in exenatide, thereby producing a complex in a site-specific, stable and environmentally friendly manner And it is very easy to separate and purify the produced complex.

In particular, the production method of the present invention is characterized in that an aqueous buffer can be used without using an organic solvent in the step of reacting a peptide with a biocompatible polymer to form a complex. The above-mentioned water-soluble buffer solution is not limited to the types of buffer solutions conventionally used in the field to which the present invention belongs. Preferably, a buffer solution containing EDTA can be used.

For example, the aqueous buffer may be a buffer selected from the group consisting of sodium phosphate buffer (SPB), phosphate buffered saline (PBS), Tri HCl buffer, and combinations thereof. no. As described above, when a water-soluble buffer is used as a reaction solvent, it is possible to solve the problem that the peptide is denatured due to toxicity due to the organic solvent, and the process of removing the organic solvent after the preparation of the peptide-polymer complex can be omitted, It is very advantageous from the side.

Further, the present invention is characterized in that a water-soluble solvent can be used in place of the mobile phase of the organic solvent in the process of separating and purifying the peptide-polymer complex, thus being eco-friendly and easy to separate and purify.

In particular, the mobile phase solvent (elution buffer) which may be used in the cation exchange chromatography of the present invention may be an aqueous solution containing sodium chloride, or a buffer. The peptide-polymer complex is dissociated by dissociating the electrostatic bond according to the concentration of sodium chloride in the mobile phase solvent (elution buffer). The concentration of sodium chloride in the mobile phase solvent may vary depending on specific chromatographic conditions.

The process for preparing a polymer conjugate using the exenatide variant of the present invention is excellent in that it can facilitate the separation of a complex to be produced without using an organic solvent.

The inventors of the present invention experimentally found that the complex comprising the exenatide mutant of SEQ ID NO: 1 or SEQ ID NO: 2 and the biocompatible polymer has an effect of improving the resistance to trypsin, which is a protease, by about 50 times as much as the exenatide monomer , And it was confirmed that the biological half-life of exenatide was significantly improved as compared with the conventional exenatide. Further, when the production process of the present invention is used, it has been confirmed that the yield of the composite is at least 95%, which will be described in more detail with reference to the following examples.

The following Examples and Experiments are illustrative of the present invention and are not intended to limit the scope of the present invention.

[ Example  One] Exenatide ( Exenatide ) Mutant  synthesis

The mutant K12C, in which Lys12 of exenatide was mutated by cysteine, and the mutant K27C, in which Lys27 was mutated by Cys, was produced by a chemical synthesis method. The molecular weight of both mutants was 4,162 Da.

The amino acid sequences of the two exenatide variants synthesized are as follows:

Lt;

K27C variant: HGEGTFTSDLSKQMEEEAVRLFIEWLCNGGPSSGAPPPS-NH2.

[ Example  2] Site specific Pegylation  through Peptides - Polymer composite manufacturing

The linear structure of PEG-MA (PEG maleimide, molecular weight 10 kDa) was used to induce the covalent bond between maleimide and the cysteine residue in the middle of the mutant. The exenatide mutant prepared in Example 1 and PEG-MA were dissolved in 10 mM PBS buffer (pH 7.0) containing 10 mM EDTA, and the molar ratio of exenatide mutant to PEG-MA was 1: 1.5 The mixture was allowed to react at room temperature for 16 hours or longer.

As shown in FIGS. 1A and 1B, the reaction results were analyzed by SDS-PAGE and PEG staining. As a result, the reaction yield of the site-specific covalent bond reaction was as high as 95% or more.

[ Example  3] Exenatide Mutant - PEG  Separation purification of complex

The exenatide variant-PEG complex prepared in Example 2 and the unreacted PEG-MA were separated by cation exchange chromatography packed with Hi-Trap ^TM SP-HP resin (purchased from GE Healthcare). The equilibrium buffer (buffer A) contained 10 mM citric acid and was pH 3.0 and buffer B (100% to 10 mM citric acid and 1M sodium chloride, pH 3.0) was eluted from 0% to 60% for elution of the conjugate bound to the resin. . ≪ / RTI > The flow rate was 1 ml / min at room temperature and the complex in the eluate was detected using 280 nm ultraviolet absorbance.

Referring to Figure 2, the first peak (# 1 peak) is unreacted PEG-MA and the second is mono-pegylated exenatide variant-PEG complex. The peak (# 2 peak) of K12C-PEG was eluted from 22% buffer B (or 0.22 M NaCl) and the peak (# 3 peak) of K27C-PEG was eluted from 33% buffer B (or 0.33 M NaCl). A small amount of unreacted exenatide eluted in 100% Buffer B (1 M NaCl), which means that the pegylation reaction of Example 2 is nearly 100% complete.

As a result of SDS-PAGE and PEG staining, it was confirmed that the yield of the mono-pegylation reaction of both mutants was very high as shown in FIGS. 2c and 2d.

[ Example  4] Exenatide Mutant - PEG  Composite Life castle ( bioactivity ) Confirm

Measurement of immunoreactivity by competitive enzyme ELISA test showed no significant difference in immunoreactivity in both exenatide variants and exenatide variant-PEG complexes as compared to circular exenatide monomers. Therefore, it can be seen that the exenatide mutant and exenatide mutant-PEG complex according to the present invention maintain their immunoreactivity well.

Peptide type ^* Concentration by ELISA ([mu] g / ml) Ratio Exenatide (round) 0.66 + 0.11 100% K12C 0.73 + 0.04 110% K27C 0.86 ± 0.24 130.3% K12C-PEG 0.80 ± 0.28 121.2% K27C-PEG 0.88 0.15 133.3%

All concentrations of all peptides are the same: 1 μg / ml (according to the Bradford assay)

In addition, resistance to trypsin, a protease, was measured for each peptide. After dissolving trypsin (1 mg / ml) in 10 mM PBS (pH 7.4) at 37 ° C for 30 minutes, 0.5 mg / ml exenatide (circular) monomer was added to the same volume (100 μl) of trypsin solution. K12C-PEG 10kD complex; Or K27C-PEG 10kD complex were mixed and cultured at 37 ° C. 100 [mu] l of stop solution (deionized distilled water containing 0.1% TFA) was added to terminate the reaction. The dissociation of exenatide was analyzed at 0.5, 1, 2, 3, 5, 10, 30, and 60 minutes via RP-HPLC (Shim-pack GIS-ODS) column.

As shown in Fig. 3, the exenatide mutant-PEG conjugate (i.e. mono-pegylated exenatide variant), K12C-PEG and K27C-PEG, each had about 50 times more stability than the exenatide monomer Respectively. Therefore, it can be seen that excellent stability can be obtained when the exenatide mutant according to the present invention is formed with a PEG polymer complex.

[ Example  5] Exenatide ( Exenatide ) - PEG  Evaluation of bioactivity of complexes

When the exenatide ligand binds to a GPCR (G-protein coupled receptor), a second series of messengers is activated to respond to cellular signals. The Gα _s receptor can stimulate adenylate cyclase and then catalyze cAMP in ATP to induce insulin secretion. Using this, the biological activity of the exenatide variant and exenatide-PEG complex was determined using the cAMP Hunter Exendin-4 Bioassay Kit (DiscoverX Corporation, Fremont, CA, USA) to confirm peptide activity.

The CHO cells contained in the kit were cultured on a 96-well plate for 30 hours. After aspiration, the plates were incubated with various concentrations (10 ^-7 to 10 ^-12 M) of exenatide (circular), K12C-PEG 10kD, or K27C-PEG 10kD. Signals at each concentration were measured with a luminescence plate reader (Biotek Synergy ^™ MX Microplate Reader). Based on the standard concentration of the cAMP in the kit, the concentration of cAMP in each of the exenatide and exenatide variant-polymer complexes was analyzed using logistic function fitting software (Origin Pro 8). The potency (EC ₅₀ ), which means the amount required to exhibit the effect of the drug as the ability to bind to the GLP-1 receptor, and the efficacy, which signifies the maximum effect of the drug, are measured and shown in Tables 2 and 4, respectively . The efficacy was measured at a concentration of 10 ^-8 M of the drug. The experiment was repeated three times and the results were expressed as mean ± SEM.

Peptide type EC ₅₀ (M) Validity (nM) Exenatide (round) 8.6x10 ^-10 22.8 ± 5.9 K12C-PEG 2.0x10 ^-11 16.9 ± 5.8 K27C-PEG 5.3x10 ^-11 15.0 ± 1.6

As shown in Table 2 and FIG. 4, the exenatide mutant-polymer complexes K12C-PEG and K27C-PEG of the present invention had a potency (EC ₅₀ ) increased by 42.3-fold and 16.3-fold relative to the circular-exenatide, respectively Respectively, and the efficacy of cAMP production was 74% and 65%, respectively. Thus, the exenatide-polymer complex has improved receptor binding capacity.

[ Example  6] PEG  Check chain length effects

To investigate the effect of PEG molecular weight on exenatide-PEG complex, a complex was prepared using exenatide variant prepared in Example 1 and 5, 10, 20 or 40 kD PEG polymer.

The exenatide peptide and 5, 10, 20 or 40 kD PEG were each dissolved in 10 mM PBS buffer (pH 7.0) containing 10 mM EDTA, mixed at room temperature so that the molar ratio of peptide to PEG was 1: 1.3, And then pegged. In order to confirm the activity change of the complex according to the molecular weight of the PEG chain, the amount of cAMP released from CHO cells was measured in the same manner as in Example 5. The efficacy was measured at a concentration of 5 x 10 < ^-8 > M. The experiment was repeated two times and the data were expressed as mean ± SEM.

Kinds EC ₅₀ (M) Efficacy (nM) K12C-PEG 5kD 5.5x10 ^-11 18.3 ± 1.4 K12C-PEG 10kD 1.1x10 ^-10 20.9 ± 1.7 K12C-PEG 20kD 3.9x10 ^-11 19.5 ± 1.8 K12C-PEG 40kD 1.2x10 ^-10 28.5 ± 2.0

As shown in Table 3 and FIG. 5, as the molecular weight of PEG increased, the efficacy increased and the titer (EC ₅₀ ) tended to decrease, but the difference was less than 2 times, which was not a significant difference Respectively. The reason for this is that as the molecular weight of PEG increases, the affinity between the peptide-PEG complex and the receptor is reduced by blocking a larger number of amino acids. However, the PEG 40kD complex has the best efficacy to exhibit the cAMP response as compared to the 10kD or 20kD PEG complex.

[ Example  7] Homo dimer HOMODIMER PEGYLATION  through Homogeneous quantities  Manufacture of Composites

Two molecules of K12C and bifunctional PEG maleimide (MA-PEG-MA, 10 kD) were dissolved in 10 mM PBS buffer (pH 7.0). K12C (1 mg / ml) was slowly added with gentle agitation such that the molar ratio of exenatide variant: MA-PEG-MA was 2: 1. The reaction was carried out at room temperature for 16 hours to prepare a peptide-polymer complex in the form of a homodimer. To confirm that the complexes were well prepared, the results were analyzed by elution chromatogram (HiTrap SP) obtained from a linear gradient elution of 0% to 40% buffer B.

As a result, as shown in FIG. 6, three peaks were observed in each cation exchange column, and fractions of each peak were obtained and subjected to mass analysis. As a result, peak # 1 was unreacted PEG, peak # 2 was mono K12C-PEG complex (Monopegylated complex) and Peak # 3 were homozygous complex (K12C-PEG 10kD-K12C). This elution sequence is also related to the theory described above, in which the monopegylated complex is eluted first, followed by eluting a homopolymer complex in which two peptides are conjugated to one PEG molecule. Further, the molecular weights of peaks # 2 and # 3 of the K12C exenatide mutant were measured by a mass spectrometer (MALDI-TOF MS), and the mass spectra of peaks # 2 and # 3 were shown in FIGS. 7A and 7B. Peak # 2 contained a mono-pegylated K12C-PEG complex (15141 Da) and a dimer (19208 Da), and the main peak at peak # 3 was the homodimer K12C-PEG 10kD-K12C (19262 Da). The mass-to-charge (m / z) ratio of all experiments closely matched the calculated values.

CAMP production release in CHO cells was measured in the same manner as in Example 5, in order to confirm the synergistic effect of the peptide-PEG complex of the monomer form and homodimer on exenatide (prototype). The efficacy was measured at a concentration of 10 ^-8 M of the drug.

Kinds EC ₅₀ (M) Validity (nM) Circular exenatide 6.1x10 ^-10 24.4 ± 1.0 K12C-PEG 10kD 6.6x10 ^-11 20.5 ± 0.7 K12C-PEG 10kD-K12C 4.4 x ^{10 -11} 19.8 ± 0.8

8 and Table 4, the EC ₅₀ value (ability to bind to the GLP-1 receptor) indicates that the homodimer K12C-PEG 10kD-K12C has a receptor binding capacity of 1.5 (as compared to the monomer K12C-PEG 10kD) Fold and the circular exenatide, respectively. In terms of efficacy, an indicator of cAMP production capacity, all complexes had lower bioactivity than circular exenatide monomers, but no significant difference.

[ Example  8] IN VIVO Pharmacokinetics ( PHARMACOKINETICS ) Confirm

The exenatide, exenatide variant (K12C) and exenatide variant (K12C) were assayed using a fluorescent imaging assay using a nude mouse model (BALB / c male, 5 weeks; purchased from Orient Bios) and IVIS Lumina XRMS (Perkin Elmer) The stability of the exenatide-PEG conjugate in vivo was confirmed (persistence).

1 mg / ml exenatide (round); Exenatide variant (K12C); K12C-PEG 5kD complex; And K12C-PEG 10kD-K12C complexes were labeled with 5 molar equivalents of Flamma® 675 vinylsulfone in 50 mM boric acid buffer (pH 8.77) at 35 ° C with constant stirring for 16 hours at 35 ° C. Next, the mixture was purified with a PD MiniTrap G-25 (USA) desalting column and buffer exchanged with 10 mM PBS buffer (pH 7.4). The concentration of the peptide-dye conjugate was determined by the manufacturer's method. Each sample was subcutaneously injected into a nude mouse (s.c.). At 4, 8, 24, and 48 hours post injection, systemic fluorescence was detected using an in vivo imaging system (IVIS; IVIS Lumina XRMS, Perkin Elmer, USA) at excitation wavelengths of 675 nm and 691 nm And visualized at the emission wavelength.

In addition, a linear peptide-polymer complex in which a cysteine residue was added to the C-terminus of exenatide and bound to PEG (40 kD) was used as a control, and the same drug as the complex of the branched form of the present invention Dynamics (C40-PEG 40kD complex).

The quantitation of the protein-dye conjugate or the ratio of the dye to the protein was calculated by the molar concentration of the protein or protein-fluorescent conjugate measured separately based on the absorbance measurement by the UV-Vis spectrophotometer. The results of the quantitative determination of the protein-dye conjugate by absorbance measurement are shown in Table 5.

Kinds A ₂₈₀ A ₆₇₅ Protein concentration (mM) Dye / protein ratio Exenatide (circular) 1.633 2.170 0.261 0.038 K12C 1.260 1.983 0.192 0.046 K12C-PEG 5kD 0.816 2.189 0.113 0.088 K12C-PEG 40kD 0.567 2.050 0.0695 0.134 C40-PEG 40kD 0.651 2.055 0.0847 0.110 K12C-PEG 10kD-K12C 0.477 1.787 0.0575 0.141

CF = 보정계수, 280nm에서 염료에 의한 흡광도를 조정; CF = 0.09
ε = 단백질 몰 흡광 계수; ε= 5500
ε'= 형광 염료의 몰 흡광 계수(molar extinction coefficient); (ε'= 220,000)A ₂₈₀ = absorbance at 280 nm
A ₆₇₅ = absorbance at 670 nm of the Flamma dye (maximum wavelength λ _max for the dye molecule)

CF = correction factor, adjust absorbance by dye at 280 nm; CF = 0.09
ε = protein molar extinction coefficient; ? = 5500
? '= molar extinction coefficient of the fluorescent dye; (? '= 220,000)

Non-invasive imaging was performed to track the release of exenatide (prototype) and exenatide variant-PEG complex in nude mice. Strong NIR fluorescence was detected on the back of nude mice after injection. The fluorescence signal was normalized according to [Equation 1]

[Formula 1]

To calculate the half-life of each sample, the fluorescence signal after the administration of a single dose was calculated by applying Equation 2 below:

[Formula 2]

C _t = C ₀ e ^{- kt}

Where C _t is the intensity of the fluorescence signal after time t, C ₀ is the initial fluorescence signal intensity (t = 0), and k is the removal rate constant.

The relationship between the removal rate constant and the half-life (t _1/2 ) is shown in Equation 3 below:

[Formula 3]

The half life of each sample obtained by normalizing the fluorescence intensity (Flamma VS 675 dye) value measured in the nude mouse according to the above formula is shown in Table 6. Fluorescence signals were increased in all samples 1 hour after injection of the peptide or peptide-polymer complex and fluorescence after 1 hour was normalized between 0-100%.

Kinds Half-life Coefficient of determination - R ² Exenatide (circular) 1.6 h 0.969 K12C 2.5 h 0.985 K12C-PEG 5kD 1.7 h 0.977 K12C-PEG 40kD 5.5 h 0.996 C40-PEG 40kD 5.1 h 0.986 K12C-PEG 10kD-K12C 2.1 h 0.980

As shown in Table 6, Fig. 9 and Fig. 10, exenatide (circular) disappeared within 2 hours after injection. This was similar to the K12C-PEG 5kD complex. Therefore, when a complex is formed with a small molecular weight (MW) polymer having a PEG of 5 kD, the effect of increasing half-life is negligible. However, the half-life of K12C-PEG 40kD complex and C40-PEG 40kD was prolonged to over 5 hours, which is a 3.5-fold increase in half-life compared to circular-exenatide. The effect of the homodimeric K12C-PEG 10kD-K12C was also insignificant but the half-life was increased compared to the exenatide prototype. The reason for the short half-life extension effect is expected to be that the molecular weight of the homodimeric K12C-PEG 10kD-K12C is about 19 kD, which is still lower than 20 kD and is relatively easily removed by renal filtration. However, the half-life of the homodimers showed only a slight increase, but it was confirmed that the effect was more excellent in the effectiveness. Thus, complexes with a PEG 40kD polymer and complexes in the form of homodimers were found to be effective in reducing the number of exenatide administrations due to efficacy or prolonged half-life.

<110> Industry-University Cooperation Foundation Hanyang University ERICA Campus <120> NOVEL EXENATIDE VARIANTS-POLYMER COMPLEX <130> P18U22C0073 <150> KR 10-2017-0011919 <151> 2017-01-25 <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> A variant of Exenatide <400> 1 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Cys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Ser          35 <210> 2 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> A variant of Exenatide <400> 2 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Cys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Ser          35 <210> 3 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Exenatide sequence <400> 3 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu   1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser              20 25 30 Ser Gly Ala Pro Pro Ser          35

Claims (12)

A peptide of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; And
A peptide-polymer complex comprising a biocompatible polymer. The method according to claim 1,
Wherein the peptide-polymer complex is a dimer comprising two peptides of the amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, for each biocompatible polymer. The method according to claim 1,
The biocompatible polymer may be selected from the group consisting of polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid ), Polylactic-glycolic acid (PLGA), lipopolymers, chitin, hyaluronic acid, and combinations thereof. The method according to claim 1,
Wherein the biocompatible polymer is a 12th cysteine (Cys) in the amino acid sequence of SEQ ID NO: 1; Or the 27th cysteine (Cys) in the amino acid sequence of SEQ ID NO: 2. The method according to claim 1,
Wherein the biocompatible polymer is polyethylene glycol (PEG). 6. The method of claim 5,
Wherein said polyethylene glycol (PEG) has a molecular weight of greater than 5 kD. 6. The method of claim 5,
Wherein the complex is linked by a covalent bond between the cysteine residue of the peptide and the maleimide residue of the polymer. A peptide of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and a biocompatible polymer in a molar ratio of 1: 1 to 3; And
And separating and purifying the desired peptide-polymer complex from the reaction mixture using cation exchange chromatography. 9. The method of claim 8,
Wherein the mixing reaction is performed in an aqueous buffer selected from the group consisting of sodium phosphate buffer (SPB), phosphate buffered saline (PBS), Tri HCl buffer, and combinations thereof. &Lt; / RTI &gt; 9. The method of claim 8,
Wherein the biocompatible polymer is polyethylene glycol or a derivative thereof. 9. The method of claim 8,
Wherein said cation exchange chromatography is carried out using a mobile phase solvent comprising sodium chloride. Wherein the 12th lysine in the exenatide peptide of the amino acid sequence of SEQ ID NO: 3 is substituted with cysteine (K12C) or the 27th lysine is replaced with cysteine (K27C).

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