KR20200131721A - Pharmaceutical composition for treating or preventing diabetic complications - Google Patents

Abstract

The present invention relates to a composition for preventing or treating diabetic complications, which comprises a C-peptide combined with an elastin-like polypeptide (ELP), thereby being able to effectively prevent or treat diseases caused by hyperglycemia, which is a main cause of diabetes, such as diabetic retinopathy, etc.

Description

Pharmaceutical composition for preventing or treating diabetic complications {PHARMACEUTICAL COMPOSITION FOR TREATING OR PREVENTING DIABETIC COMPLICATIONS}

The present invention relates to a pharmaceutical composition for the prevention or treatment of diabetic complications, and more particularly, for the prevention or treatment of diabetic complications comprising a C-peptide coupled with a temperature sensitive biopolymer (elastin-like polypeptide) It relates to a pharmaceutical composition.

Diabetes refers to a metabolic disorder of multiple etiologies characterized by chronic hyperglycemia caused by a defect in secretion of insulin or a defect in insulin action. Chronic metabolism when an abnormally high level of blood glucose is maintained for a long time. Various complications occur due to disorders and subsequent chronic vascular damage.

According to a recent study, it has been reported that diabetic angiopathy is mainly due to apoptosis due to elevated levels of reactive oxygen species (ROS) and activation of transglutaminase 2 (TGase 2).

Conventional treatment of diabetic complications caused by chronic hyperglycemia has been achieved through passive treatment in which blood sugar is controlled by administering a hypoglycemic drug such as insulin. However, there is a clear limitation in the prevention or treatment of long-term diabetic complications that are treated only by controlling blood sugar.

Recently, C-peptide has been reported to be effective in the treatment and prevention of diabetic vasculopathy, including diabetic retinopathy, neuropathy, nephropathy, cardiovascular disease, and delay in wound healing. Accordingly, C-peptide Is emerging as a prominent potential drug for treating diabetic complications. However, since C-peptide has a very short half-life when injected into the body, there is a limit to repeatedly injecting drugs or implanting a device such as an osmotic pump into the body. In addition, since the production of C-peptide is dependent on chemical synthesis, there is a problem that a lot of costs are incurred due to continuous injection.

In this regard, Korean Patent Laid-Open No. 10-2013-0115086 discloses a PEGylated C-peptide comprising a PEG moiety covalently attached to the N-terminus of the C-peptide in order to improve the half-life of the C-peptide. Are doing. However, long-term circulation of PEG molecules may induce an immune response, and there is a limitation in that the in vivo behavior of the conjugate cannot be regulated due to the polydispersity and poor stoichiometry of the polymer.

Republic of Korea Patent Publication No. 10-2013-0115086

An object of the present invention is to provide a pharmaceutical composition for effective prevention or treatment of diabetic complications caused by hyperglycemia.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating diabetic complications comprising a C-peptide coupled with an elastin-like polypeptide (ELP).

In the present invention, the elastin-like polypeptide includes a repetition of the pentapeptide motif VPGXG of SEQ ID NO: 1, and in the repeated pentapeptide motif VPGXG, X is each independently valine (Val; V) or lysine (Lys; K) It is selected from, at least one of which may be Lysine (Lys; K).

The elastin-like polypeptide may include at least one repeating unit of the amino acid sequence of SEQ ID NO: 2.

The C-peptide may be composed of the amino acid sequence of SEQ ID NO: 4.

In the present invention, the diabetic complication is caused by blood vessel leakage, diabetic retinopathy, diabetic stroke, diabetic cardiovascular disease, diabetic kidney disease, diabetic lung disease, diabetic peripheral neurosis, delayed treatment of diabetic wounds, or It may be diabetic cancer metastasis.

In the present invention, the pharmaceutical composition for preventing or treating diabetic complications inhibits apoptosis, increases intracellular reactive oxygen species (ROS) production, and inhibits transglutaminase 2 (TGase 2) activity. can do.

In the present invention, the C-peptide combined with the elastin-like polypeptide may have a phase transition behavior.

The pharmaceutical composition for preventing or treating diabetic complications of the present invention includes C-peptide coupled with an elastin-like polypeptide (ELP), effectively preventing diseases arising from hyperglycemia, which is the main cause of diabetes, It can be prevented and treated.

Specifically, the composition for preventing or treating diabetic complications according to the present invention can continuously release C-peptide in vivo for a long time with one injection. In addition, compared to the conventional C-peptide production method dependent on chemical synthesis, the production cost can be significantly reduced, and side effects of existing chemical compounds can be minimized.

1 is a diagram showing a sequence of K8 and K9-C-peptide according to an embodiment of the present invention, a cycle for expressing and purifying K9-C-peptide, and an agarose gel electrophoresis image of K9-C-peptide.
2 is a diagram showing the properties of K9-C-peptide according to an embodiment of the present invention using Experimental Example 3.
3 and 4 are diagrams showing the release pattern of C-peptide released from K9-C-peptide according to an embodiment of the present invention using Experimental Example 4.
5 is a view measuring the change in active oxygen concentration and TGase activity according to Experimental Example 7 and the evaluation of cytotoxicity according to Experimental Example 8. FIG.
6 is a diagram showing the results of cell death evaluation according to Experimental Example 9.
7 is a diagram showing the distribution results of K9-C-peptide in the rat eye according to Experimental Example 10.
8 is a view showing the results of the inhibitory effect of K9-C-peptide on the blood vessel leakage phenomenon caused by diabetes in the diabetic rat retina according to Experimental Example 11.
9 is a diagram showing the inhibitory effect of K9-C-peptide in the blood vessel leakage phenomenon occurring in the kidney of a diabetic rat according to Experimental Example 12.
10 is a diagram showing the inhibitory effect of K9-C-peptide in the vascular leakage phenomenon occurring in the lungs of diabetes-induced rats according to Experimental Example 13. FIG.
11 is a diagram showing the inhibitory effect of K9-C-peptide on endothelial cell death occurring in a diabetic rat aorta according to Experimental Example 14. FIG.
12 is a diagram showing the inhibitory effect of K9-C-peptide in the delayed wound healing phenomenon occurring in diabetic mice according to Experimental Example 15.
13 is a diagram showing the inhibitory effect of K9-C-peptide in cancer metastasis occurring in diabetic mice according to Experimental Example 16.

The present invention relates to a pharmaceutical composition for preventing or treating diabetic complications comprising a C-peptide coupled with an elastin-like polypeptide (ELP), or a method of treatment.

The present inventors combine C-peptide, which is known to be effective in the prevention and treatment of diabetic complications, with an elastin-like polypeptide (ELP) of a temperature-sensitive biopolymer by a gene recombination method to achieve a new form of temperature. A sensitive biopolymer-conjugated C-peptide was designed, and the temperature-sensitive biopolymer-conjugated C-peptide can continuously release C-peptide from the body for a long time with a single injection, thus preventing, treating or improving diabetic complications. The present invention was completed by experimentally confirming that it has an effect.

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

The pharmaceutical composition for the prevention or treatment of diabetic complications of the present invention comprises a C-peptide bound to an elastin-like polypeptide (ELP), thereby causing active oxygen species (ROS) from hyperglycemia. It was experimentally confirmed that hyperglycemia-induced vascular dysfunction caused by suppression of endothelial cell death can be prevented through inhibition of production and activation of transglutaminase 2 (TGase2).

In the present invention, a temperature-sensitive biopolymer called C-peptide and elastin-like polypeptides (ELPs) are combined through genetic engineering to enable continuous release and delivery of C-peptide drugs in vivo for a long time. The gene expressed in the state is cloned, the gene is expressed in E. coli and purely isolated, and it can be effectively applied to the prevention and treatment of diabetic complications.

The elastin-like polypeptides (ELPs), which are sensitive to heat, are a kind of recombinant biopolymer useful for drug delivery. The elastin-like polypeptides (ELPs) are composed of repeated pentapeptide motifs.

In the present invention, the elastin-like polypeptide includes SEQ ID NO: 1 of VPGXG, a repeating unit of pentapeptide.

SEQ ID NO: 1 is VPGXG (Val-Pro-Gly-Xaa-Gly), where X (or Xaa) is a guest residue.

The elastin-like polypeptide may be represented by the following formula.

[Equation 1]

[SEQ ID NO: 1] _n

Wherein n means the number of repetitions of SEQ ID NO: 1, and is an integer of 1 or more, preferably an integer of 60 to 221.

In the repeated pentapeptide motif VPGXG, X may be independently selected from valine (Val; V) or lysine (Lys; K), and at least one includes lysine (Lys; K).

Abbreviations such as Val(V), Pro(P), Gly(G), and Lys(K) used in the present specification are amino acid abbreviations. Val is the abbreviation for valine, Pro for proline, Gly for glycine, and Lys for lysine. Also, valine is expressed as V, proline is expressed as P, glycine is expressed as G, and lysine is expressed as K. The abbreviation is a widely used expression in this technical field.

The elastin-like polypeptides (ELPs) according to the present invention may form a polypeptide block in which pentapeptide is repeated.

For example, the elastin-like polypeptides (ELPs) may form a [(VPGVG) ₁₁ (VPGKG) ₁ (VPGVG) ₁ ] amino acid sequence repeating unit of SEQ ID NO: 2.

In the present invention, the physicochemical properties of elastin-like polypeptides (ELPs) such as temperature-sensitive sol-gel transfer ensure the spatiotemporal controlled release of the drug, so that the elastin-like polypeptides (ELPs)-binding C-peptide of the present invention can be used for a long time in vivo. Can continuously release C-peptide. The temperature at which the sol-gel transition is possible can be easily adjusted by changing the number and sequence of ELP units, and the genetic conjugation of proteins or peptides and ELPs is performed using recombinant DNA technology to determine the physicochemical properties and stoichiometric properties between proteins and polymers. You can strictly control the loan.

For example, in the present invention, elastin-like polypeptides (ELPs) contain lysine (K), and at least one amino acid sequence repeating unit of [(VPGVG) ₁₁ (VPGKG) ₁ (VPGVG) ₁ ] of SEQ ID NO: 2 As the ELPs to be included, preferably, 5 to 17 amino acid sequence repeat units of [(VPGVG) ₁₁ (VPGKG) ₁ (VPGVG) ₁ ] of SEQ ID NO: 2 may be included.

In some embodiments, the present invention may provide elastin-like polypeptides (ELPs) of SEQ ID NO: 3, including 9 repeating units of the amino acid sequence of SEQ ID NO: 2.

The SEQ ID NO: 3 consists of [(VPGVG) ₁₁ (VPGKG) ₁ (VPGVG) ₁ ] ₉ .

In one embodiment of the present invention, the C-peptide may be the EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ sequence, that is, SEQ ID NO: 4, but is not limited thereto.

SEQ ID NO: 4 is Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro -Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln.

In the present specification, the [(VPGVG) ₁₁ (VPGKG) ₁ (VPGVG) ₁ ] of SEQ ID NO: 2 C-peptide combined with an elastin-like polypeptide having 9 repeating amino acid sequence repeat units is "K9-C-peptide ( K9-C-peptide)" may be used in the same sense.

In some embodiments, the binding of the ELPs and the C-peptide may be formed through an additionally included linker, and the ELPs and the C-peptide may be bonded by the linker.

The linker includes the amino acid sequence of DPNYPRGH (Asp-Pro-Asn-Tyr-Pro-Arg-Gly-His) of SEQ ID NO: 5.

In one embodiment, the C-peptide combined with the elastin-like polypeptide contained in the pharmaceutical composition for the prevention or treatment of diabetic complications of the present invention, for example, K9-C-peptide, has a sol-gel transition under body temperature. As possible, the phase transition can have behavior.

The pharmaceutical composition for preventing or treating diabetic complications according to the present invention contains C-peptide bound to an elastin-like polypeptide (ELP), so that the C-peptide can be used for a long time through phase transition with one injection. Since it can be released continuously, it can exhibit a preventive or therapeutic effect on diabetic complications caused by high blood sugar.

In particular, when the K9-C-peptide according to the present invention is injected once, C-peptide having a therapeutic effect on diabetic complications is released from the K9-C-peptide for 20 days, and can be released up to 30 days. , Increased intracellular reactive oxygen species (ROS) levels caused by hyperglycemia, increased transglutaminase 2 (TGase 2) activity and inhibited apoptosis.

The pharmaceutical composition for preventing or treating diabetic complications of the present invention may be applied without particular limitation as long as it is a disease known as diabetic complication, but, for example, diabetic retinopathy, diabetic stroke, diabetic cardiovascular disease, diabetic It can be applied to diabetic complications such as kidney disease, diabetic lung disease, diabetic peripheral neurosis, diabetic wound treatment delay, diabetic cancer metastasis, among which diabetic retinopathy, diabetic lung disease, diabetic kidney disease, diabetes It can be applied more effectively to delayed treatment of sexual wounds, metastasis of diabetic cancer or diabetic cardiovascular disease.

For example, the cardiovascular disease may include hypertension, dyslipidemia, angina, arterial spasms, deep veins, cardiac hypertrophy, cerebral infarction, congestive heart failure, arteriosclerosis, coronary heart disease, myocardial infarction, and the like, and is particularly limited thereto. It is not.

The pharmaceutical composition for preventing or treating diabetic complications according to the present invention may be administered in an "effective amount" or "a pharmaceutically effective amount". "Effective amount" or "pharmaceutically effective amount" means an amount sufficient to exhibit a preventive or therapeutic effect for diabetic complications and an amount sufficient to not cause side effects or serious or excessive immune responses, and an effective dose level The disorder to be treated, the severity of the disorder, the activity of a particular compound, the route of administration, the rate of elimination, the duration of treatment, drugs used in combination or simultaneously, the age, weight, sex, diet, general health condition of the subject, and the pharmaceutical industry and It may depend on a variety of factors, including factors known in the medical field. Various general considerations are known to those of skill in the art when determining such "effective amount" or "pharmaceutically effective amount".

Preferably, the pharmaceutical composition for preventing or treating diabetic complications according to the present invention may be for one or more of ocular administration, intravitreal injection of the eye, intraperitoneal injection, subcutaneous injection, intradermal injection, intramuscular injection, and patch administration. have.

The pharmaceutical composition for preventing or treating diabetic complications according to the present invention can be appropriately administered according to the disease and weight of the subject.

For example, in the case of preventing or treating diabetic complications in humans using K9-C-peptide, a single dose using the intravitreal injection method of the present invention is 1.27㎍ to 1.27mg, subcutaneous injection ( Subcutaneous injection) method or intraperitoneal injection (intraperitoneal injection) method using a single dose of 1mg / kg to 1g / kg, intradermal injection (Intradermal injection) 41.5㎍ to 41.5mg can be.

Meanwhile, the composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent as a pharmaceutical composition. The term "pharmaceutically acceptable carrier, excipient or diluent" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Carriers, excipients, and diluents that may be included in pharmaceutical compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

If necessary, the composition of the present invention can also be used with biopolymers such as chitosan, poly-γ-glutamic acid, polylactic acid, alginate, etc. of C-peptide bound to a temperature-sensitive biopolymer (elastin-like polypeptide). It is possible.

In addition, the composition of the present invention may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. and sterile injectable solutions according to a conventional method. In the case of formulation, it can be prepared using diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient such as starch, calcium carbonate, and sucrose in the lecithin-like emulsifier. (sucrose) or lactose (lactose), it can be prepared by mixing gelatin.

In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used. As liquid preparations for oral administration, suspensions, solvents, emulsions, syrups, etc. can be used.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives are included. I can. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous agents, suspensions, emulsions, and freeze-dried preparations. As the non-aqueous preparation and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.

The present invention includes a pharmaceutical composition for preventing or treating diabetic complications, a method for preventing or treating diabetic complications using the same, and uses thereof.

Hereinafter, the present invention will be described in more detail using examples and experimental examples. However, the following Examples and Experimental Examples are intended to illustrate the present invention, and the present invention is not limited to the following Examples and Experimental Examples and may be variously modified and changed.

Experimental Example 1: Construction of plasmids for K8 and K9-C-peptide production

A synthetic gene synthesizing K1[(VPGVG) ₁₁ (VPGKG) ₁ (VPGVG) ₁ ] ELP and K1-C-peptide containing the cleavable linker sequence DPNYPRGH was synthesized and bound to the pBSC (KS+) vector.

Using a synthetic recursive directional ligation method, as shown in Fig. 1A, the gene encoding the ELP consisting of 8 repeats of K1 [(VPGVG) ₁₁ (VPGKG) ₁ (VPGVG) ₁ ] is Was created. The gene encoding K8[(VPGVG) ₁₁ (VPGKG) ₁ (VPGVG) ₁ ] ₈ was fused to the gene encoding K1-C-peptide at the 5'-end. The resulting K9-C-peptide gene was inserted into the pET25b+ vector for polypeptide expression. All completed constructs were checked for inserted sequences through DNA sequencing.

Experimental Example 2: Peptide expression and purification

Referring to FIG. 1B, in order to prepare K8 and K9-C-peptide, expression vectors encoding K8 and K9-C-peptide were transformed into E. coli BL21 strain (DE3) and expressed. And inverse transition cycling (ITC) was performed.

-D-1-thiogalactopyranoside로 유도한 후 37℃에서 0.4 내지 0.6의 OD₆₀₀으로 성장시켰다. 박테리아 세포를 4℃에서 9,000 rpm으로 10 분 동안 원심 분리하여 수확하고, 인산염 완충 식염수 [PBS; 8.1 mmol/L Na₂HPO₄, 1.2 mmol/L KH₂PO₄, 138 mmol/L NaCl, 2.7 mmol/L KCl (pH 8.0)]에 재현탁하고 얼음 위에서 각각 10 초 동안 30 회 초음파 처리하였다. DNA와 불용성 부분을 얼음과 30 분간 10 % 폴리에틸렌이민(polyethyleneimine) 배양 후 18,000g에서 15 분 동안 4 ℃에서 원심 분리하여 침전시켰다. NaCl을 가용성 부분(최종농도 3 mol/L) 에 첨가하고 50 ℃에서 10 분간 배양하였다. 혼탁한 현탁액을 18,000 g에서 15 분 동안 50 ℃에서 원심 분리 하였다 (hot spin). 상등액을 제거하고 펠릿을 얼음으로 냉각 된 PBS에 현탁시켰다. 생성된 용액을 18,000 g에서 10 분 동안 4 ℃에서 원심 분리하고 상등액을 보관하였다(cold spin). ITC 공정을 한 번 더 반복하여 정제물을 얻었다. 생성된 상청액을 milli-Q 물에 대해 24 시간 투석하고 동결 건조시켰다.Specifically, the transformed cells were 1 mmol/L isopropyl at 37°C for 4 hours.

After induction with -D-1-thiogalactopyranoside, it was grown at an OD ₆₀₀ of 0.4 to 0.6 at 37°C. Bacterial cells were harvested by centrifugation at 9,000 rpm for 10 minutes at 4° C., and phosphate buffered saline [PBS; 8.1 mmol/L Na ₂ HPO ₄ , 1.2 mmol/L KH ₂ PO ₄ , 138 mmol/L NaCl, 2.7 mmol/L KCl (pH 8.0)] and sonicated 30 times on ice for 10 seconds each. The DNA and the insoluble portion were incubated with 10% polyethyleneimine on ice for 30 minutes and then centrifuged at 18,000 g for 15 minutes at 4° C. to precipitate. NaCl was added to the soluble portion (final concentration 3 mol/L) and incubated at 50° C. for 10 minutes. The cloudy suspension was centrifuged at 18,000 g for 15 minutes at 50 °C (hot spin). The supernatant was removed and the pellet was suspended in ice-cooled PBS. The resulting solution was centrifuged at 18,000 g for 10 minutes at 4° C. and the supernatant was stored (cold spin). The ITC process was repeated once more to obtain a purified product. The resulting supernatant was dialyzed against milli-Q water for 24 hours and lyophilized.

The genes obtained by the ITC process (K1, K2, K4 and K8) and the genes obtained by the fusion of K8 and K1-C-peptide were double-decomposed by BamHI and HindIII, and then confirmed through agarose gel electrophoresis. (Fig. 1C). The sequences of the K8 and K9-C-peptide genes were confirmed through DNA sequencing.

Experimental Example 3: In vitro characterization of K9-C-peptide

In order to confirm the generation and purification of K9-C-peptide, samples collected from each ITC process were loaded onto a 10% polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie blue dye and imaged using a ChemiDoc ^™ scanner.

To characterize the reverse transition temperature of the K9-C-peptide, the turbidity of a solution of 2.5-100 mmol/L K9-C-peptide in PBS was measured with SpectraMax M5 Multi- at a wavelength of 350 nm as a function of temperature at a heating rate of 1 °C/min. It was monitored with a Mode microplate reader (Molecular Devices, Sunnyvale, CA).

K8 and K9-C-peptide were purified from E. coli using ITC, and the yield was 10 mg per 1 liter of culture solution. SDS-PAGE was used to measure the purity and molecular weight of the K9-C-peptide (Fig. 2A). Samples maintained throughout the ITC process were visualized on an SDS-PAGE gel stained with Coomassie blue, where the 60 kDa band protein was identified as a K9-C-peptide with an estimated molecular weight of 53 kDa. Western blot analysis showed that this 60 kDa band consisted of C-peptide. This result showed that the K9-C-peptide was successfully purified by ITC, and that it was sufficient to purify this K9-C-peptide in the second ITC process cycle.

The thermal reaction of the K9-C-peptide was observed by measuring the turbidity (as a function of temperature, ranging from 2.5 μmol/L to 100 μmol/L) of the K9-C-peptide solution. The turbidity of the K9-C-peptide solution increased in a temperature dependent manner, suggesting that coacervate was produced by the sol-gel transition (FIG. 2B). The transition temperature of the K9-C-peptide solution decreased from 45.4° C. to 28.1° C. in a concentration dependent manner (FIG. 2C). These results indicate that the present inventors have successfully prepared a C-peptide and a K9-C-peptide consisting of 9 ELP unit repeats, and that the conjugate can be used as an injectable depot of C-peptide.

Experimental Example 4: Conjugation of K9-C-peptide with Cy3-NHS ester

The K9-C-peptide was labeled with Cy3 NHS-ester.

Specifically, 1 ml of a 20 mg/ml K9-C-peptide solution in 100 mmol/L bicarbonate buffer (pH 8.3) was mixed with 13 μL of 5 mg/ml Cy3 mono NHS-ester in 10% dimethyl sulfoxide. And incubated for 2 hours on ice. To stop the reaction, 50 μl of 1 mol/L Tris-HCl (pH 8.0) was added to the reaction solution. The reaction mixture was loaded onto a 1.5 ml Sephadex G-25 column, and Cy3-conjugated K9-C-peptide was eluted by centrifugation at 1050 x g for 3 minutes.

Experimental Example 5: On-chip degradation profiling of K9-C-peptide hydrogel

Amine-modified glass slices were prepared as follows.

Glass slides (75 Х 25 mm) were washed with H ₂ O ₂ /NH ₄ OH/H ₂ O (1:1:5, v/v) solution, and 1.5% (v/v) 3-aminopropyltrimethoxysilane in 95% ethanol Was soaked for 2 hours and baked at 110° C. overnight. A well-type amine array was prepared by mounting a polydimethylsiloxane gasket on an amine-modified glass slice.

Enzymatic digestion profiling of K9-C-peptide by four cleavage enzymes including collagenase-2, elastase-2, pepsin and trypsin Was performed as follows.

A 10 mg/ml aliquot of Cy3-conjugated K9-C-peptide was applied to a well-type amine array at 40° C. for 2 hours to form a hydrogel. The array was buffer A (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 5 mmol/L CaCl ₂ , 0.2 mmol/L NaN ₃ , and 0.002% Brij-35, pH 7.6) at 37° C. for 2 hours In collagenase-2, buffer B (100 mmol/L Tris-HCl and 100 mmol/L NaCl, pH 7.5) in elastase-2, buffer C (10 mmol/ L HCl, pH 2.0) in pepsin and buffer D (50 mmol/L Tris-HCl, 20 mmol/L CaCl ₂ , and 0.02% Tween-20, pH 8.1) in trypsin at the indicated concentration. Cultured.

The array was prepared in Tris-buffered saline containing 0.1% Tween-20 [TBS; 50 mmol/L Tris-HCl (pH 7.5) and 140 mmol/L NaCl] for 10 minutes, then with Milli-Q water for 5 minutes, drying under air and using a confocal microscope equipped with a motorized sample stage. And analyzed.

The amount of K9-C-peptide remaining on the array surface was calculated using a standard curve, which was plotted as a sigmoidal fit using the Origin program as follows:

(1),

(One),

In the above formula (1), x represents the K9-C-peptide concentration; x ₀ represents the K9-C-peptide concentration at half of the maximum fluorescence intensity; A ₁ and A ₂ represent the upper and lower asymptotes, respectively. y represents the sample fluorescence intensity, and P represents the power. Then, using GraphPad PRISM8 (GraphPad Software; San Diego, CA), the half-maximal effective concentration (EC ₅₀ ) for cleavage enzyme was calculated by the following modified langmuir isotherm:

F _obs = (F _max Х[enzyme]/EC ₅₀ +[enzyme]) + background (2)

In the above equation (2), F _obs is the fluorescence intensity of the triple spot, F _max is the maximum fluorescence in the field, [enzyme] is the enzyme concentration, and EC ₅₀ is the apparent maximum half effective concentration.

The enzyme concentration of each protease is as follows in U/ml: One unit of collagenase-2 releases 1 micromole of L-leucine from collagen for 5 hours (pH 7.5 at 37°C), and elastase. One unit of -2 will hydrolyze 1 mmol of MeO-Suc-APV-pNA per minute (pH 7.5 at 25°C), and one unit of pepsin will release an amount of Tyr, increasing the absorbance of 0.001 per minute at 280 nm. (PH 2 at 37°C), one unit of trypsin produces an increase in absorbance of 0.001 per minute at 253 nm of benzoyl L-arginine ethyl ester (pH 7.6 at 25°C).

The degradation of the K9-C-peptide hydrogel was observed by an on-chip digestion assay, because this assay allows ultra-fast analysis using a small volume of sample. In order to visualize and quantify the degradation of K9-C-peptide by protease, K9-C-peptide was covalently conjugated with Cy3 NHS-ester, and Cy3-conjugated K9-C-peptide hydrogel array was well After fabrication using a type amine array, four proteases, collagenase-2, elastase-2, pepsin, and trypsin were incubated together at the indicated concentrations (Figure 3A).

The EC ₅₀ value for each enzyme was determined by plotting the rate of degradation based on the amount of Cy3-conjugated K9-C-peptide fragment away from spots on the array, and plotting against the protease concentration. All four of these enzymes increased K9-C-peptide degradation in an enzyme concentration dependent manner (FIG. 3B to FIG. 3F). The EC ₅₀ values obtained for the four enzymes were 0.17 U/ml, 0.23 U/ml, 510.00 U/ml and 124.60 U/ml, respectively. These results demonstrate that collagenase-2 and elastase-2 cleave K9-C-peptide more efficiently than pepsin and trypsin.

In order to simulate the release of C-peptide by hydrogel degradation in the presence of protease, the present inventors applied Elastase-2 to a hydrogel array for a specified period of time to use a on-chip digestion assay using Cy3-conjugated K9-C. -The time-dependent degradation of the peptide hydrogel was observed. The degradation of K9-C-peptide hydrogel by elastase-2 was increased in a time-dependent manner, where the highest degradation rate was 72.3% at 120 minutes, and a degradation rate of less than 10% was achieved with the reaction buffer alone (Fig. 4 of A). Then, the present inventors confirmed that the Cy3 conjugated K9-C-peptide hydrogel was gradually degraded by repeated treatment of elastase-2 performed every 30 minutes. This result showed that 48.6 + 0.8% of the hydrogel was decomposed after the first treatment, and the decomposition rate of the hydrogel increased with the number of treatments, but the maximum decomposition rate (89.7 + 0.2%) was achieved after the fifth treatment. (Fig. 4B). In the absence of elastase-2, the degradation rate of K9-C-peptide hydrogel varies from 1.3 + 0.6% (i.e. after the first treatment) to 27.9 + 2.5% (after the fifth treatment) depending on the number of buffer treatments. Increased, suggesting that repeated washing with detergent-containing buffers caused partial degradation of the hydrogel. These results suggest that the C-peptide bound to the ELP hydrogel can be released by circulating proteases in the blood, including elastase-2.

Experimental Example 6: Cell culture and treatment

Human aortic endothelial cells (HAECs) purchased from PromoCell (Heidelberg, Germany) were harvested in 2% gelatin-coated medium with 20% FBS, 3 ng/mL bFGF, 5 U/mL heparin, and 100 U/mL penicillin ( penicillin) and 100mg/mL streptomycin (streptomycin) containing M19 medium.

For the experiments described below, the HRECs cells were incubated for 6 hours in a low serum medium containing 2% FBS, 0.1 ng/ml bFGF and antibiotics, and then treated with 30 mmol/L D-glucose.

Experimental Example 7: Measurement of intracellular ROS and TGase activity levels

Intracellular ROS levels were measured using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (ThermoFisher Scientific, Waltham, MA). HAECs were treated with human C-peptide, K8 and K9-C-peptide at each concentration (0nM, 0.5nM, 1.0nM, 1.5nM, 2.0nM), incubated for 30 minutes, and then incubated with 10 mmol/LH ₂ DCFDA. Treated for 10 minutes. The labeled cells were analyzed using a confocal microscope (K1-Fluo). Single cell fluorescence intensity was measured for 30 randomly selected cells/experiment. Intracellular ROS was measured by comparing the fluorescence intensity of the treated cells with that of the control cells.

Intracellular TGase activity was measured by introducing 5-(biotinamido)pentylamine (BAPA) into the cell. HAECs were pretreated with human C-peptide, K8 and K9-C-peptide at each concentration (0nM, 0.5nM, 1.0nM, 1.5nM, 2.0nM) for 30 minutes, and 1 mmol/L BAPA for 60 minutes I did. After incorporation of BAPA into TGase2 mediated cells, irradiation with FITC-conjugated streptavidin (1:200; MillipooreSigma, Burlington, MA, USA) for 1 hour after fixation and permeabilization of cells I did. The fluorescence intensity of the stained cells was measured with a confocal microscope (K1-fluo) for 30 cells/experiment selected at random.

Hyperglycemia increased intracellular ROS levels by approximately 2.1-fold, which was prevented by K9-C-peptide treatment in a concentration-dependent manner, where the maximum effect was achieved at 1 nM (FIG. 5B and FIG. 5C. ). Human C-peptide showed a hyperglycemic-induced ROS production inhibitory effect similar to that of K9-C-peptide, but K8 did not show any inhibitory effect. Then, the present inventors studied the effect of K9-C-peptide and human C-peptide on hyperglycemic-induced TGase2 activation in HAEC. Hyperglycemia increased in situ TGase activity by approximately 1.7-fold, and this result was suppressed by transfection of TGM2-specific siRNA (Fig. 5D). TGM-2 siRNA inhibited TGase2 protein expression, suggesting that TGase2, not any other member of the TGase family, contributes overwhelmingly to hyperglycemic-induced TGase activation in HAEC. Both K9-C-peptide and human C-peptide inhibited hyperglycemic-induced TGase2 activation in a similar manner (in a concentration dependent manner), in which case the maximum effect was achieved at 1 nM, whereas K8 had no effect. It was not seen (FIG. 5E).

Experimental Example 8: Evaluating cytotoxicity of C-peptide derivatives

The cytotoxicity of the C-peptide derivative was evaluated as follows.

HAECs were cultured in 24-well plates and treated with 0nM, 1nM, 2nM, 3nM and 5nM concentrations of human C-peptide, K8 and K9-C-peptide for 24 hours. After replacing the medium with a new medium, 200 µl of 1 mg/ml 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide solution was added to each well and incubated for 4 hours. The generated formazan crystals were dissolved in dimethyl sulfoxide, and absorbance of each well was obtained at 570 nm.

Referring to FIG. 5A, the K9-C-peptide did not show any cytotoxicity at a concentration ranging from 1 nmol/L to 5 nmol/L. The negative control biopolymers of human C-peptide and K8, ie K9-C-peptide without C-peptide, also showed no cytotoxicity. Therefore, K9-C-peptide is biocompatible and can be used to study the effect of this K9-C-peptide on hyperglycemic-induced intracellular phenomena in HAEC.

Experimental Example 9: Measurement of apoptosis

Apoptotic cells were measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using the APO-BrdU TUNEL assay kit (BD Bioscience; San Jose, CA).

Specifically, cells were fixed with 1% (w/v) paraformaldehyde in PBS for 20 minutes and then treated with 70% (v/v) ethanol on ice for 30 minutes. The immobilized cells were incubated with a DNA labeling solution containing terminal deoxynucleotidyl transferase and 5-bromo-2-deoxyuridine in reaction buffer at 37°C for 1 hour. Subsequently, the cells were cultured by treatment with FITC-labeled 5-bromo-2-deoxyuridine antibody for 30 minutes and 1 μg/ml DAPI for 10 minutes. Mounted cells were observed using a confocal microscope (K1-Fluo).

Hyperglycemia increased the number of TUNEL-positive cells, and this result was inhibited by both K9-C-peptide and human C-peptide, but not by K8 (Fig. 6A and Fig. 6B. ).

Experimental Example 10: The distribution and retention of K9-C-peptide in the mouse eyes

In order to confirm the duration of K9-C-peptide retention when K9-C-peptide was injected into the eye, mice were injected with 10 νg K9-C-peptide with fluorescence (FITC) attached and PBS as a control.

Referring to FIG. 7, when FITC-K9-C-peptide was injected into the rat eye, it could be confirmed that K9-C-peptide was present for about 20 days.

From this, if K9-C-peptide is injected once, it can be expected that C-peptide having therapeutic effect will be released from K9-C-peptide for 20 days.

Experimental Example 11: Effect of K9-C-peptide on hyperglycemia-induced vascular leakage in the retinas of diabetic mice on vascular leakage in diabetic mice

To confirm the inhibitory effect of 9-C-peptide on blood vessel leakage in the diabetic rat retina, 10 νg K9-C-peptide, PBS, 10 νg K8, and 6 ng C-peptide were injected into the rat eye.

Referring to FIG. 8, a blood vessel leakage phenomenon occurring in the rat retina inducing diabetes was confirmed, and K9-C-peptide, PBS, K8, (9-C-peptide negative control), and C-peptide were injected into the eyeball 14 As a result of observation after one day, it was observed that K8 and PBS did not affect the vascular leakage, and C-peptide alone showed a therapeutic effect for about a day after injection, but did not show a therapeutic effect after that. On the other hand, K9-C-peptide was confirmed to have an inhibitory effect on blood vessel leakage even 14 days after injection.

From this, K9-C-peptide exhibited an inhibitory effect for a longer period of time compared to C-peptide alone on the vascular leakage phenomenon, and K9-C-peptide remained in the eyeball for 14 days, resulting in c-peptide from K9-C-pepetide. It can be seen that is continuously released and shows a therapeutic effect.

Experimental Example 12: Experiment of the inhibitory effect of K9-C-peptide on diabetic kidney disease

A 6-week-old male C57BL/6 mouse was prepared, and a group of normal mice (hereinafter referred to as a normal group), a group of diabetic mice (hereinafter referred to as a diabetic group), and a group in which K9-C-peptide was injected into a diabetic mouse (hereinafter, referred to as , K9C treatment group) was made. At this time, the diabetic group made a diabetes model by injecting 150 mg/kg streptozotocin (STZ) into the abdominal cavity. The K9C-treated group made a diabetes model in the same manner as the diabetic group, and after 2 weeks, 100mg/kg of K9-C-peptide was injected subcutaneously. Thereafter, after 24 hours, it was confirmed whether K9-C-peptide had an inhibitory effect on the leakage of renal blood vessels caused by hyperglycemia.

As a result of measuring the weight of mice in each group, the normal group was 23.7 ± 0.6 g, the diabetic group was 19.9 ± 0.8 g, and the K9C group was 19.1 ± 0.7 g. In addition, as a result of measuring the blood glucose of each group of mice, the normal group was 122.5 ± 4.5 mg / dL, the diabetic group was 504.0 ± 10.0 mg / dL, the K9C treatment group was measured as 505.3 ± 8.2 mg / dL.

Meanwhile, 1.25 mg of FITC-dextran was injected into the right ventricle of each group of mice in order to observe the blood vessel leakage in the kidney, and then circulated for 5 minutes, and then the kidney was dissected, fixed and embedded in an OCT compound. The kidney tissue was stained with DAPI after cryosection and observed through a confocal microscope (confocal).

From this, as shown in Fig. 9A, representative renal blood vessel leak images of each group were confirmed.

In addition, the fluorescence intensity of FITC-dextran in each group of mouse kidneys was measured to quantitatively analyze the vascular leakage phenomenon, and the results are shown in Fig. 9B (normal group, diabetic group and K9C-treated group; n = 4).

Referring to FIG. 9B, it was confirmed that FITC-dextran leaking blood vessels were more observed in the diabetic group than in the normal group, and the blood vessel leaking phenomenon was suppressed in the KC9 treatment group.

In conclusion, it was experimentally confirmed that the vascular leakage of the kidney was inhibited by administration of K9-C-peptide, and that K9-C-peptide effectively inhibited nephropathy in diabetic mice.

Experimental Example 13: Inhibitory Effect of K9-C-peptide on Diabetic Lung Disease

A 6-week-old male C57BL/6 mouse was prepared, and a group of normal mice (hereinafter referred to as a normal group), a group of diabetic mice (hereinafter referred to as a diabetic group), and a group in which K9-C-peptide was injected into a diabetic mouse (hereinafter, referred to as , K9C treatment group) was made. At this time, the diabetic group made a diabetes model by injecting 150 mg/kg streptozotocin (STZ) into the abdominal cavity. The K9C-treated group made a diabetes model in the same manner as the diabetic group, and after 2 weeks, 100mg/kg of K9-C-peptide was injected subcutaneously. Thereafter, 24 hours later, it was confirmed whether K9-C-peptide had an inhibitory effect on the leakage of pulmonary blood vessels caused by hyperglycemia.

As a result of measuring the weight of mice in each group, the normal group was 23.7 ± 0.6 g, the diabetic group was 19.9 ± 0.8 g, and the K9C treated group was 19.1 ± 0.7 g. In addition, as a result of measuring the blood glucose of each group of mice, the normal group was 122.5 ± 4.5 mg / dL, the diabetic group was 504.0 ± 10.0 mg / dL, the K9C treatment group was measured as 505.3 ± 8.2 mg / dL.

Meanwhile, in order to observe vascular leakage in the lungs, 1.25 mg of FITC-dextran was injected into the right ventricle of the mouse and then circulated for 5 minutes, and the lungs were dissected, fixed, and embedded in an OCT compound. Lung tissue was stained with DAPI after cryosection and observed through a confocal microscope (confocal).

From this, as shown in Fig. 10A, representative images of pulmonary blood vessel leakage in each group were confirmed.

In addition, the fluorescence intensity of FITC-dextran in each group of mouse lungs was measured to quantitatively analyze the vascular leakage phenomenon, and the results are shown in Fig. 10B (normal group and diabetic group; n = 4, K9C-treated group; n = 6).

Referring to FIG. 10B, it was confirmed that FITC-dextran leaking blood vessels were more observed in the diabetic group than in the normal group, and the blood vessel leaking phenomenon was suppressed in the KC9 treatment group.

In conclusion, it was experimentally confirmed that pulmonary vascular leakage was inhibited by administration of K9-C-peptide, and that K9-C-peptide effectively inhibited lung disease in diabetic mice.

Experimental Example 14: Experiment of the inhibitory effect of K9-C-peptide on diabetic cardiovascular disease

A 6-week-old male C57BL/6 mouse was prepared, and a group of normal mice (hereinafter referred to as a normal group), a group of diabetic mice (hereinafter referred to as a diabetic group), and a group in which K9-C-peptide was injected into a diabetic mouse (hereinafter, referred to as , K9C treatment group) was made. At this time, the diabetic group made a diabetes model by injecting 150 mg/kg streptozotocin (STZ) into the abdominal cavity. The K9C-treated group made a diabetes model in the same manner as the diabetic group, and after 2 weeks, 100mg/kg of K9-C-peptide was injected subcutaneously. Two weeks later, it was confirmed that K9-C-peptide was effective in killing aortic endothelial cells caused by hyperglycemia.

As a result of measuring the weight of mice in each group, the normal group was 25.9 ± 0.9 g, the diabetic group was 20.2 ± 0.3 g, the K9C treatment group was measured to be 21.3 ± 0.6 g. In addition, as a result of measuring the blood glucose of each group of mice, the normal group was 128.5 ± 9.5 mg / dL, the diabetic group was 553.0 ± 39.0 mg / dL, the K9C treatment group was measured to be 548.0 ± 14.7 mg / dL.

On the other hand, in order to observe the aortic endothelial cell killing effect, the abdominal cavity of the mouse was incised, the aorta was separated, cut lengthwise and unfolded, fixed for 15 minutes with 1% paraformaldehyde, and then further fixed in 70% ethanol for 30 minutes. Cells with apoptosis were stained using the APO-BrdU TUNEL Assay Kit, and the nuclei were stained using DAPI. This was observed through a confocal microscope (confocal).

From this, as in Fig. 11A, a representative image of endothelial cell death of each group was confirmed.

In addition, apoptosis results in aortic endothelial cells of each group of mice were quantitatively analyzed through TUNEL assay, and the results are shown in Fig. 11B (normal group and diabetic group; n = 4, K9C-treated group; n = 6).

Referring to FIG. 11B, it was confirmed that endothelial cell death was increased in the diabetic group than in the normal group, and the cell death in the aortic endothelial cells was suppressed in the KC9-treated group.

In conclusion, it was experimentally confirmed that endothelial cell death caused by hyperglycemia in the aorta caused by pulmonary vascular leakage was inhibited by administration of K9-C-peptide, and that K9-C-peptide effectively inhibited endothelial cell death in diabetic mice. I did.

Experimental Example 15: Effect of K9-C-peptide on delaying inhibition of diabetic wound healing

A 6-week-old male C57BL/6 mouse was prepared, and a group of normal mice (hereinafter referred to as a normal group), a group of diabetic mice (hereinafter referred to as a diabetic group), and a group in which K9-C-peptide was injected into a diabetic mouse (hereinafter, referred to as , K9C treatment group) was made. At this time, the diabetic group made a diabetes model by injecting 150 mg/kg streptozotocin (STZ) into the abdominal cavity. The K9C-treated group made a diabetes model in the same manner as the diabetic group, and after 2 weeks, 100mg/kg of K9-C-peptide was injected subcutaneously. Thereafter, after 24 hours, it was confirmed whether K9-C-peptide had an inhibitory effect on the delay in wound healing caused by hyperglycemia.

As a result of measuring the weight of the mice in each group, the normal group was 26.0 ± 0.1 g, the diabetic group was 19.9 ± 0.1 g, the K9C treatment group was measured as 19.3 ± 0.4 g. In addition, as a result of measuring the blood glucose of each group of mice, the normal group was 116.0 ± 14.0 mg / dL, the diabetic group was 503.5 ± 4.5 mg / dL, the K9C treatment group was measured as 505.0 ± 3.0 mg / dL.

On the other hand, in order to observe the delay in wound healing, a 6 mm wound was made on the back skin of the mouse. Then, the wound was photographed with a camera and the size was measured using a vernier caliper.

From this, as in Fig. 12A, an image of a representative wound size of each group was confirmed.

In addition, the wound size of each group was directly measured to quantitatively analyze the wound healing rate, and the results are shown in 12B (normal group, diabetic group and K9C treated group; n = 4).

Referring to FIG. 12B, it was confirmed that wound healing was delayed in the diabetic group than in the normal group, and it was confirmed that the delay in wound healing was suppressed in the KC9-treated group.

In conclusion, it was experimentally confirmed that the wound healing delay caused by hyperglycemia was inhibited by administration of K9-C-peptide, and that K9-C-peptide effectively promoted wound healing in diabetic mice.

Experimental Example 16: Effect of K9-C-peptide to inhibit diabetic cancer metastasis

A 6-week-old male C57BL/6 mouse was prepared, and a group of normal mice (hereinafter referred to as a normal group), a group of diabetic mice (hereinafter referred to as a diabetic group), and a group in which K9-C-peptide was injected into a diabetic mouse (hereinafter, referred to as , K9C treatment group) was made. At this time, the diabetic group made a diabetes model by injecting 150 mg/kg streptozotocin (STZ) into the abdominal cavity. The K9C-treated group made a diabetes model in the same manner as the diabetic group, and after 2 weeks, 100mg/kg of K9-C-peptide was injected subcutaneously. After 24 hours, B16F10 (melanoma cells) of 5X10 ⁵ cells was injected intravenously into each group to make a cancer metastasis model, and it was confirmed that K9-C-peptide has an inhibitory effect on cancer metastasis occurring in diabetic mice. .

As a result of measuring the weight of each group of mice, the normal group was 26.6 ± 0.4 g, the diabetic group was 20.4 ± 0.4 g, and the K9C treatment group was measured as 20.4 ± 0.4 g. In addition, as a result of measuring the blood glucose of each group of mice, the normal group was 127.7 ± 3.5 mg / dL, the diabetic group was 557.8 ± 7.0 mg / dL, K9C treatment group was measured to be 543.7 ± 24.8 mg / dL.

Meanwhile, in order to confirm cancer metastasis, the lungs of the mice were separated 2 weeks after the intravenous injection into each group group, immersed in Feket solution, and the tissues were bleached.

Thereafter, as shown in FIG. 13A, a representative image of cancer metastasis of each group was confirmed.

In addition, cancer metastasis was quantitatively analyzed by counting tumor nodules in the lungs through a microscope, and the results are shown in 13B (normal group and diabetic group n = 6, K9C treatment group; n = 7).

13B, it was confirmed that cancer metastasis was increased in the diabetic group compared to the normal group, and it was confirmed that cancer metastasis was suppressed in the KC9 treatment group.

In conclusion, it was experimentally confirmed that cancer metastasis in diabetic mice was inhibited by administration of K9-C-peptide, and that K9-C-peptide effectively inhibited cancer metastasis in diabetic mice.

<110> KANGWON NATIONAL UNIVERSITY UNIVERSITY-INDUSTRY COOPERATION FOUNDATION <120> PHARMACEUTICAL COMPOSITION FOR TREATING OR PREVENTING DIABETIC COMPLICATIONS <130> HY190066 <150> US 61/680,184 <151> 2012-08-06 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> elastin-like polypeptide <400> 1 Val Pro Gly Xaa Gly 1 5 <210> 2 <211> 65 <212> PRT <213> Artificial Sequence <220> <223> elastin-like polypeptides <400> 2 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 1 5 10 15 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 20 25 30 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 35 40 45 Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val 50 55 60 Gly 65 <210> 3 <211> 585 <212> PRT <213> Artificial Sequence <220> <223> elastin-like polypeptides <400> 3 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 1 5 10 15 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 20 25 30 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 35 40 45 Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val 50 55 60 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 65 70 75 80 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 85 90 95 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 100 105 110 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly 115 120 125 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 130 135 140 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 145 150 155 160 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 165 170 175 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro 180 185 190 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 195 200 205 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 210 215 220 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 225 230 235 240 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val 245 250 255 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 260 265 270 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 275 280 285 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 290 295 300 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly 305 310 315 320 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 325 330 335 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 340 345 350 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 355 360 365 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys 370 375 380 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 385 390 395 400 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 405 410 415 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 420 425 430 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 435 440 445 Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 450 455 460 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 465 470 475 480 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 485 490 495 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 500 505 510 Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 515 520 525 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 530 535 540 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 545 550 555 560 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 565 570 575 Pro Gly Lys Gly Val Pro Gly Val Gly 580 585 <210> 4 <211> 31 <212> PRT <213> C-peptide of Homo sapiens <400> 4 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln 20 25 30 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 5 Asp Pro Asn Tyr Pro Arg Gly His 1 5

Claims (10)

Elastin-like polypeptides (elastin-like polypeptides; ELPs) containing a C-peptide coupled, a pharmaceutical composition for the prevention or treatment of diabetic complications. The method according to claim 1, wherein the elastin-like polypeptide comprises a repetition of the pentapeptide motif VPGXG of SEQ ID NO: 1,
In the repeated pentapeptide motif VPGXG, X is each independently selected from valine (Val; V) or lysine (Lys; K), and at least one of which contains lysine (Lys; K), diabetic complications Pharmaceutical composition for the prevention or treatment of. The method according to claim 2, wherein the elastin-like polypeptide comprises a repeating unit of the amino acid sequence of SEQ ID NO: 2, a pharmaceutical composition for preventing or treating diabetic complications. The method according to claim 1, wherein the C-peptide is consisting of the amino acid sequence of SEQ ID NO: 4, a pharmaceutical composition for preventing or treating diabetic complications. The pharmaceutical composition for preventing or treating diabetic complications according to claim 1, wherein the elastin-like polypeptide and C-peptide are directly linked or linked by a linker. The method of claim 5, wherein the linker comprises the amino acid sequence of SEQ ID NO: 5, the pharmaceutical composition for preventing or treating diabetic complications. The method according to claim 1, wherein the diabetic complication is diabetic retinopathy, diabetic stroke, diabetic cardiovascular disease, diabetic kidney disease, diabetic lung disease, diabetic peripheral neurosis, delayed treatment of diabetic wounds, or diabetic cancer metastasis. Phosphorus, a pharmaceutical composition for the prevention or treatment of diabetic complications. The method of claim 7, wherein the diabetic complication is diabetic retinopathy, diabetic lung disease, diabetic kidney disease, delayed treatment of diabetic wounds, diabetic cancer metastasis or diabetic cardiovascular disease, a drug for preventing or treating diabetic complications Composition. The pharmaceutical composition for preventing or treating diabetic complications according to claim 1, which inhibits intracellular reactive oxygen species (ROS) levels and transglutaminase 2 (TGase 2) activity. The method according to claim 1, wherein the C-peptide combined with the elastin-like polypeptide has a phase transition behavior, a pharmaceutical composition for preventing or treating diabetic complications.

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