KR100550206B1 - mono-PEGylated epidermal growth factor and the preparation method of thereof - Google Patents

Abstract

The present invention relates to a conjugate of epithelial growth factor and polyethylene glycol conjugated with a single molecule selectively to a specific site of polyethylene glycol (PEG), a biocompatible polymer, and a method for producing the same.

The present invention provides a single molecule of polyethylene glycol, which is selectively biocompatible to the N-terminus of the epidermal growth factor (EGF), in a single molecule, so that its physiological activity is not reduced at all compared with that of the non-conjugated epidermal growth factor. It is an object of the present invention to provide a conjugate of epithelial growth factor and polyethylene glycol and a method for preparing the same.

Description

Conjugates of epithelial growth factor and polyethylene glycol and preparation method thereof {mono-PEGylated epidermal growth factor and the preparation method of             

1 is a conceptual diagram showing a process for preparing a conjugate of epidermal growth factor and polyethylene glycol of the present invention.

Figure 2 (a) is a gel gel chromatogram conjugate of the polyethylene glycol and epidermal growth factor containing an aldehyde group having a molecular weight of 2000.

Figure 2 (b) is a gel-gel chromatogram conjugate of the polyethylene glycol and epidermal growth factor containing an aldehyde group having a molecular weight of 5000.

Figure 2 (c) is a chromatogram showing the changes before and after the dialysis of the conjugate of the epithelial growth factor n-hydroxy succinimide-polyethylene glycol having a molecular weight of 3400.

FIG. 3 shows the mass of a conjugate of polyethylene glycol and epidermal growth factor conjugated with an aldehyde group having a molecular weight of 2000 and a conjugate of polyethylene glycol and epidermal growth factor containing an aldehyde group having a molecular weight of 5000.

Figure 4 is a reversed phase HPLC graph of the digestion fragments of the epithelial growth factor conjugated with polyethylene glycol by trypsin treatment according to Test Example 3.

Figure 5 measures the cell growth of the polyethylene glycol epithelial cell growth factor conjugate of Test Example 4.

6 shows that when the polyethylene glycol-epithelial growth factor conjugate is bound to the epidermal growth factor receptor expressed on the cell surface according to Test Example 5, intracellular delivery activity is not conjugated and there is no difference between the activity of the epidermal growth factor. It is a picture showing that.

FIG. 7 is a graph of time-dependent analysis of the residual amount in vivo after administration of a polyethylene glycol containing an aldehyde group having a molecular weight of 2000 and 5000, an epithelial growth factor conjugate, and a natural epithelial growth factor to a mouse by intravenous injection. .

Figure 8 is a mixture of polyethylene glycol containing an aldehyde group having a molecular weight of 2000 and 5000, epithelial growth factor, natural epithelial growth factor and pulverized rat kidney tissues at 37 ℃ hourly, and the residual amount of the immunoenzyme method It is analyzed.

The present invention relates to a conjugate of epithelial growth factor and polyethylene glycol conjugated with a single molecule selectively to a specific site of polyethylene glycol (PEG), a biocompatible polymer, and a method for producing the same.

There are a number of protein and peptide hormones and growth factors that affect the growth and differentiation of our bodies in vivo, particularly in blood and tissues. They specifically bind specifically to receptors present in the cell wall, which affect the growth and differentiation of cells. However, when these hormones are not present in the right amount in vivo, many problems appear. For example, when human growth hormone is present in small amounts in vivo, the result is not being tall.

In 1953, Dr. S. Cohen of the United States discovered a peptide growth factor consisting of a long chain of 53 amino acids called epidermal growth factor (S. Cohen.Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid) opening in the new-born animal. (1962) J. Biol. Chem. 237, 1555-1262). This growth factor is responsible for the growth regulation of cells located outside the tissues of our body. At present, the epidermal growth factor is used as a therapeutic agent to treat wounds and injuries of the stomach wall (Korean Patent Application No. 2000-8116), and it is also attracting attention as a therapeutic agent for foot ulcers that is likely to occur in diabetics (P. Kirkegaard, PS Olsen, SS Poulsen, E. Nexo.Epidermal growth factor inhibits cysteamine-induced duodenal ulcers. (1983) Gastroenterology 85, 1277-1283.SJ Konturek, T. Radecki, I. Piastrucki.Gastric cytoprotection by epidermal growth factor. (1981a) Gastroenterology 81, 438-443.

However, the peptidic growth factors present in these blood and tissues have very short death times (half life) of several minutes. Therefore, when epithelial growth factor is used for the treatment, the sustainability and stability of the drug in vivo is a big problem.

As one of the ways of increasing the short blood and tissue retention time, attempts have been made to conjugate biocompatible polymers, especially polyethylene glycol, to proteins and peptide drugs. (FM Veronese.Pepteide and protein PEGylation: a review of problems and solutions ( 2001) Biomaterials 22, 405-417. P. Bailon, W. Berthold.Polyethylene glycol-conjugated pharmaceutical proteins (1998) Pharm. Sci.Tech.Today 1, 352-356).

Polyethylene glycol is one of the synthetic polymers approved by the Food and Drug Administration (FDA), which does not cause immune responses in vivo. By preparing a protein conjugate using this synthetic polymer, the protein permeation due to the filtering effect in the kidney due to the increase in molecular weight of the conjugate is suppressed and the degradation by protein enzymes in vivo is ultimately achieved. This increases the half-life and stability in vivo. Proteins that enhance the efficacy in this way are a variety of human growth hormone, interferon, insulin, interleukin, calcitonin and the like.

Polyethylene glycol, which is used to improve the stability and persistence of protein drugs, reacts with several binding sites of protein drugs when they react with protein drugs, resulting in a mixture of multi-PEGylated products. include the problem of being a species. In addition, even if a mono-PEGyated conjugate in which one molecule of polyethylene glycol is prepared, the problem is that the biological or in vivo activity of the drug is significantly affected by the binding site.

So far, attempts have been made to conjugate polyethylene glycol to epidermal growth factor. Polyethyleneglycol used here has N-hydroxy succinimide or N-succinimidyl propionate, which is a substance that reacts specifically with the primary amine of a protein, to one end. Methoxy polyethylene glycol derivatives were used.

Since epithelial growth factor contains two epsilon primary amine groups (Lys28 and Lys48) and one N-terminal primary amine group, the number of conjugated polyethylene glycols is several per molecule of epithelial growth factor. Sticking is a common phenomenon. In this case, polyethyleneglycol, a polymer in vivo, may cause a decrease in activity by steric hindrance of epithelial growth factor receptor binding (TH Kim, H. Lee, TG Park. PEGylated recombinant human epidermal) growth factor (rhEGF) for sustained release from biodegradable PLGA microspheres. (2001) Biomaterials in press.IB Lee, Dong H. Na, CJ Park, BH Lee, SS Lee, SD Lee, KC Lee.Increased stability of PEGylated recombinant human epidermal growth factor.M2232, AAPS annual meeting and exposition, October 2001, Denver, CO.).

The inventors of the present invention, while studying to solve the above problems, when the polyethylene glycol derivative is conjugated to the epidermal growth factor, the activity of the epithelial growth factor in terms of cell growth and growth inhibition, intracellular signal transduction of the change in activity according to the junction position It has been found that conjugation of a polyethylene glycol derivative containing an aldehyde group to a N-terminal group with a single molecule has the best effect.

Therefore, the present invention conjugates a polyethylene glycol derivative including an aldehyde group in polyethylene glycol having excellent biocompatibility selectively to the N-terminus of the epidermal growth factor (EGF) with a single molecule, and compared its physiological activity with that of the natural epidermal growth factor. The present invention aims to provide a conjugate of epithelial growth factor and polyethylene glycol and a method for preparing the same, while not being reduced at all, and having increased sustainability and stability in vivo.

In the method for producing an epithelial cell growth factor having an N-terminal group and a polyethylene glycol conjugate, the epithelial cell growth factor having an N-terminal group is dissolved in a buffer solution, and the polyethylene glycol derivative is epithelial cell having an N-terminal group. Adding the growth factor to the dissolved buffer and reacting in the presence of a reducing agent;

After the reaction is characterized in that it comprises the step of removing the unreacted epithelial growth factor, polyethylene glycol and reducing agent.

The epidermal growth factor used in the present invention is produced by a known method of genetic recombination, which may be extracted from a mammal, or may use a product obtained from expression of a prokaryotic or eukaryotic host after cloning the gene into a DNA vector. have.

The prokaryotic host is Escherichia coli (Escherichia coli), the eukaryotic host is Hansenula Polymorpha (Hansenula Polymorpha), Saccharomyces. Saccharomyces cerevisiae, and mammals can use epidermal growth factor extracted from rats, dogs, and humans.

As a solvent for dissolving epidermal growth factor, any one selected from among morpholino ethanesulfonic acid, acetated sodium acid, citric acid, phosphate buffer solution and acetamido imadiacetic acid can be used. The pH of the solution is maintained at 5-6.

If the pH of the buffer solution is less than 5, there is a problem in the stability of the protein, and if the pH is more than 6, there is a problem in that the specific properties of the N-terminal conjugation of epithelial growth factor, which is a site that binds to polyethylene glycol, decreases. The pH of the buffer solution used at is preferably maintained at 5 to 6, especially pH 5.5.

On the other hand, polyethylene glycol derivatives that bind to epidermal growth factor having N-terminal groups include aldehyde polyethylene glycol, succinimide propionate polyethylene glycol, succinimide butyrate polyethylene glycol, and polyethylene glycol, in which two molecules are bonded to each other. Polyethylene glycol succinimide, succinimidyl succinate polyethylene glycol, benzotriazole carboxylic acid polyethylene glycol, epoxide polyethylene glycol, carbonyl imidazole polyethylene glycol, p-nitrophenyl carboxylic acid polyethylene glycol, isocyanate polyethylene glycol It is preferable to use an aldehyde polyethylene glycol having excellent binding properties at the N-terminus selectively in a low acidity buffer solution when conjugation with various proteins. All.

Meanwhile, in the present invention, the molecular weight of the polyethylene glycol derivative is 500 to 50000, and more preferably, the molecular weight is 2000 to 10000 because of the difficulty of securing the reaction efficiency and the polyethylene glycol derivative having a molecular weight of 10000 or more.

If the molecular weight of the polyethylene glycol derivative is less than 500, polyethylene glycol is in a liquid form, and there is a problem due to physical properties. If it exceeds 50,000, there is a manufacturing problem. The molecular weight of the polyethylene glycol derivative used in the present invention is 500 to 50000. Good to do.

Reducing agents that use polyethylene glycol derivatives in the buffer to react with epidermal growth factor having N-terminal groups are sodium cyanobromide (NaCNBH ₃ ), sodium cyanide, and catecholalane (1,3,2) Benzodioxaluminole) or a mixture of these homogeneously mixed may be used.

In addition, the reaction between the epithelial growth factor and the polyethylene glycol derivative in the buffer solution containing the reducing agent is preferably performed at 4 ° C. to 25 ° C. for 2 to 100 hours. If the reaction time is less than 2 hours, there is a problem in the conjugation efficiency of polyethylene glycol. If it exceeds 100 hours, there is a problem of stability of protein due to long exposure to acidic solution, if the reaction temperature is less than 4 ℃, the reaction rate is too slow, if it exceeds 25 ℃ 3 of the epidermal growth factor Because of the difficulty in maintaining the secondary structure, the reaction between the epidermal growth factor and the polyethylene glycol derivative in the buffer solution containing the reducing agent is preferably performed at 4 ° C. to 25 ° C. for 2 to 100 hours.

Hereinafter, the present invention will be described by the following examples. However, it will be apparent to those skilled in the art that they are intended to illustrate the present invention and do not limit the scope of the present invention.

Example 1 Conjugation between an aldehyde-methoxy polyethylene glycol derivative having a molecular weight of 2000 and an epidermal growth factor.

After cloning the gene into the DNA vector, the epidermal growth factor obtained from the expression of the E. Coli host was lyophilized, followed by 10 mg of lyophilized epidermal growth factor and 10 mg of aldehyde polyethylene glycol having a molecular weight of 2000 mM 2.5 mM sodium cyanoborohydride. A polyethylene glycol-epithelial growth factor conjugate was prepared by reacting (NaCNBH ₃ ) -containing acetate buffer (50 mM, pH 5.5) at 25 ° C. for 10 hours.

The epithelial growth factor conjugated with polyethylene glycol could not escape on the semi-dialysis membrane having a molecular weight of 10000. Thus, the unreacted polyethylene glycol, epithelial growth factor, and NaCNBH ₃ were removed using the semi-dialysis membrane.

<Example 2> Conjugation of an aldehyde-methoxy polyethylene glycol derivative having a molecular weight of 5000 with an epidermal growth factor.

After cloning the gene into the DNA vector, the epidermal growth factor obtained from the expression of the E. Coli host was lyophilized, and then 10 mg of the lyophilized epidermal growth factor and 25 mg of aldehyde polyethylene glycol having a molecular weight of 5,000 mg of 2.5 mM sodium cyanoborohydride. A polyethylene glycol-epithelial growth factor conjugate was prepared by reacting (NaCNBH ₃ ) -containing acetate buffer (50 mM, pH 5.5) at 25 ° C. for 10 hours.

The epithelial growth factor conjugated with polyethylene glycol could not escape on the semi-dialysis membrane having a molecular weight of 10000. Thus, the unreacted polyethylene glycol, epithelial growth factor, and NaCNBH ₃ were removed using the semi-dialysis membrane.

<Example 3> Conjugation of N-hydroxy succinimide polyethylene glycol with epidermal growth factor.

After cloning the gene into the DNA vector, the epidermal growth factor obtained from the expression of E. Coli host was lyophilized, followed by phosphate buffer solution containing 8 mg of lyophilized epidermal growth factor and 140 mg of N-hydroxy succinimide polyethylene glycol. (50 mM, pH 8.0) was reacted for 12 hours at 25 ℃ to prepare a polyethylene glycol epidermal growth factor conjugate.

The epithelial growth factor conjugated with polyethylene glycol could not escape on the semi-dialysis membrane having a molecular weight of 10000. Thus, the unreacted polyethylene glycol and epithelial growth factor were separated and removed using the semi-dialysis membrane.

Test Example 1 Gel Filtration High Performance Liquid Chromatography Analysis (SEC-HPLC)

Each of the polyethylene glycol-epithelial growth factor conjugates prepared in Examples 1 to 3 was analyzed by gel filtration high performance liquid chromatography (HPLC, Waters 660E).

The column used was KW-800 (Showa Denko, Japan) that can be analyzed by the protein size of the aqueous phase. As the mobile phase, 50 mM phosphate buffer solution (acidity = 6) containing 50 mM NaCl was used under the condition of 1 ml per minute. In order to identify the epidermal growth factor in the mobile phase, it was detected by 280 nm ultraviolet. In addition, in order to purify only the part where the epidermal growth factor is conjugated, only the first part on the chromatography was purified separately, and the sample was concentrated by vacuum rotation.

The analysis results of SEC-HPLC are shown in Figure 2 (a) (b) (c).

Figure 2 (a) is analyzed by gel filtration chromatography of the aldehyde polyethylene glycol epidermal growth factor conjugate of Example 1, Figure 2 (b) is a gel of the aldehyde polyethylene glycol-epidermal growth factor conjugate of Example 2 It was analyzed by manure chromatography, and Fig. 2 (c) was analyzed by gel manure chromatography of the N-hydroxy succinimide polyethylene glycol-epithelial growth factor conjugate of Example 3.

2 (a) (b), when the aldehyde polyethylene glycol derivative was reacted with the epidermal growth factor, it can be seen that one polyethylene glycol was attached even after 24 hours. On the other hand, in the case of FIG. 2 (c) reacted with N-hydroxy succinimide-polyethylene glycol derivative, it was analyzed that the number of conjugated polyethylene glycol is one, two, three.

As a result, when polyethylene glycol using an aldehyde derivative was used, a homogeneous single molecule of polyethylene glycol was selectively conjugated to the N-terminal amine group of epithelial growth factor, and the result of using N-hydroxy succinimide polyethylene glycol All three amine residues of epithelial growth factor (lysine 28, lysine 48, N-terminus) were reacted.

<Test Example 2> Flight time measurement laser mass spectrometry (MALDI-TOF)

Flight time-measuring laser mass spectrometer (MALDI), which is a device for measuring the mass of the ionized molecules and analyzing the mass of the epithelial growth factor conjugated with polyethylene glycol in Examples 1 and 2 -TOF, Voyager DE-STR Perkin-Elmer PerSeptive Biosystem (PEG2k-EGF), conjugated polyethylene glycol with epithelial growth factor (PEG2k-EGF) and conjugated polyethylene glycol with epithelial cell growth factor (PEG5k) -EGF) was measured and the results are shown in FIG. 3.

The crystal of the sample was obtained by the following method. The solution (Matrix) was dissolved in a solution of 70% acetonitrile and 30% water at a concentration of 10 mg / ml. 1 uL of the sample and 1 uL of the support solution were dropped onto the mass spectrometer plate and waited for about 2 minutes for the solution to evaporate. Sinapinic acid (Sigma) was used as the support, and the mass of the sample was confirmed by applying a voltage difference of 25000 V under the cation detection mode.

As shown in FIG. 3, the molecular weight of the epidermal growth factor is about 6100 Daltons, but a signal is detected around about 8100 mass, indicating that polyethylene glycol having a molecular weight of 200 is conjugated into a single molecule. Similarly, polyethylene glycols with a molecular weight of 5000 could detect signals around 11000 mass. Mass spectrometry also revealed that only one polyethylene glycol was attached to the epidermal growth factor.

<Test Example 3> Reversed phase chromatogram (RP-HPLC chromatography)

In Example 1 and Example 2, epithelial cell growth factor was digested with trypsin to analyze the position of polyethylene glycol attached to the epithelial cell growth factor conjugated with polyethylene glycol. After dissolving 5 mg of epidermal growth factor in Tris buffer solution containing 0.1M guanidine hydrochloride (0.1M pH 8.0), diosistitol (DTT) solution, which can act to stop the sulfur binding of proteins, It was finally put to 10mM.

In order to prevent regeneration of broken bonds, iodine acetate solution was added to make 100 mM, and the reaction was performed at room temperature for 6 hours. To remove unreacted reagents, dialysis was carried out using a semi-dialysis membrane through which a substance having a molecular weight of 3500 or less was permeated in 5 mM Tris buffer (pH 8.0), and the solution was concentrated to 1 mL by a rotary vacuum method. .

The buffer solution for trypsin digestion was reacted for 12 hours using 50 mM Tris (pH 8.0) and CaCl ₂ 1 mM, and the epidermal growth factor / trypsin weight ratio was 100/1. 50 μL of the reaction solution was taken and subjected to reverse phase chromatography (Waters Spherisorb 4.6 × 250 mm) using a C-18 column.

Mobile phase-1 was distilled water to which 0.1% tri-fluoroacetic acid (TFA) was added, and mobile phase-2 was acetonitrile of 0.1% TFA. Mobile phase-2 was programmed to flow at 2% for the first 10 minutes and mobile phase-2 was initially increased to 60% for the next 30 minutes.

Figure 4 (a) (b) is a chromatogram showing the fragments resulting from this digestion of trypsin. 4 (a) shows a case where an aldehyde polyethylene glycol having a molecular weight of 5000 is used, and FIG. 4 (b) shows a case where an aldehyde polyethylene glycol having a molecular weight of 2000 is used.

Epithelial growth factor has a total of four amino acid residues that trypsin specifically recognizes in the molecule (lysine 28 and 48, arginine 41 and 45). Therefore, as a result, five peaks can be seen on the chromatogram. When polyethylene glycol is conjugated with a lysine residue located in the middle of epithelial growth factor as described above, trypsin is not specifically recognized. Will be formed.

As shown in FIG. 4, five peaks are evidence showing that the conjugation by polyethylene glycol occurs specifically at the N-terminus.

Test Example 4 Measurement of Cell Growth Rate (NRK49F) Using Radioisotope ( ³ H-thymidine)

Rat soybean cells (ATCC, NRK49F) were seeded in 24 well cell culture plates at a concentration of 5 × 10 ³ and then grown in a cell culture (5% CO ₂ environment). After 24 hours, the cells were replaced with serum-free medium and incubated for 12 hours, followed by no treatment with epidermal growth factor (PEG2k-EGF, PEG5k-EGF) conjugated with aldehyde polyethylene glycol having molecular weights of 2000 and 5000, respectively. Natural epithelial growth factor (NGF) was added to the solution contained in the medium at a concentration of 0.01 to 100 nM. After growing for 8 hours, radioisotope ( ³ H thymidine) was added again for 12 hours.

After bursting, the cells were treated with 20% trichloroacetic acid to form a precipitate, which was then transferred to nitrocellulose paper to measure the growth of cells by measuring the amount of ³ H thymidine contained in the DNA using a radioisotope counter (Beckman LS 6500). The measurement results are shown in FIG. 5.

In the case of natural epithelial growth factor, as shown in Figure 5, the cells began to grow at around 100 pM and the concentration of epithelial growth factor did not affect the growth of cells from about 10 nM. In the case of aldehyde polyethylene glycol attached (PEG2k-EGF, PEG5k-EGF), the activity of epidermal growth factor was about the same.

This result is a positive result indicating that when the polymer is attached to the epithelial growth factor at the N-terminus, there is almost no decrease in activity.

Test Example 5 Measurement of Phosphorylation of Epidermal Growth Factor Receptor and Signaling Molecule Using Specific Antibody

African green monkey kidney cells (acquired at ATCC, COS-7 cells) were inoculated with 2.5 X 10 ⁵ cells per well, and then cultured in a 37 ° C 5% CO ₂ incubator for one day, and then with serum-free medium. After replacement, the cells were incubated for one day. The epidermal growth factor and polyethylene glycol epithelial growth factor conjugate were treated with 1 ml of 0.1 mM Na ₃ VO ₄ for 10 minutes before treatment. The epithelial growth factor and polyethylene glycol epithelial growth factor conjugates were treated for 0.5 minutes, 1 minute, 2 minutes, and 5 minutes, and then washed with cold phosphate buffer solution at 4 ° C. To break the cells, 0.5ml Lysis buffer was added and sonicated for 30 seconds to completely destroy the cells.

Prior to SDS-PAGE electrophoresis, Bradford protein quantification was performed to ensure the same protein concentration in each sample in order to add the same amount of protein solution to each gel line. 10uL of protein was added to the gel. After electrophoresis at 15 mV for 3 hours, the protein was electrically transferred to nitrocellulose paper, followed by reaction for 2 hours using specific phosphate-tyrosine and phosphate-ERK antibodies as primary antibodies. After reacting for 2 hours using a secondary antibody peroxidase (peroxidase) to wash, the substrate was put in a flash to see the band.

Figure 6 shows the results of the above experiment.

In Figure 6, the first line is a natural epidermal growth factor (NGF), an aldehyde polyethylene glycol having a molecular weight of 2000 and an epidermal growth factor conjugate (PEG2k-EGF), an aldehyde polyethylene glycol having a molecular weight of 5000 and an epidermal growth factor. Phosphorylation of tyrosine residues of epidermal growth factor receptor by conjugate (PEG5k-EGF) is shown.

The intensity of phosphorylation of the conjugated epithelial growth factor and the original epidermal growth factor receptor on the receptor shows a change in concentration. It is shown that there is no difference in phosphorylation of the receptor by the epidermal growth factor. But most importantly, the epidermal growth factor is attached to the receptor and enters the cell, so whether the signal from the receptor goes to the underlying signaling protein. So the choice in the present invention is a transporter called ERK. ERK is also a protein involved in the signaling that is also phosphorylated by the phosphorylation of epidermal growth factor receptors. In particular, ERK is known to occur only when endocytosis of epidermal growth factor occurs into cells.

The second line of Figure 6 shows this. Phosphoric acid-ERK antibodies show that phosphorylation occurs in ERK at different concentrations. As a control experiment, the third row shows the total amount of ERK in cells. Comparing lines 2 and 3 shows that the amount of ERK in the cells remains unchanged, but phosphorylation occurs equally in the polyethylene glycol-epithelial growth factor conjugate and epithelial growth factor.

The last line in Figure 6 shows that the amount of protein loaded on acrylamide gel at the western blot using the antibody specific to actin.

As described above, the polyethylene glycol-epithelial growth factor conjugate and the epidermal growth factor show no difference in intracellular signaling, and furthermore, the endocytosis also occurs equally well in the conjugate. there was.

Experimental Example 6 Measurement of Biological Stability of Polyethyleneglycol-Epithelial Growth Factor Conjugate and Epidermal Growth Factor by Intravenous Injection of Rat (ICR)

A polyethylene glycol-epithelial growth factor conjugate and an epithelial growth factor conjugate (1.2 μg 0.1 ml) incorporating an aldehyde polyethylene glycol having a molecular weight of 2000 and 5000 and epithelial growth factor were injected into a 7-week-old male ICR mice vein. The blood was collected from the heart at 5 minutes to 4 hours), and then transferred to a tube to which heparin (2 μl) was added, followed by centrifugation at 12,000 rpm to obtain plasma.

The obtained plasma was diluted between 10 ² and 10 ⁵ to fall within the immunoassay range (0.6 to 20.7 pM), and then the amount of the polyethylene glycol-epithelial growth factor conjugate and the epidermal growth factor contained in the plasma was measured. The number of mice used for each hour was five.

7 shows the results for the tests conducted above. The epithelial growth factor conjugate conjugated with polyethylene glycol was shown to have excellent persistence in the mouse model. When the polyethylene glycol is not conjugated, the half-life is 6.71 minutes, whereas the aldehyde polyethylene glycol having a molecular weight of 2000 is 15.59 minutes when the single molecule is conjugated, and the aldehyde polyethylene glycol having a molecular weight of 5000 is conjugated to the epidermal growth factor. The half life of was calculated to be 24.36 minutes. In the case of aldehyde polyethylene glycol conjugated epithelial growth factor having a molecular weight of 5000, the sustained increase was about 4 times higher than that of the non-conjugated epithelial growth factor. It is due to the lower molecular weight of polyethylene glycol that the in vivo duration is not increased much more than other polyethylene glycol modified proteins.

Test Example 7 Measurement of Biological Stability of Epithelial Growth Factor and Polyethylene Glycol-Epithelial Growth Factor Conjugate Using Rat Kidney Tissue

1.5g of rat kidneys were obtained, washed with phosphate buffer solution, and then, in the presence of 10-fold diluted phosphate buffer solution, the kidney tissue was pulverized with a manual cell crusher, and the supernatant obtained by centrifugation at 12,000 rpm for 30 minutes was taken. Was quantified by the Bradford method (5.3 mg / ml).

2 ml of the obtained supernatant, 0.5 μg 20 μl of epithelial cell growth factor, polyethylene glycol having molecular weight of 2000 and 5000 and polyethylene glycol epithelial cell growth factor conjugate solution combined with epithelial cell growth factor were mixed and reacted at 37 ° C. for a predetermined time ( 10 ml, 30 minutes, 60 minutes, 120 minutes, 240 minutes, 550 minutes) was taken 0.1 ml each. The amount of remaining epidermal growth factor was quantified by an immunoenzyme reaction method and the results are shown in FIG. 8.

As shown in FIG. 8, the polyethylene glycol-epithelial growth factor conjugate, in which the aldehyde polyethylene glycol having a molecular weight of 2000 and 5000 and the epidermal growth factor, respectively, showed significantly improved resistance to proteolytic enzymes contained in biological tissues than the epidermal growth factor. And it was found.

As described in detail above, when the polyethylene glycol derivative used in the present invention is conjugated with a single molecule of polyethylene glycol at the N-terminus of the epidermal growth factor, the biological duration is long without any change in its biological efficacy. It can have the effect of increasing the losing effect and stability. As a result, the effects of the present invention on the preparation of polymer conjugates of epidermal growth factor with better wound healing effect.

Claims (12)

Aldehyde polyethylene glycol, succinimide propionate polyethylene glycol, succinimide butanoic acid polyethylene glycol, polyethylene glycol di-polyethylene glycol succinimide, succinimidyl succinate polyethylene glycol, benzotriazole carboxylic acid polyethylene glycol , Polyethylene glycol derivatives selected from epoxide polyethyleneglycol, carbonyl imidazole polyethyleneglycol, p-nitrophenyl carboxylic acid polyethyleneglycol, isocyanate polyethyleneglycol and a single molecule at the N-terminus of epidermal growth factor A conjugate of epithelial cell growth factor and polyethylene glycol optionally covalently bonded. delete The conjugate of epidermal growth factor and polyethylene glycol according to claim 1, wherein the polyethylene glycol derivative has a molecular weight of 500 to 50000. The epithelial growth factor and polyethylene according to claim 1, wherein the epidermal growth factor is a product recombination obtained by expression of the E. Coli, Hansenula polymorpha, or S. cerevisiae gene after cloning into a DNA vector. Conjugates of glycols. A di-polyethylene glycol succinate in which an epithelial growth factor having an N-terminal group was dissolved in a buffer solution and aldehyde polyethylene glycol, succinimide propionate polyethylene glycol, succinimide butyrate polyethylene glycol, and polyethylene glycol was bonded to two molecules. Mid, succinimidyl succinate polyethylene glycol, benzotriazole carboxylic acid polyethylene glycol, epoxide polyethylene glycol, carbonyl imidazole polyethylene glycol, any one selected from p-nitrophenyl carboxylic acid polyethylene glycol, isocyanate polyethylene glycol Adding a polyethylene glycol derivative of to a buffer solution in which epithelial growth factor having an N-terminal group is dissolved and reacting in the presence of a reducing agent; Method for producing a conjugate of epidermal growth factor and polyethylene glycol, characterized in that it comprises the step of removing the unreacted epithelial growth factor, polyethylene glycol and a reducing agent after the reaction. 6. The epidermal growth factor of claim 5, wherein the epidermal growth factor is a product recombination obtained after cloning of the E. Coli, Hanshenula polymorpha, or S. cerevisiae gene into a DNA vector or directly extracted from mammals other than humans. Method for producing a conjugate of epidermal growth factor and polyethylene glycol, characterized in that. 6. The conjugate of epidermal growth factor and polyethylene glycol according to claim 5, wherein the buffer solution is any one selected from morpholino ethanesulfonic acid, acetated sodium acid, citric acid, phosphate buffer solution and acetamido imadiacetic acid. Manufacturing method The method of claim 5, wherein the buffer solution is characterized in that the pH of 5 to 6 epithelial cell growth conjugate and polyethylene glycol conjugate method delete The method for preparing a conjugate of epidermal growth factor and polyethylene glycol according to claim 5, wherein the polyethylene glycol derivative has a molecular weight of 500 to 50000. The method of claim 5, wherein the reducing agent is one selected from sodium cyanohydrogen bromide, sodium cyanide, and catechol allele or a mixture of these homogeneously mixed. According to claim 5, wherein the epithelial growth factor and the polyethylene glycol derivative in a buffer solution with a reducing agent is prepared for the conjugate of the epithelial growth factor and polyethylene glycol, characterized in that for 2 to 100 hours to react at 4 ℃ to 25 ℃ Way

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