KR20090118879A - Human growth hormone derivative linked with polyethyleneglycol, method for the preparation thereof and pharmaceutical composition comprising the same - Google Patents

Abstract

PURPOSE: A PEG-human growth hormone complex is provided to minimize biological activation and last activation in a body. CONSTITUTION: A PEG-human growth hormone complex contains a human growth hormone and polyethyleneglycol(PEG) of 20 kDa-50 kDa. The PEG is methoxyPEG aldehyde(Mpeg-ALD). A pharmaceutical composition for treating dwarfism and growth hormone deficiency or diabetic ulcer contains PEG-human growth hormone complex.

Description

Human growth hormone derivative linked with polyethyleneglycol, method for the preparation sugar and pharmaceutical composition comprising the same}

The present invention relates to a conjugate obtained by chemically modifying human growth hormone (PEG), which is currently widely used as a therapeutic protein.

Human growth hormone is an unglycosylated peptide hormone secreted from the anterior pituitary gland, and is known to bind to receptors of various tissues of the human body to promote the secretion of other growth factors or to directly act to promote the growth of various parts of the human body. After the growth hormone from human hypothalamus was found to be effective in the treatment of pituitary dysplasia, the demand for human growth hormone increased explosively, but the amount of the growth hormone extracted from human pituitary gland was very limited. A child who received human growth hormone extracted from a dead human pituitary gland died of a Creutsfelt-Jacob Disease caused by a contaminated virus during growth hormone extraction and died in the US Food and Drug Administration (FDA). The use of purified hormones from the pituitary gland has been banned (Science, 234, 22 (1986)).

However, after human growth hormone synthesized in Escherichia coli by Genentech's genetic recombination technology was approved by the FDA as a rare drug, it was also used by Eli Lilly, Nordisk, Denmark, and others. This became commercial. Currently, it is used for the treatment of dwarfism caused by growth hormone deficiency and Turner syndrome, and the treatment of growth hormone deficiency and chronic renal failure (CRI) in adults, but it is also used for osteoporosis, wounds, burns and infertility. Clinical trials are underway and the number of diseases that apply human growth hormone will increase significantly. Human growth hormone must be administered to the patient three times a week or once daily to maintain proper serum levels. Frequent subcutaneous injections for patients chronically receiving human growth hormone may degrade the patient's quality of life and may cause complications such as upper body formation or differential organ growth (such as J. Clin.Invest, 0.93, 1163 (1994)).

Therefore, various studies have been conducted to increase the in vivo residence time while maintaining the biological activity of human growth hormone to solve the problems related to the repeated injection of human growth hormone. Among them, a study has been attempted to produce a sustained release composition which encapsulates human growth hormone in a biocompatible, biodegradable polymer matrix such as polylactide and polyglycolide and releases it in vivo over one week. Biodegradable polymer combinations and encapsulation studies were conducted, and Genentech sold it as a product called Nutropin Depot. However, these controlled release devices have the disadvantage of overdosing human growth hormones, and are often released in vivo initially and then less frequently, the concentration of human growth hormones in these controlled release devices When higher, hormone molecules aggregate after several days, reducing biological activity, causing problems of immunogen in vivo.

Meanwhile, a method of combining a protein such as PEG with a protein has been introduced to increase the in vivo retention period of protein pharmaceuticals including human growth hormone. Combining protein drugs with polymers can alter the surface properties of protein molecules to increase their solubility in water or organic solvents, increase biocompatibility, reduce immune responsiveness, increase in vivo safety, and intestinal system Clearance by the kidneys, spleen or liver can be made more slowly. However, if the molecular weight of the protein is small, it may be lost by filtration in the intestinal system or kidney, but can be prevented by binding a polymer such as PEG (J. Biol. Chem., 263, 15064 (1988)). PEG is a high-molecular compound having a structure of HO-(-CH2CH2O-) n-H. Since PEG is strongly hydrophilic, binding to a pharmaceutical protein can increase its solubility. It also increases the molecular weight of the bound protein while maintaining key biological functions such as enzymatic activity and receptor binding, thereby reducing kidney filtration, protecting the protein from cells and antibodies that recognize external antigens, and Degradation can also be reduced. In addition, the molecular weight range of PEG that can bind to protein is approximately 1,000 to 100,000, and when the molecular weight of PEG is 1,000 or more, toxicity is known to be relatively low. The molecular weight range of PEG to 1,000 to 6,000 is distributed throughout the body, and the kidney It is known to be metabolized, and in particular, PEG with a molecular weight of 40,000 is distributed in organs including blood and liver. Metabolism is known to occur in the liver.

In addition, medical and pharmacologically useful proteins administered through the parenteral route have been studied to overcome the disadvantages of having antigenicity, low water solubility, and short duration of life in the body. In US Pat. No. 4179337, therapeutic agents such as proteins and enzymes coupled with PEG are known which have the effect of reducing antigenicity, increasing water solubility, increasing retention time in the body, and the like. In addition, attempts have been made to overcome the shortcomings by combining bioactive proteins with PEG. For example, ribonuclease and superoxide dismutase have been combined with PEG (Veronese). et al., 1985). In addition, U.S. Patent No. 4766106 and U.S. Patent No. 4917888 increase the water solubility of the protein by binding a polymer containing PEG to the protein. In U.S. Patent No. 4902502, PEG or other polymers are recombined. Binding to proteins reduced antigenicity and increased duration of life in the body.

On the other hand, there are disadvantages to the PEG-protein binding in addition to the above advantages, PEG is usually covalently bonded to one or more free lysine (Lys) residues of the protein to be bound, wherein the surface portion of the protein When a site directly related to the activity of the heavy protein binds to PEG, the site is no longer able to perform biological functions, thereby reducing the activity of the protein. In addition, the binding of PEG and lysine residues usually occurs randomly, so that many kinds of PEG-protein conjugates exist as a mixture depending on the binding position, thereby making the process of pure separation of the desired combination complicated and difficult. do. As such, human growth hormone has ten free lysine residues on its surface, and interest in maintaining the biological activity of human growth hormone when PEG binds to any of these residues. In Genentech's paper (Clark et al., The Journal of Biological Chemistry, 271, 21969 (1996)) and International Publication No. WO 93/00109), PEG-modified forms of the immune response promoter of human growth hormone Various PEGylation methods are known, in which PEG is coupled to human growth hormone [P. McGoff et al., Chem. Pharm. Bull., 36, 3070 (1988); Chamow, S.M. et al., Bioconjugate Chem., 5, 133 (1994); Teh, L.C. et al., Biochem. Biophys. Res. Comm., 150, 391 (1988)] In this case, the PEG used had a molecular weight of 5,000 kDa, and the PEG bound to one molecule of human growth hormone was analyzed by cell proliferation assay using FDC-P1, a premyeloid cell line. It was confirmed that the activity decreased as the number of molecules increased.

U.S. Patent No. 5766897 and International Publication No. W000 / 42175 also disclose the use of PEG-maleimide to bind PEG with human growth hormone to avoid reacting with the active site of human growth hormone. PEG was allowed to react selectively with cysteine. However, when the PEG is used, there should be free cysteine residues that are not involved in disulfide bonds in human growth hormone, but all four cysteine residues in human growth hormone participate in disulfide bonds. The PEGylation reaction was performed using the human growth hormone variant inserted therein. However, this method has a problem in that it is difficult to obtain a raw material because the human growth hormone used in the PEGylation reaction is not a natural type and the process of expressing and purifying a protein in which free cysteine is present is usually not easy.

On the other hand, the process of chemically conjugating PEG to proteins and peptides to produce new active substances is a very important factor of the chemical structure of PEG. Most protein drugs show the effects of the drug through intracellular and extracellular signaling after binding to cellular receptors. As shown in FIG. 1, when binding to PEG, which has excellent water solubility on the surface of the protein and has a very high motion under an aqueous solution, receptor binding is very disturbed. Therefore, when PEG is conjugated to a protein, it can lead to a very serious deterioration of activity, requiring much consideration of the binding site and the molecular structure of the PEG to be bound.

Thereby, the present inventors have prepared a conjugate (mono-PEG-hGH) in which one PEG molecule is bonded to the amino terminus to a natural human growth hormone to overcome the above problems, and the linear, bi-branched, and Various forms such as triple branched form are introduced to derive the optimized form of PEG conjugates.

It is an object of the present invention to selectively bind a molecule of PEG to a specific site of human growth hormone, specifically, an amino terminus, thereby increasing the safety and retention time of human growth hormone while minimizing degradation of biological activity, thereby sustaining in vivo activity. To provide a PEG-human growth hormone conjugate.

It is also to provide a PEG-human growth hormone conjugate that is most optimized for activity and function in the PEG conjugate.

In accordance with this object, the present invention provides a PEG-human growth hormone conjugate comprising human growth hormone and polyethylene glycol (PEG) having a molecular weight of 20 kDa to 50 kDa.

The present invention also provides a method for producing a PEG-human growth hormone conjugate, characterized in that the human growth hormone and PEG having a molecular weight of 20 kDa to 50 kDa are reacted in the presence of a reducing agent.

The present invention also provides a pharmaceutical composition for treating dwarfism and growth hormone deficiency comprising the PEG-human growth hormone conjugate as an active ingredient.

The present invention also provides a pharmaceutical composition for treating diabetic ulcer disease, comprising the PEG-human growth hormone conjugate as an active ingredient.

In order for a human growth hormone to play its role in vivo, the human growth hormone must bind to a human growth hormone receptor present in the membrane of the target cell, and the binding of the human growth hormone to the receptor binds to two receptor binding sites in the human growth hormone. (Cunninghum et al., Proc. Natl. Acad. Sci. USA 88, 3401 (1991)). The amino terminus of human growth hormone is not known to affect binding to the receptor (Recent Progress In Hormone Research, 48, 253 (1992)).

Preferably, the PEG polymer molecules used in the present invention may be selected from water soluble PEG polymers, and typically the ε-amino group of the lysine residue, cysteine residue or histidine, depending on the activator of the PEG when chemically modifying the protein with the PEG polymer. Can bind to a residue. However, using PEG polymers with these activating groups, one or more PEG molecules bind to human growth hormone. PEG molecules bound to various sites may lose their activity by structurally interfering with the binding of human growth hormone and its receptor. Thus, in order to bind only one molecule of PEG to one molecule of human growth hormone, it must be aqueous so as not to precipitate in the physiological environment, and must have a single reaction aldehyde group to specifically bind to the α-amino group of the amino terminus of the human growth hormone. do. In addition, the molecular weight of the PEG polymer is not limited, but 23 kDa is most preferred.

Preferably, the molecular form of PEG may be variously selected from linear, bi-branched, triple-branched, and multi-branched, etc., and considering the molecular structure of PEG, the triple-branched form is considered to be the most preferable.

Preferably, the PEGylation reaction of human growth hormone with alkoxy-PEG-aldehyde can be carried out in the presence of a reducing agent. At this time, the alkoxy-PEG-aldehyde is methoxy-PEG-aldehyde and the reducing agent is NaCNBH ₃ . After the PEGylation reaction, the difference in apparent charge was compared by anion exchange chromatography to obtain a mono-PEG-human growth hormone conjugate to isolate the conjugate bound to the mono-PEG-human growth hormone at the amino terminus of human growth hormone. The separated peaks may be identified by mono-PEG-human growth hormone through SDS-PAGE.

Preferably, in the mono-PEG-human growth hormone conjugate, a peptide map is prepared to determine whether PEG specifically binds to the amino terminus of human growth hormone, thereby producing a peptide map of the unmodified human growth hormone and a known human growth hormone. The reaction status and PEG position were compared to the peak patterns of the peptide maps (Journal of chromatography A, 808, 121 (1998); Analytical Biochemistry, 122, 348 (1982); Biotechnology and Applied Biochemistry, 10, 326 (1988)). You can check it. In other words, when PEG is bound, the HPLC peak pattern of the protein cleaved by the protease is different from that when unmodified human growth hormone is cleaved with the same protease. The generated difference represents the peak pattern by PEG binding to any one of the protease cleavage, and the difference in the peak pattern can confirm whether the PEG binding and the binding position.

Preferably, the protease according to the present invention may be used trypsin, subtilisin or endoproteinase Glu-C, and trypsin is most preferred.

Preferably, the measurement of the biological activity of the mono-PEG-human growth hormone according to the present invention is known from Acta Endocrinologica., 119, 326 (1988); J. Biolo. Chem., 271 (36), 21969 (1996). J. endocrinol.Invest., 21, 768 (1998)), and have an NB2 cell line, and cell proliferation assay to measure the activity in vitro.

According to the present invention by the above-mentioned means for solving the problem, in vivo activity is sustained by selectively binding a molecule of PEG to the amino terminal to minimize biological activity degradation, increase the safety and human residence time of human growth hormone.

Hereinafter, the present invention will be described in detail with reference to the following examples, but the following examples are not intended to limit the scope of the present invention only by exemplifying the present invention in detail.

Example 1 Preparation of Human Growth Hormone Conjugates PEGylated at the Amino Terminals

After preparing the human growth hormone to 5 mg / mL in 100 mM potassium phosphate buffer solution (pH 6.0), the four activated methoxy with average molecular weight of 5, 10, 20 (double branch) and 23 kDa (triple branch) -PEG-aldehyde (Shearwater and NOF) was injected with the solution so that the molar ratio of human growth hormone: methoxy-PEG-aldehyde was 1: 2.5, 1: 5, 1: 7.5 and 1:10. To the reaction solution was added a reducing agent NaCNBH ₃ (Sigma, USA) to the final 20 mM, and then reacted at 4 ℃ for 4 hours.

As a pretreatment step, a HiPrep 26/10 Desalting column (GE Healthcare, Inc.) was used to terminate the reaction and remove the low molecular weight material. First, the column was equilibrated with 2CV (column volume) 20 mM Tris buffer (pH 7.5), and then the reaction mixture was dropped and detected with an ultraviolet photometer having a wavelength of 280 nm. On the other hand, the low molecular material removal method is as follows.

Elution with the same buffer solution allowed the removal of low molecular weight material because the larger PEG-modified human growth hormone was eluted first and then the unreacted material was later eluted.

Example 2: Isolation of Mono-PEG-Human Growth Hormone Conjugate at the Amino Terminal

In order to isolate and purify the human growth hormone modified with PEG from the elution solution obtained in Example 1, it was carried out as follows.

The HPLC instrument (600s, Waters) was equilibrated in an ion exchange column (10 × 100 mm, Source15Q handpacked) with 20 mM Tris-HCl (pH 7.5) buffer. The PEGylated reaction mixture was loaded on a column and then multi-PEG-human growth hormone, mono-PEG-human using an automatic concentration gradient method and a salt concentration gradient method from 0-100% for 20 minutes with 1M NaCl buffer. Eluted with growth hormone and unmodified human growth hormone.

Figure 2 shows the separation of mono-PEG-human growth hormone using ion exchange chromatography. The PEG conjugation reaction is selectively performed at the N-terminus of human growth hormone, but some PEGs are conjugated to a portion other than the N-terminus to produce one or more PEG-conjugated human growth hormones as by-products. As shown in Figure 2b, through SDS-PAGE it was confirmed that a ~ c is a multi-PEG-human growth hormone.

Figure 3 shows the ion exchange chromatography results of PEG (molecular weight 5kDa, 10kDa, 20kDa and 23kDa) and human growth hormone binding reactants. As shown in FIG. 3, it was confirmed that PEG-human growth hormone was eluted with increasing molecular weight of PEG under low NaCl concentration, which is considered to be due to high fluidity under aqueous solution due to PEG molecules. In particular, 23 kDa PEG was observed to have very high flowability compared to 20 kDa PEG.

Figure 4a shows the SDS-PAGE gel results of PEG 5kDa-human growth hormone conjugate, Figure 4b is to find the most optimized reaction rate, the yield of mono-PEG-human growth hormone according to the ratio of PEG 5kDa The graph shown. As shown in Figure 4a, as the ratio of PEG / hGH increased it was confirmed that more mono-PEG-human growth hormone is produced. On the other hand, it was found that the increase of the reaction ratio leads to the increase of the side product, and therefore, the optimization of the reaction ratio is necessary. In the case of PEG 5 kDa, the reaction ratio of 5 to 10 was optimized through this experiment.

Figure 5a shows the SDS-PAGE gel results of PEG 10kDa-human growth hormone conjugate, Figure 5b is a graph showing the yield of mono-PEG-human growth hormone according to the ratio of PEG 10kDa. In the case of conjugation of PEG 10kDa to human growth hormone, the reaction ratio of about 5 to 10 was the same as the PEG 5 kDa.

Figure 6a shows the SDS-PAGE gel results of PEG 20kDa-human growth hormone conjugate, Figure 6b is a graph showing the yield of mono-PEG-human growth hormone according to the ratio of PEG 20kDa.

Figure 7a shows the SDS-PAGE gel results of PEG 23kDa-human growth hormone conjugate, Figure 7b is a graph showing the yield of mono-PEG-human growth hormone according to the ratio of PEG 23kDa. The difference in reactivity between the low molecular weight PEG and the high molecular weight PEG was not confirmed, and the reaction ratio of 5 to 10 was found to be an optimization ratio.

Example 3: Analysis of Isolated Mono-PEG-Human Growth Hormone Conjugates

In order to confirm the properties of the mono-PEG-human growth hormone conjugate prepared through Example 2, the following experiment was carried out.

<3-1> Gel Permeation Chromatography

Mono-PEG-human growth hormone was analyzed using size exclusion chromatography. As shown in Figure 8a, it was confirmed that the smaller the elution volume as the molecular weight of the conjugated PEG increases.

<3-2> SDS Protein Electrophoresis Experiment (SDS-PAGE)

Mono-PEG-human growth hormone was analyzed via electrophoresis (SDS-PAGE), where 12% gel was used. As shown in Figure 8b, as the molecular weight of the PEG increased the gel phase movement was shortened, especially in the case of the 23 kDa PEG conjugate gel size is similar to the 75 kDa standard, the increase in size is very good I could confirm it.

<3-3> Peptide Mapping

Mono-PEG-human growth hormone was identified by PEG mapping via PEG mapping. The peaks of the peptide maps of human growth hormone have been referred to known literature ( Journal of Chromatography A , 808, 121 (1998); Analytical Biochemistry , 122, 348 (1982); and Biotechnology and Applied Biochemistry , 10, 326 (1988)). .

Mono-PEG-human growth hormone and unmodified human growth hormone were prepared to have a concentration of 1.5 mg / ml in 0.5M Tris-HCl (pH7.5) buffer solution. 22.5 ul of 1 mg / ml trypsin (Roche, Switzerland) in the above buffer was mixed with 1 ml of each growth hormone solution, and reacted for 4 hours in a 37 ° C incubator to cleave each protein. Protein cleavage was separated using a C18 HPLC column, using acetonitrile linear gradient in 0.1% trifluoroacetic acid as a separation solvent.

As shown in FIG. 9, in the case of the unmodified human growth hormone, the N-terminal trypsin digest was eluted at about 27 minutes, but in the case of mono-PEG-human growth hormone, the elution peak disappeared at the same time. This confirmed that the PEG molecule is conjugated to the N-terminus of human growth hormone. In addition, it was confirmed that the PEG-conjugated human growth hormone N-terminal degradation product was eluted within about 57 to 61 minutes, and the elution time was increased as the molecular weight of PEG increased.

Example 4: In Vitro Bioactivities

In vitro activity measurement of amino-terminated mono-PEG-human growth hormone was assessed in rat node lymphoma cell line NB2 cells (ECACC, European Collection of Cell Cultures, ECACC # 97041101), which are human growth hormone-dependent mitosis cells. In vitro analysis was performed using Fischer's medium was incubated with 10% fetal bovine serum (FBS), 0.075% NaCO ₃ , 0.05 mM 2-mercaptoethanol and 2 mM glutamine in Fisher's medium and then 10% for analysis. 1% FBS culture was used in FBS. After injecting about 20,000 cells per well of a 96 well plate, each of the amino terminal mono-PEGylated human growth hormone and the unmodified human growth hormone (Native hGH) isolated in the above example was diluted and added in different concentrations. After incubation for 24 hours at 37 ℃, CO ₂ incubator. Subsequently, to measure the growth of the cells, 10 μL of cell dye (Cell titer 96 Aqueous One Solution, Promega, USA) was added to each well, followed by incubation for 2 hours, and then the absorbance of each sample was measured by measuring 590 nm absorbance. Calculated.

As shown in FIG. 10, as the concentration of the drug was increased, the proliferation of cells was induced, indicating that a greater number of cells were present in the incubator. Based on the results, the value of EC50 was calculated. For each calculated EC50 value, human growth hormone 0.36 ng / ml, PEG5k-human growth hormone 0.76 ng / ml, PEG10k-human growth hormone 0.85 ng / ml, PEG20k-human growth hormone 1.63 ng / ml and PEG23k-human growth hormone 0.99 ng / ml was confirmed.

In addition, PEG23k-human growth hormone was found to increase the titer even if the molecular weight is increased through comparison with PEG20k-human growth hormone. As described above, it may be determined that the defect with the receptor of the cell is easier because the molecular structure of PEG conjugated to the human growth hormone is triple branched.

Example 5: Pharmacokinetics in vivo

For pharmacokinetic testing, a 6-week-old subcutaneous subcutaneous amino acid PEG-human growth hormone and each mono PEG-human growth hormone conjugate was injected into a 26G syringe (1 mL, Ltd.) for pharmacokinetic testing. Korean vaccine), 100 μg / kg rat, 200 mL was administered once for all groups.

As shown in FIGS. 11A and 11B, comparing the unmodified hGH and mono PEG-human growth hormone conjugates, the unmodified hGH has a peak blood concentration (Cmax) about 8 times higher than the mono PEG-human growth hormone conjugate, but has a half-life in the body. Half-life and mean residence time (MRT) were lost within a short period of time, 1 hour and 1.6 hours. In addition, when comparing 20 kDa and 23 kDa mono-PEG-hGH, it can be seen that the half-life and average residence time of 23 kDa mono-PEG-hGH increased. Therefore, it can be seen that administration of mono-PEG-hGH, in particular 23 kDa mono-PEG-hGH, can increase the duration of the body than unmodified hGH, which can dramatically change the economic and therapeutic aspects of protein treatment.

Example 6: In Vivo Activity Measurement

In order to measure the in vivo activity of the mono-PEG-human growth hormone conjugate, the following experiment was performed using a 5-week old male rat (Hypsectomized Sprague Dawley rat, SLC, Japan).

<6-1> Weight increase and decrease measurement

Weight gain tests were performed on 5 week-old male rats whose pituitary glands were removed. Solvent control and unmodified hGH, mono-20 kDa, 23 kDa PEG-hGH conjugates were administered subcutaneously in rats, and the concentrations and doses are shown in Table 1.

group Medication Dosage Dosing method One PBS (Control) 500 ul Once / week, 2 weeks 2 hGH (Daily) 30 μg / time, 200 ul Once / day, 2 weeks 3 hGH (Bolus) 210 μg / time, 500 ul Once / week, 2 weeks 4 20 kDa PEG-hGH 210 μg / time, 500 ul Once / week, 2 weeks 5 23 kDa PEG-hGH 630 μg / time, 500 ul Once / week, 2 weeks

12 shows the results of weight increase and decrease after administration of each sample. The unmodified human growth hormone used as a standard, even if a week (7 days) dose is injected once, the weight gain effect lasts for about a day, then rapidly decreases, and returns to the state before the injection after 7 days. Later, after the second drug was injected, there was a similar tendency and there was no continuous activity. However, when administered once daily, an increase in body weight was observed, confirming that unmodified human growth hormone should be administered daily in order to have continuous in vivo activity.

On the other hand, when mono 20 kDa and 23 kDa PEG-hGH were administered once, they maintained the same activity as the group that received unmodified hGH daily for 4 and 7 days after administration, in particular, 23 kDa PEG-hGH was observed after 7 days. After the second infusion, the activity was maintained up to two weeks better than the group that received daily unmodified hGH.

Thus, mono 20 kDa and 23 kDa PEG-hGH has superior sustained activity compared to unmodified hGH, it was confirmed that the sustained activity of 23 kDa PEG-hGH increased than 20 kDa. Through this, it is possible to solve the therapeutic limitation of the unmodified hGH which is currently used only once daily / injection type.

<6-2> growth plate increase and decrease measurement

As shown in FIGS. 13A and 13B, rats removed from the pituitary gland after drug administration for 2 weeks in Example <6-1> were opened, the growth plates were extracted, images of geological sections, and the results were statistically analyzed. The growth plate cross-section of rats injected with unmodified hGH showed the same growth plate thickness as the control group injected with no drug, indicating that no growth was achieved. The growth plate thickness of rats injected with unmodified hGH daily was About 1.5 times thicker than the control group was confirmed.

However, 20 kDa PEG-hGH improved the thickness of the growth plate from the first injection, especially 23 kDa PEG-hGH showed a statistically significant improvement of the thickness than the rat injected with the daily unmodified hGH.

Therefore, it was confirmed that the mono-PEG-human growth hormone conjugate is substantially effective in improving the thickness of the growth plate tissue as well as improving the weight.

<6-3> Body weight increase and decrease of mono-PEG-human growth hormone conjugate according to the dose

As shown in Examples <6-1> and <6-2>, 5-week-old male rats whose pituitary glands were removed to measure in vivo activity according to the high dose of 23 kDa PEG-hGH The weight increase and decrease was measured. At the dosages and doses shown in Table 2 below, solvent controls, unmodified human growth hormone, and 23 kDa PEG-hGH conjugates were administered in 26G syringes subcutaneously in rats.

group Medication Dosage Dosing method One PBS (Control) 500 ul Once / week, 2 weeks 2 hGH (Daily) 30 μg / time, 200 ul Once / day, 2 weeks 3 23 kDa PEG-hGH 70 μg / time, 500 ul Once / week, 2 weeks 4 23 kDa PEG-hGH 210 μg / time, 500 ul Once / week, 2 weeks 5 23 kDa PEG-hGH 630 μg / time, 500 ul Once / week, 2 weeks

As shown in FIG. 14, the same unchanged hGH as in Example <6-1> showed a continuous increase in body weight after daily injection, and the increase in weight of 23 kDa PEG-hGH at various doses was increased in rats. I also gained weight.

However, 210 ug and 630 ug administered 23 kDa PEG-hGH was found to remain at the same body weight as the group that received the unmodified hGH daily after 7 days, and injected once a week (7 days) in a similar in vivo Since the activity is shown, it was confirmed that an excessive injection amount was not required.

In addition, when comparing Examples <6-1> (FIG. 12) and <6-3> (FIG. 14), a similar weight gain tendency was observed in a 210 ug group administered with 20 kDa PEG-hGH and a 70 ug group administered with 23 kDa PEG-hGH. It was found that the 23kDa PEG-hGH showed an enhanced body activity of about three times.

1 relates to a process for separating mono-PEG-hGH from the result obtained after conjugation of hGH and PEG.

FIG. 2A is a schematic diagram of the chromatogram of ion exchange chromatography of a binder modified by molar ratio of 5 kDa PEG to hGH (hGH: PEG = 1: 2.5, 1: 5, 1: 7.5 and 1:10).

FIG. 2B is a photograph of SDS-polyacrylamide gel of peaks a to f of FIG. 2A, with lanes a to c as multi-PEG-hGH, lane d as mono-PEG-hGH, lane e as hGH unreacted and lane f, respectively. Relates to unreacted hGH aggregates.

Figure 3 relates to ion exchange chromatography of PEG-hGH binding reactants with PEG modified with 5 kDa, 10 kDa, 20 kDa and 23 kDa in hGH.

FIG. 4A relates to SDS-polyacrylamide gel results of conjugates modified with the ratio of PEG 5 kDa to hGH (1: 2.5, 1: 5, 1: 7.5 and 1:10).

FIG. 4B shows unreacted hGH, other product (Side Product) and PEG5k-hGH conjugates of modified conjugates with PEG 5 kDa ratios (1: 2.5, 1: 5, 1: 7.5 and 1:10) in hGH It shows the yield of and relates to the optimal reaction rate.

Figure 5a relates to a SDS-polyacrylamide gel picture of the conjugate modified by the ratio of PEG 10 kDa to hGH.

Figure 5b shows the yield of unreacted materials, other substances and PEG5k-hGH conjugate of the conjugate modified by the ratio of PEG 10 kDa to hGH in relation to the optimum reaction ratio.

FIG. 6A relates to a SDS-polyacrylamide gel photograph of a conjugate in which the ratio of 20 kDa PEG to hGH is modified.

Figure 6b shows the yield of unreacted, other substances and PEG5k-hGH conjugate of the conjugate modified by the ratio of PEG 20 kDa to hGH in relation to the optimum reaction ratio.

FIG. 7A relates to a SDS-polyacrylamide gel photograph of a conjugate in which the ratio of tri-branched PEG 23 kDa to hGH is modified in proportion.

Figure 7b shows the yield of unreacted, other substances and PEG5k-hGH conjugate of the conjugate modified by the ratio of PEG 23 kDa to hGH relative to the optimum reaction ratio.

FIG. 8A is a chromatogram of unmodified hGH and size exclusion chromatography (GPC, Gel Permi Chromatography) of mono-PEG-hGH in PEG-hGH conjugates modified with 5 kDa, 10 kDa, 20 kDa and 23 kDa PEG It is shown.

FIG. 8B relates to the SDS-polyacrylamide gel photograph of mono-PEG-hGH finally isolated in unmodified hGH and PEG-hGH conjugate modified PEG at 5 kDa, 10 kDa, 20 kDa and 23 kDa.

9 is a peptide mapping of unmodified hGH and unmodified hGH with 5 kDa, 10 kDa, 20 kDa, 23 kDa mono-PEG-hGH.

FIG. 10 shows the in vitro activity of unmodified hGH, 5 kDa, 10 kDa, 20 kDa, 23 kDa mono-PEG-hGH administered to NB2 cells, and cell growth according to concentration.

11a shows the highest blood concentration (Cmax), highest blood drug concentration time (Tmax), and blood drug concentration curve area of hGH with unmodified hGH, 20 kDa, 23 kDa mono-PEG-hGH administered once to SD rats. (AUC), half-life, loss (CL / F), mean residence time (MRT), and in vivo pharmacokinetic measurements.

FIG. 11B shows the pharmacokinetic characteristics of hGH over time as unmodified hGH, 20 kDa, and 23 kDa mono-PEG-hGH were administered to SD rats.

Figure 12 relates to weight gain over time by administering a solvent control, unmodified hGH, 20 kDA, 23 kDa mono-PEG-hGH to rats without pituitary gland, and unmodified hGH was injected daily with Bolus injection 20 kDA, 23 kDa mono-PEG-hGH is measured in vivo for two injections.

FIG. 13A shows a bone skeletal cross section of the rat after 2 weeks of administration of a solvent control group, unmodified hGH, 20 kDA, and 23 kDa mono-PEG-hGH to the pituitary gland from which rats were measured, and measured in vivo.

Figure 13b is a statistical representation of the bone skeletal cross-section of the rat 2 weeks after the administration of the control group, unmodified hGH, 20 kDA, 23 kDa mono-PEG-hGH in the pituitary gland removed rats.

Figure 14 relates to weight gain over time by administering a solvent control, unmodified hGH and various doses of 23 kDa mono-PEG-hGH to rats without pituitary gland, where unmodified hGH was injected daily and 23 kDa Mono-PEG-hGH is measured in vivo for two injections.

Figure 15 shows the problem of binding along the molecular structure and binding site of PEG in protein-PEG binding.

Claims (17)

A PEG-human growth hormone conjugate comprising human growth hormone and polyethylene glycol (PEG) having a molecular weight of 20 kDa to 50 kDa. The method of claim 1, PEG is a PEG-human growth hormone conjugate, characterized in that methoxypolyethylene glycol aldehyde (methoxyPEG aldehyde, mPEG-ALD). The method of claim 1, PEG-human growth hormone conjugate, characterized in that the PEG is in multiple forms. The method of claim 1, PEG-human growth hormone conjugate, characterized in that the PEG is in three forms. The method of claim 1, PEG-human growth hormone conjugate, wherein PEG has a molecular weight of 23 kDa. The method of claim 1, PEG-human growth hormone conjugate, characterized in that PEG is mono PEG. A method for producing a PEG-human growth hormone conjugate, characterized by reacting human growth hormone and PEG having a molecular weight of 20 kDa to 50 kDa in the presence of a reducing agent. The method of claim 7, wherein PEG is a methoxy polyethylene glycol aldehyde (methoxyPEG aldehyde, mPEG-ALD), characterized in that the PEG-human growth hormone conjugate. The method of claim 7, wherein Method for producing a PEG-human growth hormone conjugate, characterized in that the PEG in multiple forms. The method of claim 7, wherein Method for producing a PEG-human growth hormone conjugate, characterized in that the PEG is in three forms. The method of claim 7, wherein A process for preparing a PEG-human growth hormone conjugate, wherein PEG has a molecular weight of 23 kDa. The method of claim 7, wherein Method for producing a PEG-human growth hormone conjugate, characterized in that the PEG is a mono PEG. The method of claim 7, wherein Method for producing PEG-human growth hormone, characterized in that the reaction molar ratio of PEG-human growth hormone is 2 to 20. The method of claim 7, wherein A method of producing a PEG-human growth hormone conjugate, characterized in that the reducing agent is NaCNBH ₃ . The method of claim 7, wherein Method for producing a PEG-human growth hormone conjugate, characterized in that the reaction mixture of PEG-human growth hormone is reacted for 1 to 24 hours in a buffer solution of pH 5.0 ~ 7.0. A pharmaceutical composition for treating dwarfism and growth hormone deficiency, comprising any one of claims 1 to 6 as an active ingredient. A pharmaceutical composition for treating diabetic ulcer disease, comprising any one of claims 1 to 6 as an active ingredient.

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