Hyaluronidase polypeptides and uses thereof

Abstract

The invention relates to hyaluronidase polypeptides and uses thereof.

Description

Technical Field

[0001] The present invention relates to hyaluronidase polypeptides and uses thereof. Background Art

[0002] Hyaluronidase is a general term for the enzyme family that catalyzes the degradation of hyaluronic acid. Initially, Duran-Reynals introduced hyaluronidase as a diffusion factor. Later, it has been called hyaluronidase because it was observed to show potent activity on hyaluronic acid. According to its enzymatic mechanism, the enzyme is divided into the following three types: hyaluronan 4-glycan hydrolase (EC 3.2.1.35) distributed in testis, lysosome and bee venom, hyaluronan 3-glycan hydrolase (EC3.2.1.36) found in extracts and hyaluronan lyase (EC 4.2.2.1) found in bacteria.

[0003] In particular, hyaluronidase (PH-20) in the testis is a glycosylphosphatidylinositol (GPI) anchoring enzyme present on the acrosomal portion of sperm and is an important enzyme that causes fertilization by degrading the thick outer wall of the egg. In addition, PH-20 is known to cleave the β (1-4) bond between D-glucuronic acid and N-acetyl-D-glucosamine found in hyaluronic acid, chondroitin, and chondroitin sulfate in glycosaminoglycans distributed in mammalian skin. The general molecular formula of these enzymes is C 2455 H 3775 N 617 O 704 S 21 The molecular weight is 53870.9 g / mol. In the human body, there are six genes related to enzymes including HYAL1, HYAL2, HYAL3 and PH-20 / SPAM1.

[0004] Since the 1950s, the widespread use of hyaluronidase has been comprehensively reviewed. The first application is the subcutaneous injection of parenteral fluids. In addition, the enzyme has been used in the fields of orthopedics, ophthalmology, plastic surgery, dentistry, oral surgery, gynecology and otolaryngology, such as for infiltration and blocking anesthesia to increase the diffusion of local anesthetics and steroids, the dispersion of collected body fluids (e.g., hematomas), the prevention of peritoneal adhesions and stone formation, and the treatment of infertility.

[0005] The currently available hyaluronidase on the market is extracted from sheep or bovine testicles, as exemplified by Vitrase (ISTA Pharmaceuticals, sheep source) and Amphadase (Amphastar Pharmaceuticals, bovine source). It is commercialized by loading untreated hyaluronidase of suitable concentration into vials and then freeze-drying. The commercial products of animal-derived hyaluronidase leave many problems when applied to different fields, because the foreign proteins contained therein can cause allergic reactions and their biological activity is reduced due to the reduction of stability over time.

[0006] To overcome such problems, recombinant hyaluronidase has been studied. Recombinant proteins can be expressed in different types of cells including Escherichia coli, yeast, insect cells, animal cells, etc. Especially for hyaluronidase, its activity is affected by the glycosylation produced during the post-translational modification process. This is because polysaccharides may affect the antigenicity, structural folding, solubility and stability of glycoproteins. According to this view, among different types of expression cells, animal cells are suitable, among which the Chinese hamster ovary (CHO) cells that have been guaranteed to be safe are most preferred, because the yeast or insect cells where glycosylation occurs are different from mammalian cells in the post-translational modification process.

[0007] The first recombinant hyaluronidase of PH-20 was developed by Halozyme Therapeutic and has been sold under the trade name Hylenex, and is being developed for different uses such as subcutaneous injection, vitrectomy and eye diseases. However, hyaluronidase is still poor in yield or stability and the supply is insufficient compared to demand, so there is a need for hyaluronidase with improved yield or stability. Summary of the invention

[0008] Technical issues

[0009] One aspect of the present invention is to provide hyaluronidase polypeptides having excellent stability and improved activity.

[0010] Another aspect of the present invention is to provide a composition for topical administration, comprising a polypeptide according to one aspect of the present invention.

[0011] Another aspect of the present invention is to provide a drug delivery carrier comprising the polypeptide according to one aspect of the present invention.

[0012] Still another aspect of the present invention is to provide a composition for preventing or treating edema, comprising the polypeptide according to one aspect of the present invention.

[0013] Technical Solution

[0014] One aspect of the present invention relates to a polypeptide wherein the C-terminal region is deleted from the amino acid sequence of a wild-type hyaluronidase.

[0015] Another aspect of the present invention relates to a polypeptide comprising at least one continuous amino acid stretch of 1 to 203 amino acids deleted from the C-terminus of the amino acid sequence of a wild-type hyaluronidase.

[0016] Yet another aspect of the present invention relates to a composition for topical administration, comprising a polypeptide.

[0017] Yet another aspect of the present invention relates to a drug delivery carrier comprising a polypeptide.

[0018] Still another aspect of the present invention relates to a composition for preventing or treating edema, comprising a polypeptide.

[0019] Hereinafter, a detailed description of the present invention will be given.

[0020] One aspect of the present invention relates to a polypeptide having 90% or higher sequence homology with a polypeptide whose C-terminal region is deleted from the amino acid sequence of a wild-type hyaluronidase. The wild-type hyaluronidase may be a polypeptide consisting of the amino acid sequence of SEQ ID NO:1.

[0021] The polypeptide according to one aspect of the present invention has hyaluronidase activity and may have at least one of the following characteristics (1) to (6):

[0022] (1) stable at pH 3 to pH 10, specifically, the enzyme activity is 57% or more of the initial activity after storage for 4 weeks at a pH in the range between higher than 3 and 10 or lower, or the enzyme activity is 32% or more of the initial activity after storage for 4 weeks at a pH of pH 3 to lower than pH 5,

[0023] (2) stable at a temperature of -20°C to 45°C, specifically, the enzyme activity is 63% or more of the initial activity after storage at a temperature below 0°C for 4 weeks, or 83% or more of the initial activity after storage at a temperature of 0°C to 40°C for 4 weeks, or 52% or more of the initial activity after storage at a temperature of 40°C or higher for 4 weeks,

[0024] (3) high hyaluronidase activity, specifically, the hyaluronidase activity is more than 1 to 3 times that of wild-type hyaluronidase,

[0025] (4) having a potency that is 1 to 3 times greater than the potency of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1,

[0026] (5) having an activity of 120,000 to 150,000 IU / mg, and

[0027] (6) One or more amino acid residues are glycosylated.

[0028] In detail, in an example of the present invention, a polypeptide is prepared by deleting the C-terminal region of the amino acid sequence of a sheep-derived wild-type hyaluronidase (CAS No. 488712-31-8). It is observed that the stability and activity of the polypeptide thus obtained are significantly higher than those of the wild-type hyaluronidase, and even when used in smaller amounts, the polypeptide exhibits the equivalent efficacy of drug absorption, drug diffusion promotion efficacy, and body fluid reabsorption efficacy compared to the polypeptide of the wild-type hyaluronidase. Therefore, a polypeptide according to one embodiment of the present invention may be a hyaluronidase.

[0029] In detail, the polypeptide according to one embodiment of the present invention may have 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more or 99.9% or more sequence homology with a polypeptide comprising a continuous amino acid sequence of at least one 1 to 203 amino acids missing from the C-terminus of the amino acid sequence of a wild-type hyaluronidase. In this regard, the polypeptide according to one embodiment of the present invention is not a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. In addition, the polypeptide according to one embodiment of the present invention may retain the catalytic activity of a wild-type hyaluronidase. Specifically, the activity and / or stability of the polypeptide according to one embodiment of the present invention may be equal to or greater than the activity and / or stability of a wild-type hyaluronidase. A wild-type hyaluronidase may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0030] Specifically, the polypeptide according to one embodiment of the present invention may be a C-terminal region deletion variant, in which n amino acids from the C-terminus of the amino acid sequence of the wild-type hyaluronidase are deleted (wherein n is a natural number from 1 to 203).

[0031] For example, the polypeptide according to one embodiment of the present invention may delete at least one segment of 1 to 203, 1 to 170, 1 to 136, 1 to 102, 1 to 68, 34 to 203, 34 to 170, 34 to 136, 34 to 102, 34 to 68, 68 to 203, 68 to 170, 68 to 136, 68 to 102, 34, 68, 102, 136 or 170 amino acids from the C-terminus of the amino acid sequence of the wild-type hyaluronidase. The polypeptide according to one embodiment of the present invention may further delete the first amino acid from the N-terminus of the amino acid sequence of the wild-type hyaluronidase. The amino acid sequence of the wild-type hyaluronidase may be the amino acid sequence of SEQ ID NO: 1.

[0032] For example, the polypeptide according to one embodiment of the present invention may consist of an amino acid sequence extending from the first amino acid at the N-terminus to the m-th amino acid or from the second amino acid to the m-th amino acid of the amino acid sequence of the wild-type hyaluronidase (wherein m is a natural number from 315 to 517). The amino acid sequence of the wild-type hyaluronidase may be the amino acid sequence of SEQ ID NO: 1.

[0033] For example, the polypeptide according to one embodiment of the present invention may consist of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0034] For example, the polypeptide according to one embodiment of the present invention can be expressed using animal cells as hosts. In this regard, the polypeptide according to one embodiment of the present invention can be expressed in animal cells as hosts, and glycosylated by a post-translational modification (PTM) process during expression.

[0035] The polypeptide according to one embodiment of the present invention may be stable at pH 3 to 10. Specifically, the enzymatic activity of the polypeptide according to one embodiment of the present invention may be reduced to a lesser extent when stored at a pH of 3 to 10 compared to a wild-type hyaluronidase.

[0036] In the examples of the present invention, it was observed that the polypeptide according to one embodiment of the present invention retained enzymatic activity after storage at a pH of 3 to 10 for four weeks.

[0037] For example, after storage for 4 weeks at a pH between pH 3 and below pH 5, or at a pH in the range of pH 3 to 4 (e.g., pH 3), a polypeptide according to one embodiment of the present invention may have an enzyme activity totaling 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, or 55% or more of its initial activity. In this case, the polypeptide can be stored at a temperature of 5° C. or 37° C.

[0038] For example, after storage for 4 weeks at a pH (e.g., pH 5, pH 7, or pH 10) between higher than pH 3 and pH 10 or lower, pH 4 to 10, or pH 5 to 10, the polypeptide according to one embodiment of the present invention may have an enzyme activity amounting to 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, or 65% or more of its initial activity. In this case, the polypeptide can be stored at a temperature of 5° C. or 37° C.

[0039] For example, a pH in the range of between above pH 3 and below pH 10, between above pH 3 and pH 9 or below, between above pH 3 and pH 8 or below, between above pH 3 and pH 7 or below, between pH 4 or above and below pH 10, pH 4 to 9, pH 4 to 8, pH 4 to 7, between pH 5 and below pH 10, pH 5 to 9, pH 5 to 8, pH 5 to 7 (e.g., pH 5 or pH 7). 7) after 4 weeks of storage at room temperature. The polypeptide according to one embodiment of the present invention may have 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more or 82% or more of its initial activity. In this case, the polypeptide can be stored at a temperature of 5°C or 37°C.

[0040] The polypeptide according to one embodiment of the present invention can be stable under freezing, refrigeration and high temperature conditions. In detail, when stored under freezing, refrigeration and high temperature conditions, the degree of reduction of the enzymatic activity of the polypeptide according to one embodiment of the present invention is lower than that of the wild-type hyaluronidase.

[0041] In examples of the present invention, it was observed that the polypeptide according to one embodiment of the present invention retained enzymatic activity after storage under frozen (e.g., -18 to -20°C), refrigerated (e.g., 2 to 8°C) and high temperature (e.g., 40 to 45°C) conditions for 4 weeks.

[0042] For example, after storage at a frozen temperature, for example, at a temperature of less than 0°C, -20 to less than 0°C, -20 to -10°C or -20 to -18°C (for example, at a temperature of -20°C) for 4 weeks, the polypeptide according to one embodiment of the present invention may have a total of 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more of the initial activity. In some embodiments, the polypeptide may be stored at a pH of 5 or 7. In some embodiments, the polypeptide may be stored at a pH of 5 or 7. In some embodiments, the polypeptide may be stored at a pH of 5 or 7.

[0043] For example, at refrigerated temperature, for example, 0 to 40 ℃, 0 to 10 ℃ or 2 to 8 ℃ of temperature (for example, 5 ℃) after storage for 4 weeks, the polypeptide according to one embodiment of the present invention can have 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more of the initial activity. In this regard, the polypeptide can be stored at pH 5 or 7.

[0044] For example, at high temperature, for example, 40 ℃ or higher, 40 to 50 ℃ or 40 to 45 ℃ (for example, 40 ℃) temperature after storage for 4 weeks, the polypeptide according to one embodiment of the present invention can have 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more enzyme activity of the initial activity. In this regard, the polypeptide can be stored at a pH of 5 or 7.

[0045] A polypeptide according to one embodiment of the present invention may have a titer higher than the titer of a wild-type hyaluronidase. For example, a polypeptide according to one embodiment of the present invention may have a titer higher than 1 to 3 times, higher than 1 to 2.5 times, higher than 1 to 2 times, higher than 1 to 1.9 times, higher than 1 to 1.8 times, higher than 1 to 1.7 times, 1.1 to 3 times, 1.1 to 2.5 times, 1.1 to 2 times, 1.1 to 1.9 times, 1.1 to 1.8 times, 1.1 to 1.7 times, 1.2 to 3 times, 1.2 to 2.5 times, 1.2 to 2 times, 1.2 to 1.9 times, 1.2 ...8 times, 1.1 to 1.7 times, 1.2 to 3 times, 1.2 to 2.5 times, 1.2 to 2 times, 1.2 to 1.9 times, 1.2 to 1.8 times, 1.1 to 1.8 times, 1.1 to 1.7 times, 1.2 or 1.5 to 1.7 times the titer.

[0046] For example, a polypeptide according to one embodiment of the present invention may have an IU / mg of 120,000 to 150,000, 120,000 to 145,000, 120,000 to 140,000, 120,000 to 135,000, 121,000 to 150,000, 121,000 to 145,000, IU / mg, 121,000 to 140,000 IU / mg, 121,000 to 135,000 IU / mg, 122,000 to 150,000 IU / mg, 122,000 to 145,000 IU / mg, 122,000 to 140,000 IU / mg, 122,000 to 135,000 IU / mg.

[0047] Another aspect of the present invention relates to a nucleic acid molecule encoding a polypeptide according to an embodiment of the present invention, a vector comprising the nucleic acid molecule, and a cell comprising the vector. The cell can be selected from the group consisting of: bacteria including Escherichia coli and actinomycetes, yeast, fungi, insect cells, animal cells, mammalian cells, algae cells, and plant cells. Mammalian cells can be selected from the group consisting of: CHO, NS0, HEK293, BHK, Per.C6, MDCK, Vero, MRC, HeLa, IMR, and Sp2 / 0. CHO cells can be selected from the group consisting of: CHO-DG44, CHO-DUKX, CHO-S, CHO-K1, and CHO-DP12.

[0048] Another aspect of the present invention relates to a composition for topical administration, which comprises a polypeptide according to one embodiment of the present invention. The composition can be adapted for subcutaneous administration. In an example of the present invention, when topical administration is performed, it is observed that compared with wild-type hyaluronidase, the polypeptide according to one embodiment of the present invention exhibits significantly better drug absorption and diffusion promotion effects. Therefore, another embodiment of the present invention relates to a drug delivery carrier comprising a polypeptide according to one embodiment of the present invention.

[0049] In addition, in the examples of the present invention, when administered topically, it was observed that the polypeptide according to one embodiment of the present invention exhibited a significantly higher effect of promoting the reabsorption of excess body fluid compared to wild-type hyaluronidase. Therefore, another embodiment of the present invention relates to a pharmaceutical composition for preventing or treating edema, which comprises a polypeptide according to one embodiment of the present invention as an active ingredient.

[0050] The composition (eg, pharmaceutical composition) of the present invention may further comprise at least one active ingredient that exhibits equivalent or similar functions.

[0051] In addition, according to conventional techniques known to those skilled in the art, the compositions of the present invention (e.g., pharmaceutical compositions) can be formulated with pharmaceutically acceptable carriers and prepared in single-dose form or enclosed in multi-dose containers. As used herein, the term "carrier" refers to a substance that helps to incorporate the substance of interest into cells or tissues. Herein, the term "pharmaceutically acceptable" refers to a substance that is physiologically acceptable and does not cause gastrointestinal disorders, allergic reactions (such as dizziness), or similar reactions when administered to humans.

[0052] Any pharmaceutically acceptable carrier can be used, as long as it is typically used for formulation. Examples of pharmaceutically acceptable carriers include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0053] In addition to the above ingredients, the composition according to the present invention (e.g., pharmaceutical composition) may further contain additives such as fillers, anticoagulants, lubricants, moisturizers, fragrances, emulsifiers, preservatives, etc. In the present invention, the content of the additives in the composition is not particularly limited, but can be appropriately adjusted within the range of acceptable content for typical formulations.

[0054] As used herein, the term "excipient" refers to any substance, not itself a therapeutic agent, used as a carrier or vehicle for delivering a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage characteristics or to allow and facilitate unit dosage composition.

[0055] The composition (eg, pharmaceutical composition) according to the present invention can be formulated into different forms (such as sterile injections, etc.) according to the purpose and can be administered via different routes (such as topical, subcutaneous, intramuscular injection, etc.).

[0056] The preferred dosage of the composition (e.g., pharmaceutical composition) according to the present invention may vary within its range depending on the patient's state, body weight, age, sex, health status, dietary composition specificity, formulation characteristics, disease extent, administration time of the composition, administration method, administration period or interval, excretion rate and drug form, but can be appropriately selected by those skilled in the art.

[0057] As used herein, the term "effective dose of a pharmaceutical composition" refers to the amount of a composition containing active ingredients sufficient to treat a particular symptom. This may vary depending on the method of preparing the pharmaceutical composition, the mode of administration, the time of administration and / or the route of administration, etc., and may be diversified according to different factors including the type and degree of the reaction to be achieved by administering the pharmaceutical composition, the type of individual to whom the drug is administered, the age, weight, general health, symptoms or severity of the disease, sex, diet, excretion, other components of pharmaceutical compositions used for the corresponding individual at the same time or at different times, etc., as well as other similar factors well known in the pharmaceutical field, and those skilled in the art can easily determine and prescribe an effective dose for a predetermined treatment.

[0058] The pharmaceutical composition according to the present invention can be administered once a day or divided into several administrations. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Taking all the above factors into account, the pharmaceutical composition of the present invention can be administered in an amount that achieves maximum efficacy without side effects by a minimum amount.

[0059] Compared with wild-type hyaluronidase, polypeptide according to one embodiment of the present invention or compositions have higher titer, and therefore can realize equivalent effect with lower dosage.For example, polypeptide according to one embodiment of the present invention or compositions can be administered with 90% or lower, 85% or lower, 80% or lower, 75% or lower, 70% or lower or 65% or lower daily dosage of the daily dosage of wild-type hyaluronidase.Dosage can be based on weight or weight %.In addition, daily total dose can be divided and continuously or discontinuously administered as required.

[0060] Beneficial Effects

[0061] With the improvement of expression level and stability in animal cells, the hyaluronidase polypeptide according to one embodiment of the present invention exhibits an enzyme activity equal to or greater than that of mature wild-type PH-20. Therefore, when expressed in CHO cells, compared with mature wild-type PH-20, the hyaluronidase polypeptide according to one embodiment of the present invention has an increased expression level and is highly stable, which has the effect of increasing the industrial practicality of various uses.

[0062] In addition, due to the ability of degrading hyaluronic acid as a component of the intercellular space, hyaluronidase regulates tissue adhesion to promote the penetration and diffusion of drugs, and has the effect of promoting the reabsorption of excessive body fluids present in the tissue, so its coverage and application areas have gradually expanded. However, animal-derived wild-type PH-20 is very easy to infect substances derived from animal sources. The hyaluronidase polypeptide according to one embodiment of the present invention is unlikely to cause infection and is therefore safe, compared with animal-derived wild-type PH-20, the peptide shows high activity at the same dose, and can further increase the industrial practicality of various uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1a to Figure 1c is a diagram of SDS-PAGE showing the pH stability of the polypeptide according to one embodiment of the present invention compared to the wild-type hyaluronidase ( Figure 1a : Week 0 of storage, Figure 1b : 2nd week of storage, Figure 1c : 4th week of storage).

[0064] Figure 2a to Figure 2cis a diagram of SDS-PAGE showing the temperature stability of the polypeptide according to one embodiment of the present invention compared with the wild-type hyaluronidase ( Figure 2a : Week 0 of storage, Figure 2b : 2nd week of storage, Figure 2c : The 4th week of storage; frozen: frozen, refrigerated: refrigerated, high temperature: high temperature).

[0065] Figure 3 Shown are the results of a plate titer assay under different pH and temperature conditions to examine whether the hyaluronic acid solution exhibits its own activity.

[0066] Figure 4a Shown are the results of titer assays against wild-type hyaluronidase at temperatures ranging from 20 to 60°C.

[0067] Figure 4b The results of the titer measurement under the temperature condition of 20 to 60° C. for the polypeptide according to one embodiment of the present invention are shown.

[0068] Figure 5a The results of titer assays against wild-type hyaluronidase at temperatures between 35 and 40°C are shown.

[0069] Figure 5b The results of the titer assay under the temperature condition of 35 to 40° C. for the polypeptide according to one embodiment of the present invention are shown.

[0070] Figure 6a Results of a titer assay against wild-type hyaluronidase at pH 5 to 7 are shown.

[0071] Figure 6b The results of the potency assay under the conditions of pH 5 to 7 for the polypeptide according to one embodiment of the present invention are shown.

[0072] Figure 7 A graph showing blood levels of the polypeptide according to one embodiment of the present invention and Hirax in SD rats over time. DETAILED DESCRIPTION

[0073] A better understanding of the present invention may be obtained from the following examples, which are set forth for the purpose of illustrating the present invention but should not be construed as limiting the present invention.

[0074] Example 1. Preparation of hyaluronidase

[0075] Hyaluronidase is prepared by truncating 34 amino acid residues (Example 1-2), 68 amino acid residues (Example 1-3), 102 amino acid residues (Example 1-4), 136 amino acid residues (Example 1-5), 170 amino acid residues (Example 1-6) or 204 amino acid residues (Example 1-7) from the C-terminal extension region of the amino acid sequence of SEQ ID NO: 1 of sheep-derived wild-type hyaluronidase (CAS No. 488712-31-8).

[0076] First, cDNA was synthesized based on the amino acid sequence of wild-type hyaluronidase (CAS No. 488712-31-8). The gene of hyaluronidase was amplified using polymerase chain reaction (hereinafter referred to as "PCR") and its expression and activity in CHO-DG44 cells were tested with the aid of pcDNA 3.1 vector. The gene was anchored in CHO-DG44 cells by pOptiVEC vector. When CHO-DG44 cells reached 4×10 6 Up to 6×10 6 When the density of each cell, use electroporation to transform the cell with a plasmid constructed by inserting hyaluronidase cDNA into pOptiVEC vector. After conversion, CHO-DG44 cells are cultivated in Power CHO 2CD culture medium (with L-glutamine 4mM) and centrifuged at 12,000rpm for 10 minutes. Reclaim the supernatant, then purify it by different methods (including affinity chromatography, hydrophobic chromatography, ion exchange chromatography, etc.) as required. Filter purified hyaluronidase by water replacement in ultrafiltration and microfiltration. The amino acid sequence of the hyaluronidase thus obtained is provided in Table 1.

[0077] Table 1

[0078]

[0079]

[0080]

[0081] Example 2. Determination of the stability of hyaluronidase according to the deletion site

[0082] (1) Comparison of enzyme activity or content by storage pH according to the deletion site

[0083] Among the hyaluronidases prepared in Example 1, the pH stability of six hyaluronidases other than the hyaluronidases of Examples 1-7 lacking activity was determined according to the deletion site. Each amino acid sequence was expressed in transient cells and the cell culture thus obtained was concentrated in the same manner before measuring the enzyme activity. The experimental concentration was adjusted to 1,500 IU / mL (= 100%), the solution was prepared by substitution with water according to the conditions shown in Table 2, and stored at 37°C for 4 weeks under the conditions of pH 3.0, pH 5.0, pH 7.0 or pH 10.0, while titration was performed every two weeks.

[0084] According to the following scheme, the enzyme activity or content of samples with different deletion sites was measured at each time point:

[0085] 1) The activities of hyaluronidases with different deletion sites were determined by comparing the ratio with EP STD.

[0086] 1-1) Preparation of pH 6.4 phosphate buffered saline: A solution of 2.5 g of disodium hydrogen phosphate dodecahydrate, 2.5 g of sodium dihydrogen phosphate and 8.2 g of sodium chloride in 950 mL of water was adjusted with 1 M sodium hydroxide or 1 M hydrochloride to have a pH of 6.4, and water was added to form a final volume of 1,000 mL.

[0087] 1-2) Prepare diluent: Dissolve 0.140 g of gelatin reagent in a mixture of 100 mL of pH 6.4 phosphate buffered saline and 100 mL of water at 37° C. The diluent should be used within 2 hours after preparation.

[0088] 1-3) Prepare substrate solution: Add 100 mL of water little by little to 0.5 g of sodium hyaluronate while stirring. Slowly add water until the sodium hyaluronate swells. Continue stirring at 4°C for 12 hours or longer. The substrate solution thus obtained is stored at 4°C and should be used within 4 days after preparation.

[0089] 1-4) Preparation of standard solution: Dissolve the EP STD (EDQM) reference standard in diluent to obtain a concentration of about 50 IU / mL. Take exactly 3 mL of this solution and place it in a 250 mL volumetric flask, then add the diluent to the volumetric flask to form an exact volume of 250 mL. The resulting solution is used as the standard solution.

[0090] 1-5) Preparation of test solution: Hyaluronidase test solutions having different deletion sites (each having about 0.6 IU / mL) were adjusted to a predetermined pH value and then diluted before use.

[0091] 2) Manual: Test the standard and test solutions according to the following method.

[0092] 2-1) A 50 mL conical tube in which 7.5 mL of pH 6.4 phosphate buffered saline and 5.0 mL of the substrate solution had been mixed was placed in a water bath maintained at 37°C.

[0093] 2-2) Add 2.5 mL of the test solution to the conical tube containing pH 6.4 phosphate buffered saline and substrate solution and mix for 1 minute.

[0094] 2-3) Ubbelohde microviscometer (DIN 51 562, part 2, capillary MIII type, constant: about 0.1 mm 2 / s 2 or equivalent viscometer) to load all the mixed solutions in the conical tube.

[0095] 2-4) Use a second precision timer to record the time it takes for the solution to flow downward from the upper indicator line to the lower indicator line in the Ubbelohde microviscometer.

[0096] 2-5) Record the time several times during about 20 minutes.

[0097] 2-6) Repeat the above steps three times.

[0098] 3) Calculation: Calculate the potency (IU / mg) according to the equation.

[0099] 3-1) Reaction time: T1+T2 / 2

[0100] 3-2)ηr -1 :{(kxT2) / 0.6915} -1

[0101] T1: The time (seconds) it takes for the solution to rise to the upper indicator line in the Ubbelohde microviscometer

[0102] T2: T-T1

[0103] T: The time (seconds) it takes for the solution to drop to the lower indicator line in the Ubbelohde microviscometer

[0104] k: Ubbelohde microviscometer constant (mm 2 / s 2 ), refers to the specification of the Ubbelohde microviscometer 0.6915: the kinematic viscosity of the substrate solution at 37°C (mm 2 / s 2 )

[0105] 3-3) Activity calculation: (B T / B R )*(E R / ET )*A

[0106] B T : With reaction time on the x-axis and natural logarithm ηr on the y-axis -1 The slope of the regression curve drawn for the test solution in the coordinates of

[0107] BT: with reaction time on the x-axis and natural logarithm ηr on the y-axis -1 The slope of the regression curve drawn for the standard solution in the coordinates of

[0108] ET: concentration of test solution (mg / mL)

[0109] ER: concentration of standard solution (mg / mL)

[0110] A: Potency of standard solution (IU / mg)

[0111] Table 2

[0112]

[0113] The test data for pH stability are shown in Table 3 below. The enzyme activity (in IU / mL) in each test is expressed as a percentage of a reference value of 1,500 IU / mL (=100%), which is the initial enzyme activity at week 0. The enzyme activity ratio was calculated according to the following equation:

[0114] Enzyme activity ratio (%) = (enzyme activity at the measurement time) / (initial enzyme activity) * 100

[0115] Table 3

[0116]

[0117]

[0118] As shown in Table 3, the pH stability data obtained for hyaluronidases with different deletion sites for 4 weeks indicate that the enzyme activity ratio (%) in the pH range of 5.0 to 7.0 is higher than the enzyme activity ratio (%) of other pH values. The hyaluronidases of embodiments 1-2 to 1-6 show significantly higher pH stability compared with the wild-type hyaluronidase of embodiment 1-1, wherein the hyaluronidases of embodiments 1-2 to 1-4 are superior to other hyaluronidases. Values ​​exceeding 100% appear to be measurement errors, which generally occur due to larger fluctuations in the titer tests of biological products such as vaccines and recombinant proteins (cytokines, monoclonal antibodies, etc.).

[0119] (2) Comparison of enzyme activity or content according to the deletion site and storage temperature

[0120] The temperature stability of the hyaluronidase of Examples 1-1 to 1-6 was determined in the same manner as in (1) of Example 2. Solutions were prepared according to the conditions listed in Table 2, and the solutions were stored at freezing temperature (-20°C), refrigeration temperature (5°C) and high temperature (40°C) for 4 weeks, during which the potency test was performed every two weeks. The stability against temperature was measured for hyaluronidase with different truncation sites for 4 weeks and the measured values ​​are shown in Table 4.

[0121] Table 4

[0122]

[0123] As shown in Table 4, the hyaluronidases of Examples 1-2 to 1-6 exhibited greater temperature stability and retained significantly higher enzyme activity ratios (%), especially under frozen and refrigerated storage conditions. The amino acid DS hyaluronidase corresponding to Example 1-3 was truncated by about 12%, exhibiting the highest activity among the recombinant hyaluronidases, which was named BMI2004 and used in the experiment.

[0124] Example 3. Determination of the stability of wild-type hyaluronidase and BMI2004

[0125] (1) Comparison of enzyme activity or content based on pH

[0126] The pH stability between BMI2004 and wild-type hyaluronidase prepared in Example 1 was compared. BMI2004 and wild-type hyaluronidase (manufacturer: Korea BMI, trade name: Hirax, hereinafter referred to as Hirax) used for pH stability test had a purity of 95% or more and each had a concentration of 1,500 IU / mL (=100%), and solutions were prepared by substitution with water according to the conditions in Table 5, and stored at 5°C for 4 weeks under the conditions of pH 3.0, pH 5.0, pH 7.0, pH 10.0, during which potency tests were performed every two weeks. At each time point, the enzyme activity or content of the sample was measured in the same manner as in Example 2 (1).

[0127] Table 5

[0128]

[0129] The measurement results of the pH stability assay are shown in Table 6. The enzyme activity (in IU / mL) in each test was expressed as a percentage of a reference value of 1,500 IU / mL (=100%), which was the initial enzyme activity at week 0. The enzyme activity ratio was calculated according to the following equation:

[0130] Enzyme activity ratio (%) = (enzyme activity at the measurement time) / (initial enzyme activity) * 100

[0131] Table 6

[0132]

[0133] As shown in Table 6, the stability of BMI2004 and Hirax according to pH was observed during about 4 weeks. The enzyme activity ratio (%) measured at pH 5.0 and pH 7.0 was higher than the enzyme activity ratio (%) measured at pH 3.0 and pH 10.0. After the second week, the content (%) of Hirax tended to decrease rapidly compared with BMI2004. Values ​​exceeding 100% appear to be measurement errors, which generally occur due to large fluctuations in potency tests of biological products such as vaccines and recombinant proteins (cytokines, monoclonal antibodies, etc.). In order to additionally test the changes in enzyme activity (or content) for stability confirmation according to pH of Hirax and BMI2004, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis; SDS-PAGE) was performed under the conditions of Table 5 above and according to the following scheme:

[0134] 1) Prepare sample buffer (5X): Use Pierce TM Lane Marker Reducing Sample Buffer (Thermo Scientific TM , catalog number 39000).

[0135] 2) Prepare running buffer (1X): Use Novex TM Tris-Glycine SDS Running Buffer (10X) (Invitrogen, catalog number LC2675). Take exactly 100 mL of this solution and place it in a 1,000 mL volumetric flask, then add water to the volumetric flask to make an exact volume of 1,000 mL.

[0136] 3) Preparation of test solution: Modify the reference standards of Hirax and BMI2004 with water according to the conditions. Accurately take about 20 μL of each solution and 5 μL of sample buffer (5X) and mix in an EP tube.

[0137] 4) Manual: Perform the following measurements on the test solution and PageRuler Prestained Protein Ladder reference standard (Thermo, catalog number 26616).

[0138] 4-1) After removing the comb from it, clean the Novex with water. TMWedgewell TM A thin slice of 8-16% Tris-Glycine Gel (Invitrogen, catalog number XP08160BOX) or equivalent gel is used to replace the buffer.

[0139] 4-2) The washed gel was installed in a Mini Gel Tank. In the Mini Gel Tank, the cathode compartment was completely filled with running buffer (1X), while the anode compartment was filled about 2 / 3 with running buffer (1X).

[0140] 4-3) Load 7 μL PageRuler Prestained Protein Ladder standard and 25 μL test solution into the gel.

[0141] 4-4) Connect the mini gel tank to a power source and operate as follows, after which the samples are run to 90% of the gel length:

[0142] Volts: 140V

[0143] Ampere: 400mA

[0144] Time: 60 minutes (the time can be changed according to the operating conditions).

[0145] 4-5) After the run is complete, the gel is separated from the mold and washed with water.

[0146] 4-6) Immerse the washed gel in a dish containing a staining reagent (Coomassie Brilliant Blue R-250 Staining Solution, BIO-RAD, catalog number 1610436).

[0147] 4-7) Place the plate on a rocker and shake at 30 rpm for 30 minutes.

[0148] 4-8) After staining, the gel was transferred to a plate containing a destaining solution (Coomassie Brilliant Blue R-250 Destaining Solution, BIO-RAD, catalog number 1610438), and the plate was placed on a shaker and shaken at 30 rpm until the gel was destained and replaced with a new destaining solution. When destaining was carried out to a certain extent, the gel was immersed in water to remove the destaining solution.

[0149] 4-9) While removing the destaining solution therefrom, observe the gel under white light.

[0150] The test results are depicted in Figure 1a to Figure 1c.like Figure 1a to Figure 1c As shown, from the SDS-PAGE results, it can be understood that BMI2004 is more stable than Hirax with respect to pH over time, based on whether other bands are generated as the flow progresses over time in a water-substituted state.

[0151] (2) Comparison of enzyme activity or content according to storage temperature

[0152] The pH stability was compared between BMI2004 and Hirax prepared in Example 1. The purity of BMI2004 and Hirax used for the temperature stability test was 95% or more and each had a concentration of 1,500 IU / mL (=100%). The solution was prepared with water according to the conditions in Table 7, and stored at a freezing temperature (-20°C), a refrigerated temperature (5°C), and a high temperature (40°C) for 4 weeks, during which the potency test was performed every two weeks.

[0153] Table 7

[0154]

[0155] The stability of Hirax and BMI2004 against temperature was measured for 4 weeks and the measured values ​​are summarized in Table 8 and depicted in Figure 2. The highest content (%) was detected at freezing temperatures of -18°C or lower. After the 2nd week, the content (%) of BMI2004 remained significantly higher than that of Hirax under all temperature conditions.

[0156] Table 8

[0157]

[0158] In another stability assay according to temperature, changes in the contents of Hirax and BMI2004 at different temperatures were measured in the same manner as in Example 3. In this regard, SDS-PAGE was performed under the conditions of Table 7. The results of SDS-PAGE are depicted in Figure 2. As shown in Figure 2, it can be understood that BMI2004 is more stable than Hirax based on the formation of other bands that are different from the initial position of the main band under aqueous conditions over time under freezing, refrigeration, and high temperature.

[0159] Example 4. Optimal enzyme activity according to temperature

[0160] The disk titer determination in this embodiment is designed based on the disk assay method (cylinder plate assay) in the microbiological determination of antibiotics. Specifically, the peni cylinder in which a predetermined amount of hyaluronidase is distributed is placed on a solid agarose disk containing hyaluronic acid so that the hyaluronidase is diffused in the agarose disk. Through this diffusion, hyaluronic acid is degraded by hyaluronidase, and the residual undegraded hyaluronic acid is precipitated by cetylpyridinium chloride to form a clear halo. In this embodiment, the enzymatic activity of hyaluronidase can therefore be determined by the size of the halo produced by using the disk through this type of simple method. Under each condition, the disk titer analysis is carried out as follows:

[0161] 1) Preparation of hyaluronic acid solution: 0.2 g of hyaluronic acid was completely dissolved in 100 mL of water and the pH was adjusted to a pH of 7.0±0.1 with hydrochloric acid and sodium hydroxide.

[0162] 2) Preparation of 1.5% agarose: 1.5 g agarose (SIGMA, catalog number A9539) was added to 100 mL water and dissolved using microwave.

[0163] 3) Preparation of 10% cetylpyridinium chloride: Dissolve 10 g of cetylpyridinium chloride (SIGMA, catalog number C0732) in 100 mL of water.

[0164] 4) Test solution: Adjust Hirax and BMI2004 reference standards with water according to the conditions.

[0165] 5) Manual: Perform the following analysis on the test solution.

[0166] 5-1) The hyaluronic acid solution was warmed at 37°C for about 20 minutes before mixing with 1.5% agarose.

[0167] 5-2) After 100 mL of 1.5% agarose was cooled to about 60°C, 100 mL of the hyaluronic acid solution was added and mixed by stirring.

[0168] 5-3) The mixture was poured into a Petri dish (SPL, catalog number 10050) to a thickness of about 3 mm.

[0169] 5-4) After the agarose gel is completely solidified, a peni cylinder (KisanBio, catalog number KS-P0161) is placed at an angle of 90 ° on the concentric circles of the culture dish. The test solution is taken out and loaded into the peni cylinder with an amount of 20 μL, and then cultured in an incubator maintained at the temperature conditions listed in each table for 18 to 20 hours.

[0170] 5-5) After the reaction was completed, the peni cylinder was removed and 3 mL of 10% cetylpyridinium chloride was added. After 20 minutes, a clear halo was observed.

[0171] 5-6) The halo is accurately measured to a diameter (mm) of 0.5mm or less.

[0172] (1) Confirmation of the activity of hyaluronic acid solution

[0173] To test whether the hyaluronic acid solution exhibits its own activity, disk titer tests were performed at various pH values ​​and temperatures before the optimal enzyme activity determination. The tests were performed at pH 4.0, pH 7.0 or pH 10.0 at 20°C, 25°C, 30°C, 35°C and 40°C. The test results are plotted on Figure 3 In. Figure 3 As shown, it was observed that the hyaluronic acid solution had no activity of its own.

[0174] (2) Comparison of enzyme activity at temperatures between 20 and 40°C

[0175] The purity of BMI2004 and Hirax used in this assay for enzyme activity was 95% or higher, and each was adjusted with water to have a concentration of 1,500 IU / mL (=100%). Solutions were prepared according to the conditions in Table 9 and disk titer analysis was performed at 20°C, 25°C, 30°C, 35°C, 40°C, and 60°C.

[0176] Table 9

[0177]

[0178] The results of the measurements are depicted in Figure 4a and Figure 4b The enzyme activity of Hirax and BMI2004 was tested by plate titer assay at 20 to 60°C. Therefore, the halo appears larger at 35°C and 40°C. To quantify the enzyme activity, the Figure 4a and Figure 4b The diameter of the halo in and the measured values ​​are shown in the following Table 10. In Table 10, the diameter (mm) has an error deviation of ± 0.5 mm.

[0179] Table 10

[0180]

[0181] As shown in Table 10, comparison of enzyme activities at 20 to 40°C indicated that the activity of BMI2004 was higher than that of Hirax.

[0182] (3) Comparison of enzyme activity at temperatures between 35 and 40°C

[0183] Based on the results of the activity comparison between Hirax and BMI2004 measured in (2) of Example 4, additional tests were performed under subdivided temperature conditions. In this regard, solutions were prepared according to the conditions listed in Table 11 below and disc titer analysis was performed at 35°C, 37°C and 40°C.

[0184] Table 11

[0185]

[0186] The test results are shown in Figure 5a and Figure 5b The enzyme activities of Hirax and BMI2004 were tested by disk titer assay at 35°C, 37°C and 40°C. Therefore, the halo appears the largest at 37°C. The diameter of the halo is shown in Table 12 below. The diameter (mm) has an error deviation of ±0.5 mm.

[0187] Table 12

[0188]

[0189] As shown in Table 12, comparison of enzyme activities at 35°C, 37°C, and 40°C indicated that the activity of BMI2004 was higher than that of Hirax.

[0190] Example 5. Optimal enzyme activity according to pH

[0191] The purity of Hirax and BMI2004 used in the determination of the optimal enzyme activity was 95% or more, and each was adjusted with water to have a concentration of 1,500 IU / mL (=100%). The solution was prepared according to the conditions in Table 13. The enzyme activity was measured according to the determination of Example 4. Based on the activity results according to the temperature measured in (3) of Example 4, the disc titer determination according to pH was performed under the condition of 37°C, under which the maximum halo appeared.

[0192] Table 13

[0193]

[0194] The results of the measurements are depicted in Figure 6a and Figure 6b As a result of the disk titer assay of the enzyme activity of Hirax and BMI2004 at 37°C under the conditions of pH 5.0 to 10.0, the halo appeared particularly large at pH 7.0 to 10.0. A comparison of the halo diameters is shown in Table 14 below. The diameter (mm) has an error deviation of ±0.5 mm.

[0195] Table 14

[0196]

[0197] As can be seen in Table 14, comparison of enzyme activities at 37°C under conditions of pH 3.0 to 10.0 indicated that the activity of BMI2004 was superior to that of Hirax at pH 5.0 or higher, and was particularly excellent at pH 7.0 or higher.

[0198] Example 6. Determination of the titer of hyaluronidase

[0199] The titers of the hyaluronidase according to one embodiment of the present invention and the conventional enzyme Hirax were measured in the same manner as in (1) of Example 2, and the measured values ​​are shown in Table 15.

[0200] Table 15

[0201]

[0202]

[0203] As shown in Table 15, BMI2004 exhibited an activity of about 122,433 to 134,678 IU per mg protein, while Hirax exhibited an activity of about 80,538 to 81,396 IU per mg protein. Therefore, even when the hyaluronidase according to one embodiment of the present invention is used in an amount of about 63% of the amount of conventional Hirax, equivalent activity can be achieved.

[0204] Example 7. Effect of hyaluronidase on drug absorption and diffusion (1)

[0205] In order to determine the promoting effect of hyaluronidase on drug absorption and diffusion according to one embodiment of the present invention, trypan blue was used to measure the promotion of drug absorption and diffusion.

[0206] Specifically, Hirax and BMI2004 were each mixed with a 0.2% trypan blue solution and injected subcutaneously once into Balb / c nude mice, and the diffusion of trypan blue in the mice was monitored. Based on the potency listed in Table 15, all drugs were formulated to have a concentration of 10 IU / mL or 100 IU / mL under the conditions of Table 16 and administered at a dose of 0.05 mL. At each time, the diffusion area (πmm 2 ).

[0207] Table 16

[0208]

[0209] 1) Mean ± SD

[0210] 2) *p<0.05, **p<0.01, ***p<0.001, compared with saline-treated group by SPSS (one-way analysis of variance (ANOVA), LSD test)

[0211] The diffusion area was measured from 2.5 to 20 minutes as shown in Table 16. From 5 minutes after administration, Hirax and BMI2004 significantly increased the diffusion area compared to the negative control saline.

[0212] Example 8. Effect of hyaluronidase on drug absorption and diffusion (2)

[0213] In order to confirm the effect of hyaluronidase according to one embodiment of the present invention on promoting drug absorption and diffusion, the drug absorption and diffusion promotion was measured using an akinesia model.

[0214] Specifically, cynomolgus monkeys were used as a motor impotence model for testing the ability to enhance drug penetration. The test materials were BMI2004 and Hirax listed in Table 15, and saline was used as a negative control group. Lidocaine and bupivacaine were used as anesthetics to be mixed with the samples. The samples were each injected into 6 monkeys via the right and left periorbital route at a dose of 2 mL.

[0215] After the anesthetic was administered together with the sample, the time until the movement of the pupil disappeared (time to reach akinesia) and the time from pupil anesthesia to observation of pupil movement (duration of akinesia) were measured and the measured values ​​are shown in Table 17.

[0216] Table 17

[0217]

[0218] 1) Drugs: lidocaine 2% (0.9 mL), bupivacaine 0.5% (0.9 mL) and medication (0.2 mL)

[0219] As seen in Table 17, the negative control saline did not show an akinetic effect, while when administered together with an anesthetic, the peptide according to one embodiment of the present invention enhanced the penetration of the anesthetic and thus brought about an akinetic effect equivalent to that of the conventional peptide Hirax, confirming its ability to induce drug absorption and diffusion. In addition, the peptide according to one embodiment of the present invention showed equivalent activity to conventional Hirax at about 63% of the protein dose.

[0220] Example 9. Determination of the ability of hyaluronidase to promote reabsorption of excess body fluid

[0221] In order to confirm the promoting effect on the reabsorption of excess body fluid, an edema model was used. In this embodiment, the edema model was constructed by artificially causing lymphedema therein. The test materials were BMI2004 and Hirax listed in Table 15, and saline was used as a negative control group. The samples and dosages are given in Table 18 below.

[0222] Table 18

[0223] sample Lymphedema Dose( / site) way Number of animals brine + - SC 6 Hirax + 100IU (about 1.234ug) SC 6 BMI2004 + 100IU (about 0.776ug) SC 6

[0224] Due to its accuracy and ease, lymphedema induction was performed in the tail. A 2 mm wide circular cut was made from the skin 1 cm from the base of the mouse tail. For this, approximately 4 mm 2 The ventral side of the sac was not cut open. In order to test the effect of reducing edema, as listed in Table 19 below, the diameter (mm) of the tail was measured at each time point from before administration (0 hour) to the 15th day of induction after the first administration. The second administration was performed after 24 hours, and the third administration was performed after 48 hours. In this test, the diameter (mm) of the tail was measured using a caliper at the 10mm point of the defect.

[0225] Table 19

[0226] sample brine Hirax BMI2004 0 hours 4.55±0.43 4.53±0.49 4.53±0.38 1 hour 4.57±0.43 4.39±0.49 4.57±0.65 2 hours 4.48±0.36 4.45±0.40 4.50±0.37 4 hours 4.52±0.38 4.41±0.38 4.48±0.51 24 hours 4.56±0.34 4.57±0.39 4.55±0.45 25 hours 4.68±0.32 4.48±0.35 4.53±0.44 26 hours 4.56±0.32 4.43±0.38 4.45±0.47 28 hours 4.58±0.37 4.42±0.44 4.46±0.48 48 hours 4.65±0.39 4.49±0.41 4.47±0.38 49 hours 4.56±0.27 4.22±0.37 4.26±0.37 50 hours 4.58±0.25 4.13±0.38 4.09±0.36 52 hours 4.59±0.25 4.16±0.47 4.18±0.36

[0227] As shown in Table 19, administration of BMI2004 reduced lymphedema, confirming the promoting effect of the protein on the reabsorption of excess body fluid. In addition, the peptide according to one embodiment of the present invention exhibited equivalent activity compared to conventional Hirax at about 63% of the protein dose.

[0228] Example 10. Determination of in vivo stability of hyaluronidase

[0229] To confirm the in vivo stability, the hyaluronidase according to one embodiment of the present invention was injected intravenously and its pharmacokinetics were monitored. In this embodiment, SD rats were used as test animals. By intravenous infusion, Hirax and BMI2004 were administered to each animal with a total dose of about 180,000 IU in 30 minutes. Blood samples were taken before administration, 15 minutes after the start of infusion (infusion), 30 minutes after the start of infusion (end of infusion), and 31, 33, 36, 40, 45, 60, 75, 90 minutes and 2.5 hours, 4.5 hours, 24.5 hours and 48.5 hours after the start of infusion.

[0230] The pharmacokinetic variables of the polypeptide according to one embodiment of the present invention and Hirax are given in Table 20. The blood levels of the polypeptides over time are plotted in Figure 7 In. Figure 7As shown in Table 20, Hirax degrades too quickly to measure the in vivo half-life. In contrast, it was observed that the peptide according to one embodiment of the present invention had a half-life of about 0.272 hours (about 16.3 minutes). Since the half-life is longer than that of Hirax, the polypeptide according to one embodiment of the present invention has the potential to show a greater effect than Hirax by staying in the body for a long time. In addition, they differ in blood content in the body. The blood content of Hirax peaks within 0.25 hours (15 minutes) after administration, but the blood content of BMI2004 peaks within 0.5 hours (30 minutes) after administration. The polypeptide exhibits the potential to bring a greater effect than Hirax because its blood content increases more slowly and remains in the body for a longer time than Hirax.

[0231] Table 20

[0232] Test Materials Dosage (IU / kg) <![CDATA[t max (h)]]> <![CDATA[t 1 / 2 (h)]]> CL(mL / h / kg) Hirax 180000 0.250 NC NC BMI2004 180000 0.500 0.272 418

Claims

1. A polypeptide having hyaluronidase activity, comprising a polypeptide having an amino acid sequence of SEQ ID NO: 1 and at least one continuous amino acid sequence of 1 to 203 amino acids missing from the C-terminus of the polypeptide. 2 . The polypeptide according to claim 1 , comprising at least a continuous stretch of 34 to 170 amino acids deleted from the C-terminus of the amino acid sequence of SEQ ID NO:

1. 3 . The polypeptide according to claim 1 , comprising at least a continuous amino acid stretch of 34 to 68 amino acids deleted from the C-terminus of the amino acid sequence of SEQ ID NO:

1. 4 . The polypeptide according to claim 1 , comprising an amino acid sequence in which 34, 68, 102, 136 or 170 amino acids are deleted from the C-terminus of the amino acid sequence of SEQ ID NO:

1. 5 . The polypeptide according to claim 1 , further comprising deleting the first amino acid from the N-terminus of the amino acid sequence of SEQ ID NO:

1.

6. The polypeptide according to claim 1, wherein The polypeptide consists of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

7. The polypeptide according to claim 1, wherein The polypeptide is glycosylated.

8. The polypeptide according to claim 1, wherein The polypeptide is stable at pH 3 to pH 10.

9. The polypeptide according to claim 1, wherein The polypeptide is stable at temperatures between -20 and 45°C.

10. The polypeptide according to claim 1, wherein The polypeptide has an enzyme activity of 57% or more of the initial activity after storage for 4 weeks under pH conditions ranging between higher than pH 3 and pH 10 or lower.

11. The polypeptide according to claim 1, wherein The polypeptide has an enzyme activity of 32% or more of the initial activity after storage under pH conditions ranging between pH 3 or more and less than pH 5 for 4 weeks.

12. The polypeptide according to claim 1, wherein The polypeptide has an enzyme activity of 63% or more of its initial activity after storage at a temperature below 0°C for 4 weeks.

13. The polypeptide according to claim 1, wherein The polypeptide has an enzyme activity of 83% or more of its initial activity after storage at a temperature of 0 to 40° C. for 4 weeks.

14. The polypeptide according to claim 1, wherein The polypeptide has an enzyme activity of 52% or more of its initial activity after storage at a temperature of 40° C. or more for 4 weeks.

15. The polypeptide according to claim 1, wherein The polypeptide has an activity ranging between one-fold higher and three-fold or lower than the activity of the polypeptide consisting of the amino acid sequence of SEQ ID NO:

1.

16. The polypeptide according to claim 1, wherein The polypeptide has an activity of 120,000 to 150,000 IU / mg.

17. A nucleic acid molecule encoding the polypeptide according to any one of claims 1 to 16. A vector comprising the nucleic acid molecule according to claim 17 .

19. A cell comprising the vector according to claim 18.

20. The cell according to claim 19, wherein The cell is selected from the group consisting of bacteria, yeast, fungi, insect cells, animal cells, mammalian cells, algae cells and plant cells.

21. The cell according to claim 20, wherein The mammalian cells are selected from the group consisting of CHO, NS0, HEK293, BHK, Per.C6, MDCK, Vero, MRC, HeLa, IMR and Sp2 / 0.

22. A composition for topical administration comprising the polypeptide according to any one of claims 1 to 16.

23. The composition according to claim 22, wherein The composition is adapted for subcutaneous administration or intramuscular injection.

24. A drug delivery carrier comprising the polypeptide according to any one of claims 1 to 16.

25. A composition for preventing or treating edema, comprising the polypeptide according to any one of claims 1 to 16.

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