CN113144174A

CN113144174A - Medicine for treating diseases related to high uric acid

Info

Publication number: CN113144174A
Application number: CN202010075471.4A
Authority: CN
Inventors: 路伟
Original assignee: Hangzhou Grand Biologic Pharmaceutical Co ltd
Current assignee: Hangzhou Grand Biologic Pharmaceutical Co ltd
Priority date: 2020-01-22
Filing date: 2020-01-22
Publication date: 2021-07-23
Anticipated expiration: 2040-01-22
Also published as: CN113144174B

Abstract

The invention provides a medicament, which is prepared fromComprises urate oxidase, wherein at least 7 of the following amino acid sites in the urate oxidase are connected with PEG groups, K⁴、K¹⁷、K³⁰、K³⁵、K⁹⁷、K¹¹²、K¹¹⁶、K¹⁵²、K¹⁷⁹、K¹⁹⁰、K²²²、K²⁶⁶、K²⁷²、K²⁸⁵、K²⁹¹、K²⁹⁸. Compared with urate oxidase without PEG modification, the urate oxidase of the embodiment of the invention has greatly reduced immunogenicity, and the medicine containing the urate oxidase can realize the treatment of diseases with high urate correlation.

Description

Medicine for treating diseases related to high uric acid

Technical Field

The invention relates to the field of biomedicine, in particular to a medicine, a pharmaceutical composition and a pharmaceutical application.

Background

With the improvement of the quality of life and the change of diet and living habits of people, the intake of high-protein and high-purine food is increased, and the number of gout patients tends to increase year by year. In europe, the number of gout patients has increased about one-fold over the last 20 years. The current incidence rate of hyperuricemia and gout in China is increased to about 2-3%. If no clinical symptoms appear in hyperuricemia, controlling diet; when clinical symptoms caused by the disease appear, the medicine treatment is needed.

Therefore, there is still a need for researchers to further develop drugs for treating gout.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention provides a urate oxidase medicine capable of treating intractable gout.

In a first aspect of the invention, a medicament is presented. According to an embodiment of the invention, the drug comprises urate oxidase, in which at least 7 of the following amino acid sites have a PEG group attached, K⁴、K¹⁷、K³⁰、K³⁵、K⁹⁷、K¹¹²、K¹¹⁶、K¹⁵²、K¹⁷⁹、K¹⁹⁰、K²²²、K²⁶⁶、K²⁷²、K²⁸⁵、K²⁹¹、K²⁹⁸。

Compared with urate oxidase without PEG modification, the urate oxidase of the embodiment of the invention has greatly reduced immunogenicity, and the medicine containing the urate oxidase can realize the treatment of diseases with high urate correlation.

According to an embodiment of the present invention, the above-mentioned medicament may further include at least one of the following additional technical features:

according to an embodiment of the invention, at least 9 of the following amino acid positions in the urate oxidase are linked with a PEG group, K⁴、K¹⁷、K³⁰、K³⁵、K⁹⁷、K¹¹²、K¹¹⁶、K¹⁵²、K¹⁷⁹、K¹⁹⁰、K²²²、K²⁶⁶、K²⁷²、K²⁸⁵、K²⁹¹、K²⁹⁸。

According to an embodiment of the invention, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine of the following amino acid positions in the urate oxidase are linked with a PEG group, K⁴、K¹⁷、K⁹⁷、K¹⁷⁹、K¹⁹⁰、K²²²、K²⁶⁶、K²⁷²、K²⁸⁵。

According to the embodiment of the invention, the molecular weight of the PEG group is 9-11 KD.

According to an embodiment of the invention, the urate oxidase has the amino acid sequence shown in SEQ ID NO 1.

TYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:1)。

According to an embodiment of the present invention, the peptide pattern of the urate oxidase has a reduced peak area of at least 7 predetermined peptide fragments in a relative ratio of not less than 75%, preferably not less than 80%, more preferably not less than 90% compared to the peptide pattern of urate oxidase without PEG attached. Compared with the urate oxidase without PEG, the urate oxidase provided by the embodiment of the invention has low immunogenicity, and can realize effective treatment of diseases with high urate correlation.

According to an embodiment of the present invention, the peptide pattern of the urate oxidase has a reduced peak area of at least 9 predetermined peptide fragments in a relative ratio of not less than 75%, preferably not less than 80%, more preferably not less than 90% compared to the peptide pattern of urate oxidase without PEG attached.

According to an embodiment of the invention, the peptide diagram of the urate oxidase is shown in fig. 4.

In a second aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises a drug as described above or a urate oxidase as described above. The pharmaceutical composition according to the embodiment of the present invention can be used for the treatment or prevention of diseases associated with hyperuricemia.

According to an embodiment of the invention, the pharmaceutical composition further comprises at least one of the following additional technical features:

according to an embodiment of the present invention, the pharmaceutical composition further comprises other drugs for treating or preventing diseases associated with hyperuricemia.

In a third aspect of the invention, the invention proposes the use of a medicament as described above or a urate oxidase as described above or a pharmaceutical composition as described above for the preparation of a medicament for the treatment of a disease associated with hyperuricemia.

According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

according to an embodiment of the present invention, the disease associated with hyperuricemia comprises a disease selected from chronic hyperuricemia, gout, kidney disease, hyperuricemic arthritis, kidney stones, gout nodules, hypertension, diabetes, hypertriglyceridemia, metabolic syndrome, coronary heart disease, atherosclerosis, and hyperuricemia induced by cancer chemotherapy.

Drawings

FIG. 1 is a purity check chart of PHC SDS-PAGE according to an embodiment of the present invention;

FIG. 2 is a PHC RP-HPLC purity detection chart according to an embodiment of the present invention;

FIG. 3 is a Lys-C cleavage map of PHC according to an embodiment of the present invention;

FIG. 4 is a PHA Lys-C cleavage map according to an embodiment of the present invention;

FIG. 5 is a Lys-C mapping of PHC and PHA according to an embodiment of the present invention;

FIG. 6 is a graph showing the results of varying levels of uric acid (. mu. mol/L) and allantoin (. mu. mol/L) levels in serum after a single administration in rats according to an example of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

The term "urate oxidase" as used herein is to be understood in a broad sense and refers to a generic term for mixtures of urate oxidases produced in the same batch in actual production practice.

The structural modification of the protein structure sequence of the urate oxidase, such as partial amino acid substitution, deletion or addition, is carried out, so as to realize the effects of the example, including but not limited to reducing immunogenicity and the like.

The urate oxidase is not particularly limited, and may be any urate oxidase and urate oxidase analogs thereof, and representative examples include, but are not limited to, mammalian sources, microorganisms, plants, and the like.

The urate oxidase from different species can be obtained by various ways, including but not limited to natural extraction, chemical synthesis, genetic engineering recombinant expression and the like.

In another preferred embodiment, the uricase is recombinantly expressed in a host cell by recombinant techniques from the coding sequence of the uricase protein sequence (SEQ ID NO: 1).

In another preferred embodiment, the recombinant expression strain is prepared by using Escherichia coli or yeast as a host and constructing the recombinant expression strain, and more preferably, Escherichia coli is used as a host bacterium for recombinant expression.

As used herein, the polyethylene glycol (PEG), which refers to a mixture of a condensation polymer of ethylene oxide and water, is represented by the general formula H (OCH)₂CH₂) nOH represents that the polymer is a hydrophilic polymer with neutral pH, no toxicity and high water solubility, and has a linear or branched structure. To combine PEG with protein, one or more ends of PEG are activated, and corresponding modifying groups can be selected for activation according to the modified target protein, such as amino, sulfhydryl, carboxyl or hydroxyl.

In another preferred embodiment, the PEG modification site for uricase of the present invention is the epsilon amino group of a lysine residue, but there is also a small modification of the alpha amino group of the N-terminal lysine residue. The urate oxidase is covalently linked to a modification group of PEG through an amino-lipid bond, a secondary amino bond or an amide bond, preferably a polyethylene glycol molecule is coupled with urate oxidase to form an amide bond, wherein the modification group of polyethylene glycol includes but is not limited to N Hydroxysuccinimide (NHS), N hydroxysuccinimide carbonate (SC), N hydroxysuccinimide acetate (SCM), N hydroxysuccinimide propionate (SPA), N hydroxysuccinimide butyrate (SBA), N hydroxysuccinimide succinate (SS), Nitrophenylcarbonate (NPC) and the like, wherein the blocking group of polyethylene glycol includes but is not limited to monomethoxy, ethoxy, glucose or galactose, preferably monomethoxy.

In another preferred embodiment, the polyethylene glycol may be linear or branched.

In another preferred example, the molecular weight of the polyethylene glycol used by the polyethylene glycol uric acid oxidase is 9-11 KD, preferably 10 KD.

In another preferred embodiment, the modified sample is purified using a method including, but not limited to, molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, tangential flow ultrafiltration, or a combination thereof. More preferably, molecular sieve chromatography and tangential flow ultrafiltration.

In another aspect of the invention, the invention provides the uricase modified by the polyethylene glycol and application thereof. The conjugate can realize the effect of remarkably reducing the blood uric acid level in vivo, and can be used for treating hyperuricemia and ventilation.

The polyethylene glycol uricase is more suitable for being used as a medicine for treating chronic hyperuricemia or ventilation and a composition thereof. The major symptoms of hyperuricemia and ventilation include, but are not limited to, uric acid nephropathy and ventilated arthritis.

The administration routes of the polyethylene glycol uricase comprise but are not limited to intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection and the like.

Compared with uricase without PEG, the polyethylene glycol uric acid oxidase has lower in vivo immunogenicity.

The polyethylene glycol uric acid oxidase has low immunogenicity, which means that after the polyethylene glycol uric acid oxidase is injected into human or animal bodies through muscles, antibodies aiming at the urate oxidase are not generated, and the bodies do not generate antibodies of polyethylene glycol molecules or generate low-titer anti-polyethylene glycol antibodies.

According to the embodiment of the invention, the polyethylene glycol modified urate oxidase can reduce immunogenicity on the premise of ensuring enzyme activity. Thus, the polyethylene glycol-modified urate oxidase of the present invention and the pharmaceutical composition comprising the same may be administered in the treatment or prevention of diseases associated with hyperuricemia.

The term "administering" as used herein means introducing a predetermined amount of a substance into a patient by some suitable means. The polyethylene glycol-modified urate oxidase of the present invention may be administered by any common route as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary and rectal, but the invention is not limited to these exemplified modes of administration. In addition, the pharmaceutical compositions of the present invention may be administered using a specific device that delivers the active ingredient to the target cells.

The administration frequency and dose of the pharmaceutical composition of the present invention can be determined by a number of relevant factors, including the type of disease to be treated, the administration route, the age, sex, body weight and severity of the disease of the patient and the type of drug as an active ingredient.

The term "therapeutically effective amount" refers to an amount of a compound sufficient to significantly ameliorate some of the symptoms associated with a disease or condition, i.e., to provide a therapeutic effect for a given condition and administration regimen. For example, in the treatment of chronic hyperuricemia or gout, a drug or compound that reduces, prevents, delays, inhibits, or retards any symptoms of the disease or disorder should be therapeutically effective. A therapeutically effective amount of a drug or compound need not cure a disease or condition, but will provide treatment for a disease or condition such that the onset of the disease or condition in an individual is delayed, prevented or prevented, or the symptoms of the disease or condition are alleviated, or the duration of the disease or condition is altered, or the disease or condition becomes less severe, or recovery is accelerated, for example.

The term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses the treatment of diseases (primarily referred to as hyperuricosuric-related diseases) in mammals, particularly humans, including: (a) preventing disease in individuals who are susceptible to disease but have not yet been diagnosed; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of urate oxidase containing polyethylene glycol modifications described herein to an individual in need thereof.

According to embodiments of the invention, the polyethylene glycol modified urate oxidase or pharmaceutical composition of the invention may be used in combination with conventional treatment methods and/or therapies or may be used separately from conventional treatment methods and/or therapies. When the polyethylene glycol-modified urate oxidase or the pharmaceutical composition of the present invention is administered in combination therapy with other drugs, they may be administered to the individual sequentially or simultaneously. Alternatively, the pharmaceutical composition of the invention may comprise a combination of the polyethylene glycol modified urate oxidase of the invention, a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient, and other therapeutic or prophylactic agents known in the art.

The term "average degree of modification" refers to the number of PEG bound per urate oxidase monomer.

It will be appreciated by those skilled in the art that the determination of whether a particular amino acid position has PEG attached thereto can be made by routine techniques, such as those described in the section "detection of modified sites of polyethylene glycol" in example 3 of the present application. Briefly, the method comprises: 1) carrying out enzyme digestion on non-pegylated and pegylated urate oxidase by adopting one or more enzymes, for example, Lys-C or Trypsin can be adopted for carrying out single enzyme digestion, and Lys-C and Trypsin can also be adopted for carrying out double enzyme digestion; 2) separating enzyme digestion fragments by a high performance liquid chromatography method to generate chromatograms of non-pegylated and pegylated urate oxidases, namely a peptide diagram; 3) comparing the difference of the peptide graphs of the non-pegylated and pegylated urate oxidase, determining the relative proportion of the reduction or disappearance of the peak of the peptide segment where the specific amino acid site is located in the pegylated urate oxidase, and further judging whether the specific amino acid site on the peptide segment is modified by PEG. Specifically, in example 3 of the present application, the relative proportion of the decrease or disappearance of the peak area of the peptide fragment in which a specific amino acid position is located can be calculated by the following formula:

P(％)＝(A₂-A₁)/A₂×100％，

p (%) denotes the relative proportion of decrease or disappearance of the peak area of the peptide fragment in which the specific amino acid site is present, A₂The peak area of the peptide segment at the specific amino acid position in a PHC peptide chart, A₁Is the peak area of the peptide segment where the specific amino acid position is located in the modified protein peptide graph to be detected.

It is understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the various technical features described in detail below (e.g., the embodiments) can be combined with each other to constitute a new or preferred technical solution, which can be more clearly understood with reference to the following examples. The examples are for illustrative purposes only and are not intended to be limiting of the invention, to the extent it is not discourse.

EXAMPLE 1 preparation of urate oxidase (PHC)

1.1 construction of recombinant expression of urate oxidase

cDNA of uric acid oxidase protein (code number: PHC) (SEQ ID NO:1) is synthesized by adopting a whole gene synthesis mode, Nde I and BamH I are used as target gene insertion sites, pET-30a plasmid is used as an expression vector and is introduced and transformed into escherichia coli BL21(DE3), and the engineering bacteria of the urate oxidase high-expression host bacteria are obtained by using Kanamycin resistance screening.

And (3) fermenting and culturing the transformed engineering strain by adopting a fermentation tank, carrying out induction expression for 4h by using 1mmol/L IPTG, and centrifugally collecting cells. Suspending the weight of the fermentation centrifugal bacteria in 20mmol/L Tris, 5mmol/L EDTA buffer solution, homogenizing and breaking bacteria at high pressure of about 600bar to obtain urate oxidase precipitate, washing and buffering the precipitate respectively: 20mmol/L Tris, 0.1% Triton X-100, pH 8.0-8.5 and Wash buffer two: 50mmol/L NaHCO₃After washing once, the rich urate oxidase precipitate was suspended in 100mmol/L Na₂HCO₃(pH9.7-10.3) in a buffer solution at a suspension ratio of 1:50(W/V), and the mixture was stirred at room temperature overnight to be dissolved, followed by centrifugation to collect the supernatant.

The supernatant sample was dissolved with urate oxidase and first captured on a DEAE-Sepharose FF column. Using a solution containing 50mmol/L Na₂HCO₃(pH 9.7-10.3) buffer equilibration, and elution of urate oxidase with carbonate buffer containing 0.3M NaCl. Then, the polymer in urate oxidase was removed by Source 30Q. Using 50mmol/L Na₂HCO₃(pH9.7-10.3), performing buffer balance, and performing linear gradient elution by using carbonate buffer containing 0-0.5M NaCl to obtain the urate oxidase PHC with high purity. The urate oxidase PHC purity obtained after purification is shown in fig. 1: the purity is more than 95% by SDS-PAGE detection, as shown in figure 2: the purity is more than 95 percent by RP-HPLC detection, and no aggregate is formed.

Example 2 preparation of PEGylated urate oxidase (PHA)

The urate oxidase obtained by purification in example 1 was diluted to 8mg/ml with elution buffer, diluted in a ratio of 1: adding 10K-PEG-SPA dry powder into 80-120 mol ratio (urate oxidase: 10K-PEG-SPA), stirring at room temperature for reaction for more than 1 hour until the PEG coupling degree does not change with time. After the reaction is finished, removing the modified by-products through molecular sieve chromatography, then performing ultrafiltration concentration and sterile filtration to obtain the 10K modified PEGylated urate oxidase (the code is PHA).

Example 3 characterization of the enzyme uricoxidase

3.1 average modification degree and enzyme Activity detection

The protein concentration is determined by Lowry method, and the activity of the polyethylene glycol uric acid oxidase is determined by a spectrophotometer. The maximum ultraviolet absorption wavelength of urate oxidase substrate uric acid is 293nm, the maximum ultraviolet absorption wavelength of product allantoin is 224nm, and in a certain concentration range, the absorption value of uric acid at 293nm is in direct proportion to the concentration thereof, and quantitative determination of uric acid can be carried out by using a spectrophotometer method. The specific process is as follows: the UV-visible spectrophotometer was turned on, the wavelength was adjusted to 293nm, and the instrument water bath circulation system was turned on to maintain the temperature at 37 ℃. Using sodium tetraborate buffer solution as blank control, and correcting zero point; 2.95ml of substrate reaction solution (0.1mol/L sodium tetraborate, 100. mu. mol/L uric acid, pH9.5, preheating at 37 ℃) is placed in a quartz cuvette, 50. mu.l of test sample is added, the mixture is rapidly mixed, and the absorption value is measured at 293 nm. Continuously measuring the change of absorbance at 293 nm; calculating the uric acid degradation concentration according to the C-A/Epsilon L (wherein A is the light absorption value of uric acid with specific concentration at 293nm, Epsilon is the molar extinction coefficient of uric acid, L is the optical path of the cuvette, and C is the molar concentration of uric acid), and calculating the enzyme activity; definition of enzyme activity: the amount of enzyme required to convert 1. mu. mol of uric acid per minute into allantoin at an optimum reaction temperature of 37 ℃ and an optimum reaction pH of 9.5 was defined as one activity unit (U).

Detection of average modification degree of polyethylene glycol uric acid oxidase is carried out by SEC-HPLC tandem UV/RI (ultraviolet and refractive index detector combination). According to the fact that the protein has a maximum absorption peak at 280nm of ultraviolet, PEG does not absorb at the wavelength, and the absorption value of the differential refraction detector to the protein and the PEG in a certain range is in direct proportion to various concentrations of the protein and the PEG. Therefore, the respective contents of PEG and protein parts in the pegylated urate oxidase can be obtained by an external standard mode of a PEG reference substance and a PHC physical and chemical reference substance, and the number of PEG molecules on each urate oxidase monomer, namely the average modification degree, can be further obtained by the following calculation mode.

PEG urate oxidase average modification degree ═ (urate oxidase subunit relative molecular weight × amount of PEG in sample)/(PEG relative molecular weight × amount of protein in sample).

The average modification degree of the PEGylated uricosuric oxidase (PHA) obtained in the example 2 is 9.4, the enzyme activity is 10.7U/mg, and compared with the enzyme activity of unmodified uricosuric oxidase of 11.4U/mg, the retention rate of the enzyme activity of the modified PEGylated uricosuric oxidase is more than 90%.

3.2 detection of polyethylene glycol modification sites

In the following procedure, the inventors carried out detection of modification sites of the urate oxidase obtained in example 2.

The PEG modified site of the urate oxidase modified by polyethylene glycol can be determined by performing enzyme digestion on non-pegylated and pegylated urate oxidase by using one or more enzymes, and then obtaining a chromatogram, namely a peptide map, through chromatographic detection. The non-pegylated and pegylated urate oxidase can be digested by single enzyme digestion (Lys-C or Trypsin) and/or double enzyme digestion (Lys-C and Trypsin combined). Separating enzyme digestion fragments by a reverse phase column, and judging the modification sites of the polyethylene glycol uric acid oxidase by comparing the disappearance or reduction ratio of the peptide fragments.

Analysis principle of modification sites in trypsin and Lys-C double-restriction mass peptide map: Lys-C can perform specific digestion on the C-terminal of lysine (K), and trypsin performs specific digestion on the C-terminal peptide bond by taking basic amino acids of arginine (R) and lysine (K) as digestion sites. Comparing the change conditions of the peptide fragments after enzyme digestion in PHC and PHA, and analyzing and confirming the relative proportion of reduction or disappearance of the PEG modified peptide fragments. Whether the lysine site on the peptide fragment is modified by PEG and the modification proportion can be judged by the reduction or disappearance relative proportion of the peptide fragment. It should be noted that the PEG modification is a non-uniform modification, and a site with a high modification ratio is considered to be modified.

Taking Lys-C single enzyme as an example, the specific steps are as follows:

(1) sample treatment: dissolving and diluting urate oxidase and PEGylated urate oxidase with enzyme digestion buffer solution (25mmol/L Tris-HCl, 20% acetonitrile, pH9.0) to 1mg/ml, collecting 100 μ L of each, adding 2 μ L of Lys-C, performing enzyme digestion at 37 deg.C for 4 hr, and adding 10 μ L of 1mol/L hydrochloric acid solution to terminate the reaction.

(2) Analysis conditions were as follows:

the instrument comprises the following steps: thermo Ultimate 3000HPLC and MSQ Plus;

a chromatographic column: welch Materials Yueku

XB-C₁₈(4.6mm×250mm，5μm)；

Analysis conditions were as follows: solution a (aqueous solution containing 0.1% TFA), solution B (acetonitrile solution containing 0.1% TFA);

gradient: 0-70min, B from 3-70%;

LC detection wavelength: 214 nm.

An ion source: ESI;

ion type: a positive ion;

taper hole voltage: 50V;

scanning range: 300-;

scanning time: 1S;

the split was about 0.3ml/min after column.

Sample size 100. mu.l was injected and chromatogram recorded.

(3) And (4) processing a result:

comparing chromatograms (peptide maps) of urate oxidase and pegylated urate oxidase, and calculating the peak area of the corresponding PHA peptide fragment under the same concentration of PHA and PHC by using the following formula:

P(％)＝(A₂-A₁)/A₂×100％

wherein A is₂The peak area of a peptide fragment in a PHC peptide map, A₁The peak area of the peptide fragment in PHA peptide fragment.

According to the protein sequence (SEQ ID NO:1) analysis of this example, the modification potential site of urate oxidase has T¹、K³、K⁴、K¹⁷、K²¹、K³⁰、K³⁵、K⁴⁸、K⁴⁹、K⁶⁶、K⁷⁴、K⁷⁶、K⁷⁹、K⁹⁷、K¹¹²、K¹¹⁶、K¹²⁰、K¹⁵²、K¹⁵⁵、K¹⁵⁸、K¹⁶⁹、K¹⁷⁹、K¹⁹⁰、K²¹⁵、K²²²、K²³¹、K²⁶⁶、K²⁷²、K²⁸⁵、K²⁹¹、K²⁹³And 31 sites are equal.

As shown in FIG. 3, FIG. 4 and FIG. 5, FIG. 3 is a Lys-C cleavage map of PHC, FIG. 4 is a Lys-C cleavage map of PHA, and FIG. 5 is a Lys-C cleavage map of PHC and PHA. Analysis of the peptide map of the PEG-modified urate oxidase digested enzyme obtained in example 3 revealed that K is present in the corresponding site with reduced peak areas of peptide fragments digested with PHA and corresponding fragments digested with PHC⁴、K¹⁷、K³⁰、K³⁵、K⁹⁷、K¹¹²、K¹¹⁶、K¹⁵²、K¹⁷⁹、K¹⁹⁰、K²²²、K²⁶⁶、K²⁷²、K²⁸⁵、K²⁹¹、K²⁹⁸. Wherein, K is positioned at more than 75% of sites where peptide fragments disappear after PHA enzyme digestion⁴、K¹⁷、K⁹⁷、K¹⁷⁹、K¹⁹⁰、K²²²、K²⁶⁶、K²⁷²、K²⁸⁵And 9 sites are equal.

Example 4 PEG-uricase in vivo pharmacodynamic study

In this experiment, rats received a single tail vein injection of 1mg/kg peg-uricase, blood was taken from the orbit at different time points, and the allantoin content was determined by quantitative mass spectrometry and the uric acid content was determined by fluorescence. FIG. 6 shows the levels of uric acid (μmol/L) and allantoin (μmol/L) levels in serum of rats after a single administration. The results of the experiments show that the blood drug can be maintained for at least 10 days after administration, and the continuous reduction of uric acid is shown while the degradation product allantoin is correspondingly increased.

Example 5 PEG-uricase Oxosomal in vivo immunogenicity assay

Polyethylene glycol uricoxidase (PHA) is administered once a week for 4 times continuously by vein, each time is 1mg/kg, blood is collected 7 days after each administration, and an ELISA method is adopted to detect the antibody, so that the result proves that the antibody is not detected, the polyethylene glycol uricoxidase (PHA) in the serum still has enzyme activity after repeated administration is detected, and the serum always maintains low-level blood uric acid compared with a control group (normal saline group).

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> Jinan Reran Enterprise management consultation company, Inc

Lu Wei

<120> A medicine for treating diseases associated with hyperuricemia

<130> PIDC3192662

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 298

<212> PRT

<213> Artificial

<220>

<223> amino acid sequence of urate oxidase

<400> 1

Thr Tyr Lys Lys Asn Asp Glu Val Glu Phe Val Arg Thr Gly Tyr Gly

1 5 10 15

Lys Asp Met Ile Lys Val Leu His Ile Gln Arg Asp Gly Lys Tyr His

20 25 30

Ser Ile Lys Glu Val Ala Thr Thr Val Gln Leu Thr Leu Ser Ser Lys

35 40 45

Lys Asp Tyr Leu His Gly Asp Asn Ser Asp Val Ile Pro Thr Asp Thr

50 55 60

Ile Lys Asn Thr Val Asn Val Leu Ala Lys Phe Lys Gly Ile Lys Ser

65 70 75 80

Ile Glu Thr Phe Ala Val Thr Ile Cys Glu His Phe Leu Ser Ser Phe

85 90 95

Lys His Val Ile Arg Ala Gln Val Tyr Val Glu Glu Val Pro Trp Lys

100 105 110

Arg Phe Glu Lys Asn Gly Val Lys His Val His Ala Phe Ile Tyr Thr

115 120 125

Pro Thr Gly Thr His Phe Cys Glu Val Glu Gln Ile Arg Asn Gly Pro

130 135 140

Pro Val Ile His Ser Gly Ile Lys Asp Leu Lys Val Leu Lys Thr Thr

145 150 155 160

Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp Gln Phe Thr Thr Leu Pro

165 170 175

Glu Val Lys Asp Arg Cys Phe Ala Thr Gln Val Tyr Cys Lys Trp Arg

180 185 190

Tyr His Gln Gly Arg Asp Val Asp Phe Glu Ala Thr Trp Asp Thr Val

195 200 205

Arg Ser Ile Val Leu Gln Lys Phe Ala Gly Pro Tyr Asp Lys Gly Glu

210 215 220

Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr Asp Ile Gln Val Leu Thr

225 230 235 240

Leu Gly Gln Val Pro Glu Ile Glu Asp Met Glu Ile Ser Leu Pro Asn

245 250 255

Ile His Tyr Leu Asn Ile Asp Met Ser Lys Met Gly Leu Ile Asn Lys

260 265 270

Glu Glu Val Leu Leu Pro Leu Asp Asn Pro Tyr Gly Lys Ile Thr Gly

275 280 285

Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295

Claims

1. A drug characterized by comprising urate oxidase in which at least 7 of the following amino acid sites are linked with PEG groups,

K⁴、K¹⁷、K³⁰、K³⁵、K⁹⁷、K¹¹²、K¹¹⁶、K¹⁵²、K¹⁷⁹、K¹⁹⁰、K²²²、K²⁶⁶、K²⁷²、K²⁸⁵、K²⁹¹、K²⁹⁸。

2. the drug of claim 1, wherein at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine of the following amino acid positions in the urate oxidase have a PEG group attached thereto,

K⁴、K¹⁷、K⁹⁷、K¹⁷⁹、K¹⁹⁰、K²²²、K²⁶⁶、K²⁷²、K²⁸⁵。

3. the drug of claim 1, wherein the PEG group has a molecular weight of 9 to 11 KD.

4. The drug according to claim 1, wherein the urate oxidase has an amino acid sequence shown in SEQ ID NO. 1.

5. The drug according to claim 1, characterized in that the peptide pattern of the urate oxidase has a reduced peak area of at least 7 predetermined peptide fragments in relative proportion of not less than 75%, preferably not less than 80%, more preferably not less than 90% compared to the peptide pattern of urate oxidase without PEG attached.

6. The agent of claim 5, wherein the uricase has a peptide pattern as shown in figure 4.

7. A pharmaceutical composition comprising the pharmaceutical agent of any one of claims 1 to 6.

8. The pharmaceutical composition of claim 7, comprising an additional agent for treating or preventing a disease associated with hyperuricemia using a combination of two or more agents.

9. Use of the medicament of any one of claims 1 to 6 or the pharmaceutical composition of any one of claims 7 to 8 for the preparation of a medicament for the treatment of a disease associated with hyperuricemia.

10. The use according to claim 9, wherein the disease associated with hyperuricemia comprises a disease selected from the group consisting of chronic hyperuricemia, gout, kidney disease, hyperuricemic arthritis, kidney stones, gout nodules, hypertension, diabetes, hypertriglyceridemia, metabolic syndrome, coronary heart disease, atherosclerosis, and hyperuricemia induced by cancer chemotherapy.