CN113144174B - Medicine for treating hyperuricemia related diseases - Google Patents
Medicine for treating hyperuricemia related diseases Download PDFInfo
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- CN113144174B CN113144174B CN202010075471.4A CN202010075471A CN113144174B CN 113144174 B CN113144174 B CN 113144174B CN 202010075471 A CN202010075471 A CN 202010075471A CN 113144174 B CN113144174 B CN 113144174B
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/04—Drugs for disorders of the urinary system for urolithiasis
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- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C12Y—ENZYMES
- C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
- C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
- C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase
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Abstract
The invention provides a medicament comprising urate oxidase, wherein at least 7 of the following amino acid sites in the urate oxidase are connected with PEG groups, K 4 、K 17 、K 30 、K 35 、K 97 、K 112 、K 116 、K 152 、K 179 、K 190 、K 222 、K 266 、K 272 、K 285 、K 291 、K 298 . Compared with the uric acid oxidase without PEG modification, the uric acid oxidase provided by the embodiment of the invention has greatly reduced immunogenicity, and the medicament containing the uric acid oxidase can realize the treatment of hyperuricemia related diseases.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a medicine, a medicine composition and a medicine application.
Background
With the improvement of life quality and the change of diet and life habit of people, the food intake of high protein and high purine is increased, and the number of gout patients tends to increase year by year. In europe, the number of gout patients has doubled over the last 20 years. The incidence rate of the current hyperuricemia and gout in China is improved to about 2-3 percent. If the hyperuricemia does not have clinical symptoms, the diet is controlled; when clinical symptoms caused by it occur, medication is required.
Therefore, there is still a need for researchers to further develop drugs for treating gout.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, the invention provides a urate oxidase medicament capable of treating refractory gout.
In a first aspect of the invention, the invention provides a medicament. According to an embodiment of the invention, the medicament comprises urate oxidase, at least 7 of the following amino acid positions in the urate oxidase are linked with PEG groups, K 4 、K 17 、K 30 、K 35 、K 97 、K 112 、K 116 、K 152 、K 179 、K 190 、K 222 、K 266 、K 272 、K 285 、K 291 、K 298 。
Compared with the uric acid oxidase without PEG modification, the uric acid oxidase provided by the embodiment of the invention has greatly reduced immunogenicity, and the medicament containing the uric acid oxidase can realize the treatment of hyperuricemia related diseases.
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 PEG groups, K 4 、K 17 、K 30 、K 35 、K 97 、K 112 、K 116 、K 152 、K 179 、K 190 、K 222 、K 266 、K 272 、K 285 、K 291 、K 298 。
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 sites in the urate oxidase are linked with PEG groups, K 4 、K 17 、K 97 、K 179 、K 190 、K 222 、K 266 、K 272 、K 285 。
According to an 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 an amino acid sequence shown in SEQ ID NO. 1.
TYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:1)。
According to an embodiment of the present invention, the relative proportion of peak area reduction of the uricase compared to the peptide map of the uricase without PEG attached thereto is not less than 75%, preferably not less than 80%, more preferably not less than 90% of the peak area reduction of at least 7 predetermined peptide fragments. The urate oxidase according to the embodiment of the invention has low immunogenicity compared with the urate oxidase without PEG, and can realize effective treatment of hyperuricemia-related diseases.
According to an embodiment of the present invention, the relative proportion of peak area reduction of the uricase compared to the peptide map of the uricase without PEG attached thereto is not less than 75%, preferably not less than 80%, more preferably not less than 90% of the peak area reduction of at least 9 predetermined peptide fragments.
According to an embodiment of the present invention, the peptide map of urate oxidase is shown in fig. 4.
In a second aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises the medicament as described above or the urate oxidase as described above. The pharmaceutical composition according to the embodiment of the invention can be used for treating or preventing diseases related to hyperuricemia.
According to an embodiment of the present 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 hyperuricemia-related diseases.
In a third aspect of the invention, the invention provides the use of a medicament as hereinbefore described or a urate oxidase as hereinbefore described or a pharmaceutical composition as hereinbefore described in the manufacture of a medicament for the treatment of a hyperuricase-associated disease.
According to an embodiment of the present invention, the above-mentioned use may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the hyperuricemia-related disease comprises a disease selected from the group consisting of chronic hyperuricemia, gout, kidney disease, hyperuricemia arthritis, kidney stones, gout nodules, hypertension, diabetes mellitus, hypertriglyceridemia, metabolic syndrome, coronary heart disease, atherosclerosis, hyperuricemia caused by cancer chemotherapy.
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FIG. 1 is a PHC SDS-PAGE purity detection according to an embodiment of the invention;
FIG. 2 is a chart of PHC RP-HPLC purity detection according to an embodiment of the invention;
FIG. 3 is a Lys-C cleavage map of PHC according to an embodiment of the invention;
FIG. 4 is a PHA Lys-C cleavage map according to an embodiment of the present invention;
FIG. 5 is a Lys-C cleavage alignment diagram of PHC and PHA according to an embodiment of the present invention;
FIG. 6 is a graph showing the results of the change in uric acid (μmol/L) and allantoin (μmol/L) levels in serum after a single administration of rats according to an embodiment 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 by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
The term "urate oxidase" as used herein is to be understood in a broad sense and refers to the collective term for mixtures of urate oxidases produced in the same batch in actual production practice.
Structural modification such as substitution, deletion or addition of partial amino acids is performed on the protein structural sequence of urate oxidase, so that the immunogenicity and the like are reduced.
The urate oxidase is not particularly limited, and may be urate oxidase of any origin and its urate oxidase analogues, and representative examples include, but are not limited to, mammalian origin, microorganisms, plants, and the like.
The urate oxidase from different species can be obtained through various ways, including but not limited to natural extraction, chemical synthesis, recombinant expression in genetic engineering, etc.
In another preferred embodiment, the urate oxidase is recombinantly expressed in host cells by recombinant techniques using the coding sequence of the urate oxidase protein sequence (SEQ ID NO: 1).
In another preferred embodiment, the recombinant expression strain is prepared by a method of constructing a recombinant expression strain using E.coli or yeast as a host, and more preferably, E.coli is used as a host for recombinant expression.
As used herein, the polyethylene glycol (PEG), referring to a mixture of a condensation polymer of ethylene oxide and water, is represented by the general formula H (OCH) 2 CH 2 ) nOH represents a hydrophilic polymer with neutral pH, no toxicity and high water solubility, and has a linear or branched structure. The PEG needs to be combined with the protein to activate one or more ends of the PEG, and corresponding modification groups can be selected for activation according to the modified target protein, such as amino, sulfhydryl, carboxyl or hydroxyl groups.
In another preferred embodiment, the site for PEG modification of 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. Urate oxidase is covalently linked to a modifying group of PEG via an urethane bond, a secondary urethane bond or an amide bond, preferably a polyethylene glycol molecule coupled to urate oxidase to form an amide bond, the modifying group of polyethylene glycol including but not limited to N-hydroxysuccinimides, nitrobenzene including but not limited to N-hydroxysuccinimides (NHS), N-hydroxysuccinimide carbonates (SC), N-hydroxysuccinimide acetates (SCM), N-hydroxysuccinimide propionates (SPA), N-hydroxysuccinimide butyrates (SBA), N-hydroxysuccinimide succinates (SS), nitrophenyl carbonates (NPC), etc., wherein the blocking group of polyethylene glycol includes but is not limited to monomethoxy, ethoxy, glucose or galactose sugars, preferably monomethoxy.
In another preferred embodiment, the polyethylene glycol may be linear or branched.
In another preferred embodiment, polyethylene glycol urate oxidase is used with a polyethylene glycol molecular weight of 9 to 11KD, preferably 10KD.
In another preferred embodiment, purification of the modified sample employs techniques including, but not limited to, molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, tangential flow ultrafiltration, or a combination. More preferably molecular sieve chromatography, tangential flow ultrafiltration.
In another aspect of the invention, a polyethylene glycol modified urate oxidase and applications thereof are provided. The conjugate can achieve the effect of obviously reducing the uric acid level in the body, and can be used for treating hyperuricemia and ventilation.
The polyethylene glycol urate oxidase is more suitable for being used as a medicine for treating chronic hyperuricemia or ventilation and a composition thereof. The main symptoms of hyperuricemia and ventilation include, but are not limited to, uric acid nephropathy and ventilated arthritis.
The administration route of the polyethylene glycol urate oxidase includes but is not limited to intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection and the like.
The polyethylene glycol urate oxidase has lower immunogenicity in vivo than urate oxidase without PEG.
The polyethylene glycol urate oxidase has low immunogenicity, which means that after intramuscular injection of polyethylene glycol urate oxidase in human or animal body, no antibody against urate oxidase is produced, and no antibody against polyethylene glycol molecule or low titer polyethylene glycol antibody is produced by organism.
According to the embodiment of the invention, the polyethylene glycol modified urate oxidase can reduce immunogenicity on the premise of ensuring the enzyme activity. Thus, the polyethylene glycol modified urate oxidase of the present invention and the pharmaceutical composition comprising the polyethylene glycol modified urate oxidase may be administered in the treatment or prevention of hyperuricase-associated diseases.
The term "administering" as used herein refers to 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 intended 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. Furthermore, the pharmaceutical compositions of the present invention may be administered using a specific device that delivers the active ingredient to the target cells.
The frequency and dosage of administration 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 route of administration, the age, sex, 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 disorder, i.e., an amount that provides a therapeutic effect for a given disorder and dosing regimen. For example, in the treatment of chronic hyperuricemia or gout, a drug or compound that reduces, prevents, delays, inhibits or retards any symptom of the disease or disorder should be therapeutically effective. A therapeutically effective amount of the drug or compound is not required to cure the disease or disorder, but will provide treatment for the disease or disorder such that the onset of the disease or disorder in the subject is delayed, prevented or prevented, or the symptoms of the disease or disorder are alleviated, or the period of the disease or disorder is altered, or for example the disease or disorder becomes less severe, or healing is accelerated.
The term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treatment" encompasses treatment of a disease (primarily hyperuricase-associated disease) in a mammal, particularly a human, including: (a) Preventing disease in an individual susceptible to disease but not yet diagnosed with disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" or "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 a uricase comprising polyethylene glycol modification as described herein to an individual in need thereof.
According to embodiments of the present invention, the polyethylene glycol modified urate oxidase or pharmaceutical composition of the present invention may be used in combination with or 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 an individual sequentially or simultaneously. Alternatively, the pharmaceutical compositions of the present invention may comprise a polyethylene glycol modified urate oxidase of the present invention, a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient, and other therapeutic or prophylactic combinations 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 site has PEG attached thereto can be determined by conventional techniques, for example, by reference to the methods set forth in the "polyethylene glycol modified site detection" section of example 3 of the present application. Briefly, the method comprises: 1) The non-PEGylated and PEGylated urate oxidase is cleaved with one or more enzymes, such as Lys-C or Trypsin, or double cleavage with Lys-C and Trypsin; 2) Separating the enzyme sections by high performance liquid chromatography to generate chromatograms of non-PEGylated and PEGylated urate oxidase, i.e. peptide maps; 3) Comparing the differences in the peptide maps of the non-pegylated and pegylated urate oxidase to determine the relative proportion of the decrease or disappearance of the peptide Duan Feng at the specific amino acid position in the pegylated urate oxidase and further determine whether the specific amino acid position on the peptide fragment 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 where the specific amino acid site is located can be calculated by the following formula:
P(％)＝(A 2 -A 1 )/A 2 ×100％，
p (%) represents the relative proportion of decrease or disappearance of the peak area of the peptide fragment where a specific amino acid site is located, A 2 Peak area of peptide fragment in PHC peptide diagram for peptide fragment with specific amino acid site, A 1 The peak area of the peptide fragment with the specific amino acid position in the peptide map of the modified protein to be detected.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and the various technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions, which can be more clearly understood with reference to the following examples. For the sake of brevity, the examples are given for illustrative purposes only and are not intended to limit the invention.
Embodiments of the present invention will be described in further detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Example 1 preparation of urate oxidase (PHC)
1.1 recombinant expression construction of urate oxidase
cDNA of urate oxidase protein (code: PHC) (SEQ ID NO: 1) is synthesized by adopting a total 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 introduced and transformed into escherichia coli BL21 (DE 3), and high-expression host bacteria engineering bacteria of urate oxidase are obtained by Kanamycin resistance screening.
Fermenting and culturing the converted engineering strain by adopting a fermentation tank, carrying out induction expression for 4 hours by 1mmol/L IPTG, and centrifuging to collect cells. The fermentation centrifugal bacteria are resuspended in 20mmol/L Tris,5mmol/L EDTA buffer solution, and high-pressure homogenization and bacteria breaking are carried out at about 600bar to obtain urate oxidase sediment, and the sediment is respectively washed and buffered: 20mmol/L Tris,0.1% Triton X-100, pH 8.0-8.5 and wash buffer II: 50mmol/L NaHCO 3 After one wash, the enriched urate oxidase precipitate was suspended in 100mmol/L Na 2 HCO 3 (pH 9.7-10.3) in a buffer solution, the suspension ratio is 1:50 (W/V), stirring at room temperature overnight for dissolution, and then centrifuging to collect the supernatant.
The urate oxidase was taken to dissolve the supernatant, and captured first with a DEAE-Sepharose FF column. With a content of 50mmol/L Na 2 HCO 3 (pH 9.7-10.3) buffer balance, and eluting urate oxidase with carbonate buffer containing 0.3M NaCl. Then, the macromolecule in urate oxidase was removed with Source 30Q. With 50mmol/L Na 2 HCO 3 (pH9.7-10.3), and performing linear gradient elution by using carbonate buffer containing 0-0.5M NaCl to obtain high-purity urate oxidase PHC. The purity of the final urate oxidase PHC is shown in FIG. 1: SDS-PAGE showed purity greater than 95%, as shown in FIG. 2: the purity was greater than 95% by RP-HPLC, without aggregate formation.
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 at 1: and 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 is not changed with time. After the reaction is finished, removing modified byproducts through molecular sieve chromatography, and then performing ultrafiltration concentration and sterile filtration to obtain 10K modified PEGylated uricase (PHA).
Example 3 characterization of PEGylated urate oxidase
3.1 average degree of modification and enzyme Activity detection
The protein concentration was measured by Lowry method, and polyethylene glycol urate oxidase activity was measured under a spectrophotometer. The maximum ultraviolet absorption wavelength of uric acid oxidase substrate uric acid is 293nm, and the maximum ultraviolet absorption wavelength of product allantoin is 224nm, and the absorption value of uric acid at 293nm is proportional to the concentration thereof in a certain concentration range, and the quantitative determination of uric acid can be performed by a spectrophotometry. The specific process is as follows: the UV-visible spectrophotometer was turned on to adjust the wavelength to 293nm and the instrument water bath circulation system was turned on to maintain the temperature at 37 ℃. Taking sodium tetraborate buffer solution as blank control, and correcting zero points; 2.95ml of a substrate reaction solution (0.1 mol/L sodium tetraborate, 100. Mu. Mol/L uric acid, pH9.5, preheated at 37 ℃) was placed in a quartz cuvette, 50. Mu.l of a test substance was then added and rapidly mixed, and the absorption value was measured at 293 nm. Continuously measuring the absorbance change at 293 nm; calculating uric acid degradation concentration according to C=A/epsilon L (wherein A is the light absorption value of uric acid with specific concentration at 293nm, epsilon is the uric acid molar extinction coefficient, L is the cuvette optical path and C is the uric acid molar concentration), and calculating enzyme activity; definition of enzyme activity: the amount of enzyme required to convert 1. Mu. Mol uric acid to allantoin per minute at an optimum reaction temperature of 37℃and an optimum reaction pH of 9.5 was defined as one activity unit (U).
Average modification of polyethylene glycol urate oxidase was detected using SEC-HPLC tandem UV/RI (combination of ultraviolet and refractive index detector). According to the fact that the protein has the maximum absorption peak at 280nm of ultraviolet, and the PEG does not absorb at the wavelength, the absorption value of the differential refraction detector for the protein and the PEG in a certain range is proportional 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 the external standard mode of the PEG reference substance and the PHC physicochemical reference substance, and further the number of PEG molecules on each urate oxidase monomer, namely the average modification degree, can be obtained by the following calculation mode.
Average degree of modification of PEG urate oxidase= (relative molecular weight of urate oxidase subunit x amount of PEG in sample)/(relative molecular weight of PEG x amount of protein in sample).
The average modification degree of the PEGylated uric acid oxidase (PHA) obtained in the example 2 is 9.4, the enzyme activity is 10.7U/mg, and the retention rate of the modified PEGylated uric acid oxidase is more than 90% compared with the unmodified uric acid oxidase with the enzyme activity of 11.4U/mg.
3.2 detection of polyethylene glycol modification sites
In the following procedure, the inventors performed detection of modification sites for urate oxidase obtained in example 2.
The PEG modification site of polyethylene glycol modified urate oxidase can be obtained by subjecting non-PEGylated and PEGylated urate oxidase to enzyme digestion with one or more enzymes, and then subjecting to chromatographic detection to obtain chromatogram, i.e. peptide map confirmation. Non-pegylated and pegylated urate oxidases may be cleaved using single cleavage (Lys-C or Trypsin) and/or double cleavage (Lys-C and Trypsin in combination). And separating the enzyme sections by the reverse phase column, and judging the modification site of the polyethylene glycol urate oxidase by comparing the disappearance or reduction ratio of the peptide sections.
Principle of modified site analysis in trypsin and Lys-C dual cleavage mass peptide map: lys-C can specifically cleave the C-terminal of lysine (K), and trypsin specifically cleaves the C-terminal peptide bond of the lysine (K) by taking basic amino acid arginine (R) and lysine (K) as cleavage sites. The relative proportion of PEG modified peptide fragments reduced or disappeared can be analyzed and confirmed by comparing the change condition of each peptide fragment corresponding to the enzyme cutting in PHC and PHA. By the relative proportion of the reduction or disappearance of the peptide fragment, it can be determined whether the lysine site on the peptide fragment is modified by PEG and the proportion of modification. It should be noted that PEG modification is a heterogeneous modification, and a site can be considered modified if the proportion of modification at that site is high.
Taking Lys-C single enzyme digestion as an example, the following is concrete:
(1) Sample treatment: the urate oxidase and the PEGylated urate oxidase were dissolved and diluted to 1mg/ml with digestion buffer (25 mmol/L Tris-HCl,20% acetonitrile, pH 9.0), 100. Mu.l each was taken, 2. Mu.l Lys-C was added, and digestion was carried out at 37℃for 4 hours, and then 10. Mu.l 1mol/L hydrochloric acid solution was added to terminate the reaction.
(2) Analysis conditions:
instrument: thermo Ultimate 3000HPLC and MSQ Plus;
chromatographic column: yuehu Weich Materials
XB-C 18 (4.6mm×250mm，5μm)；
Analysis conditions: solution A (aqueous solution containing 0.1% TFA), solution B (acetonitrile solution containing 0.1% TFA);
gradient: 0-70min, and 3-70% of B;
LC detection wavelength: 214nm.
Ion source: ESI;
ion type: a positive ion;
taper hole voltage: 50V;
scanning range: 300-2000Da;
scanning time: 1S;
the post column split was about 0.3ml/min.
Sample volume was 100. Mu.l and the chromatogram was recorded.
(3) And (3) result processing:
comparing the chromatograms (peptide maps) of urate oxidase and pegylated urate oxidase, the peak areas of corresponding PHA peptide fragments at the same concentrations of PHA and PHC can be calculated using the following formula:
P(％)＝(A 2 -A 1 )/A 2 ×100％
wherein A is 2 Peak area of peptide fragment in PHC peptide graph, A 1 The peak area of the peptide fragment in PHA peptide fragment is shown.
Analysis of the protein sequence (SEQ ID NO: 1) according to this example reveals that the potential site for modification of urate oxidase has T 1 、K 3 、K 4 、K 17 、K 21 、K 30 、K 35 、K 48 、K 49 、K 66 、K 74 、K 76 、K 79 、K 97 、K 112 、K 116 、K 120 、K 152 、K 155 、K 158 、K 169 、K 179 、K 190 、K 215 、K 222 、K 231 、K 266 、K 272 、K 285 、K 291 、K 293 And 31 sites.
As shown in FIG. 3, FIG. 4 and FIG. 5, wherein FIG. 3 is a Lys-C cleavage peptide map of PHC, FIG. 4 is a Lys-C cleavage peptide map of PHA, and FIG. 5 is a Lys-C cleavage comparison map of PHC and PHA. From the analysis of the polyethylene glycol modified urate oxidase cleavage peptide map obtained in example 3, it was found that K was present at the corresponding site where the peak area of the peptide fragment after PHA cleavage and the peak area of the corresponding fragment after PHC cleavage were reduced 4 、K 17 、K 30 、K 35 、K 97 、K 112 、K 116 、K 152 、K 179 、K 190 、K 222 、K 266 、K 272 、K 285 、K 291 、K 298 . Wherein, the site at which the peptide segment disappears to more than 75% after PHA enzyme digestion has K 4 、K 17 、K 97 、K 179 、K 190 、K 222 、K 266 、K 272 、K 285 And 9 sites.
EXAMPLE 4 in vivo pharmacodynamics study of polyethylene glycol urate oxidase
In this experiment, rats were given 1mg/kg of polyethylene glycol urate oxidase tail intravenous injection, blood was taken from the orbit at different time points, allantoin content was measured by quantitative mass spectrometry, and uric acid content was measured by fluorescence. FIG. 6 shows the levels of uric acid (μmol/L) and allantoin (μmol/L) levels in serum after a single administration of rats. Experimental results indicate that blood level can be maintained for at least 10 days after administration, which is manifested by a sustained uric acid decrease with a corresponding increase in allantoin as a degradation product.
EXAMPLE 5 in vivo immunogenicity determination of polyethylene glycol urate oxidase
Polyethylene glycol urate oxidase (PHA) was administered once a week, continuously intravenously 4 times, 1mg/kg each time, and blood was collected 7 days after each administration, and the results of detection of antibodies by ELISA showed that no drug-resistant antibodies were detected, while polyethylene glycol urate oxidase (PHA) was still enzymatically active in serum after repeated administration, and maintained low levels of uric acid in serum as compared with the control group (physiological saline group).
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means 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 present invention. In this specification, schematic representations of the above terms are not necessarily directed 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, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
SEQUENCE LISTING
<110> Jinan Ruian company management consultation Limited
Lu Wei
<120> medicament for treating hyperuricemia-related diseases
<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 (7)
1. A medicament, which is characterized by comprising urate oxidase, wherein the following amino acid sites in the urate oxidase are connected with PEG groups,
K 4 、K 17 、K 30 、K 35 、K 97 、K 112 、K 116 、K 152 、K 179 、K 190 、K 222 、K 266 、K 272 、K 285 、K 291 、K 298 the molecular weight of the PEG group is 10KD,
the amino acid sequence of the urate oxidase is shown as SEQ ID NO. 1.
2. The medicament according to claim 1, wherein K in the urate oxidase amino acid sequence 4 、K 17 、K 97 、K 179 、K 190 、K 222 、K 266 、K 272 、K 285 The proportion of PEG modification at 9 sites is not less than 75%.
3. A pharmaceutical composition comprising a medicament according to any one of claims 1 to 2.
4. A pharmaceutical composition according to claim 3, comprising a further medicament for the treatment or prophylaxis of hyperuricemia-related diseases using a combination.
5. Use of a medicament according to any one of claims 1 to 2 or a pharmaceutical composition according to any one of claims 3 to 4 for the preparation of a medicament for the treatment of hyperuricemia-related diseases.
6. The use according to claim 5, wherein the hyperuricemia-related disease comprises chronic hyperuricemia.
7. The use according to claim 5, wherein the hyperuricemia-related disease comprises a disease selected from the group consisting of gout, kidney disease, hyperuricemia due to chemotherapy of cancer, hypertension, diabetes, metabolic syndrome, coronary heart disease, atherosclerosis.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6576235B1 (en) * | 1998-08-06 | 2003-06-10 | Mountain View Pharmaceuticals, Inc. | PEG-urate oxidase conjugates and use thereof |
| CN102260653A (en) * | 2011-06-30 | 2011-11-30 | 北京盛宏生物技术有限公司 | Preparation and application method of PEG recombinant pig-human urate oxidase fusion protein |
| CN102757945A (en) * | 2011-04-28 | 2012-10-31 | 杭州俊丰生物工程有限公司 | Human urate oxidase protein and preparation method and polyethylene glycol composite thereof |
| CN108103079A (en) * | 2017-06-20 | 2018-06-01 | 北京五加和分子医学研究所有限公司 | A kind of gene therapy medicament of hyperuricemia |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SI3321359T1 (en) * | 2005-04-11 | 2021-07-30 | Horizon Pharma Rheumatology Llc | Variant forms of urate oxidase and use thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6576235B1 (en) * | 1998-08-06 | 2003-06-10 | Mountain View Pharmaceuticals, Inc. | PEG-urate oxidase conjugates and use thereof |
| CN102757945A (en) * | 2011-04-28 | 2012-10-31 | 杭州俊丰生物工程有限公司 | Human urate oxidase protein and preparation method and polyethylene glycol composite thereof |
| CN102260653A (en) * | 2011-06-30 | 2011-11-30 | 北京盛宏生物技术有限公司 | Preparation and application method of PEG recombinant pig-human urate oxidase fusion protein |
| CN108103079A (en) * | 2017-06-20 | 2018-06-01 | 北京五加和分子医学研究所有限公司 | A kind of gene therapy medicament of hyperuricemia |
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