WO2006015512A1 - Arginine déiminase modifiée - Google Patents

Arginine déiminase modifiée Download PDF

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Publication number
WO2006015512A1
WO2006015512A1 PCT/CN2004/000933 CN2004000933W WO2006015512A1 WO 2006015512 A1 WO2006015512 A1 WO 2006015512A1 CN 2004000933 W CN2004000933 W CN 2004000933W WO 2006015512 A1 WO2006015512 A1 WO 2006015512A1
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peg
adi
arginine deiminase
compound
arginine
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PCT/CN2004/000933
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English (en)
Chinese (zh)
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Wenbo Zhang
Yong Su
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Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd.
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Priority to PCT/CN2004/000933 priority Critical patent/WO2006015512A1/fr
Publication of WO2006015512A1 publication Critical patent/WO2006015512A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Definitions

  • the present invention relates to arginine deiminase modified with polyethylene glycol and its use in the treatment of tumors. Background technique
  • liver cancer is also one of the major forms of cancer.
  • the development of drugs for the treatment of liver cancer is very urgent and has very important significance.
  • Tumor cells and normal cells have different nutritional needs. Some non-essential amino acids, normal cells can be synthesized autonomously, and tumor cells lose the ability to synthesize such amino acids. Selective "starved" tumor cells using an enzyme that degrades this type of amino acid is theoretically a highly selective, low-toxic tumor treatment. A well-known example is asparaginase, which is used to treat acute lymphoblastic leukemia. Some human tumor cells cannot synthesize arginine, so it is theoretically possible to develop anti-tumor drugs by arginine-degrading enzymes.
  • Normal cells do not require arginine for growth because they synthesize arginine from citrulline by a two-step reaction catalyzed by argininosuccinate synthase and argininosuccinate lyase.
  • argininosuccinate synthase argininosuccinate synthase
  • argininosuccinate lyase hepatoma, melanoma and some other sarcomas do not express argininosuccinate synthetase, and thus they are auxotrophs of arginine. This metabolic difference can be used to develop safe and effective therapeutic agents for the treatment of these diseases.
  • Arginine deiminase catalyzes the conversion of arginine to citrulline and can be used to remove arginine. Therefore, arginine deiminase can be used to treat hepatoma, melanoma and some other sarcomas.
  • Natural arginine deiminase can be found in a variety of microorganisms. Takaku et al.
  • arginine deiminase as a heterologous protein, is highly immunogenic in humans and is rapidly cleared in the patient's blood circulation.
  • Protein PEGylation is an effective way to overcome the immunogenicity of heterologous proteins. Covalently modified by polyethylene glycol can reduce the immunogenicity of the foreign protein and prolong the half-life. Natural arginine deiminase
  • polyethylene glycol modified arginine deiminase is more effective in treating tumors.
  • PEGylated adenosine deaminase (PEG-ADA) and asparaginase (PEG-ASP) have been used clinically for many years.
  • the polyethylene glycol (PEG) molecule must be activated by an activating group to react with a reactive group such as an amino group, a thiol group, a carboxyl group or an imidazolyl group on the surface of the protein to form a covalent bond to the protein molecule.
  • a common activating group comprises a linking group, with or without a leaving group, the leaving group is shed during the reaction, and the PEG is covalently linked to the protein via a linking group.
  • maleimide groups including succinimide succinate (SS), propionic acid succinate (SPA), succinic acid succinate (SCM), iminosuccinic succinate Amide (SSA) or N-hydroxysuccinimide (L S), epoxy, oxycarbonylimidazolyl (including, for example, carbonyldiimidazolyl (CDI)), nitrophenyl (including, for example, Nitrophenyl carbonate (NPC) or trichlorophenyl carbonate (TPC)), isocyanate group, vinylsulfone group, tyrosine group, cysteine group, histidine group or primary amine.
  • SS succinimide succinate
  • SPA propionic acid succinate
  • SCM succinic acid succinate
  • SSA iminosuccinic succinate Amide
  • L S N-hydroxysuccinimide
  • epoxy epoxy
  • oxycarbonylimidazolyl including, for example, carbonyldiimidazolyl (CDI)
  • linking group is not essential, and the presence of a linking group (1) may provide a target site for enzymatic hydrolysis or hydrolysis, resulting in instability of the linker, such as an ester bond in SS-PEG; (2)
  • the linking group may be immunogenic or potentially toxic, such as cyanuric chloride.
  • An object of the present invention is to provide a modified arginine deiminase which has an excellent antitumor effect and is more stable and has low immunogenicity.
  • a pegylated arginine deaminase compound having no linking group which has the structure of formula (I) -
  • PEG represents a polyethylene glycol having an average molecular weight of from 1000 to 20000 Da
  • ADI represents arginine Deaminase
  • - indicates a covalent bond between PEG and ADI
  • n is an integer from 2 to 30.
  • the arginine deiminase is an arginine deiminase of a microorganism of the genus Mycoplasma.
  • the arginine deiminase is an arginine deiminase of arginine mycoplasma, Mycoplasma hominis, or Mycoplasma arthritis. More preferably, the arginine deiminase is an arginine deiminase of arginine mycoplasma.
  • the PEG has a molecular weight of 4000 to 6000 Da
  • the arginine deiminase has the amino acid sequence of SEQ ID NO: 2.
  • the compound has n of 5 to 17, more preferably 7 to 15, most preferably 9 to 12.
  • a pharmaceutical composition comprising a compound of formula (I) according to the invention and a pharmaceutically acceptable carrier.
  • PEG represents a polyethylene glycol having an average molecular weight of 1000 to 20000 Da
  • ADI represents an arginine deiminase
  • - represents a covalent bond between PEG and ADI
  • n is an integer of 2 to 30.
  • the method includes the steps of:
  • mPEG is monomethoxypolyethylene glycol and X is a leaving group.
  • Figure 1 shows the arginine deiminase nucleic acid sequence. Five codons encoding tryptophan TGA are unintentional codons in E. coli and changed to TGG. (A, 606, 792, 819, 885, and 1230 bits change to G).
  • Figure 2 shows the amino acid sequence of arginine deiminase.
  • Figure 3 shows the HPLC profile of native and PEGylated arginine deiminase.
  • Figure 4A shows plasma arginine concentrations following administration of native and PEGylated arginine deiminase.
  • Figure 4B shows the plasma citrulline concentration after administration of native and PEGylated arginine deiminase.
  • Figure 5 shows the immunogenicity of native and PEGylated arginine deiminase.
  • Figure 6 shows the in vivo antitumor activity of arginine deiminase. detailed description .
  • modified ADI formed by directly linking PEG with a molecular weight of 1000-20000 Da to arginine deiminase (ADI), and existing modified groups with a linking group Compared with ADI, it has superior comprehensive properties, namely excellent anti-tumor effect, more stable chemical properties and lower immunogenicity.
  • ADI arginine deiminase
  • PEG polyethylene glycol
  • TMPEG/mPEG-tresyl trifluoroethylsulfonyl monomethoxypolyethylene glycol
  • mPEG-Cl chlorinated monomethoxy polyethylene glycol
  • polyethylene glycol or “PEG” refers to a mixture of ethylene oxide and a linear or branched form polycondensate of water, represented by the general formula H (0CH 2 CH 2 ) n 0H, This n is at least 4.
  • H 0CH 2 CH 2
  • n 0H
  • the approximate molecular weight is expressed by "polyethylene glycol” or “PEG” together with the following numerical suffix.
  • PEG 5000 refers to polyethylene glycol having an average molecular weight of about 5,000.
  • a “hepatoma” can be a malignant or benign tumor of the liver, including, for example, hepatocellular carcinoma.
  • "Patient” means an animal, preferably a mammal, more preferably a human.
  • PEG-modified arginine deiminase or "PEG-ADI” refers to a compound of formula (I): '
  • PEG represents a polyethylene glycol having an average molecular weight of from 1,000 to 20,000 Da
  • ADI represents an arginine deiminase
  • "-" represents a covalent bond between PEG and ADI 1 .
  • arginine deiminase and its gene can be obtained from any source, including recombinant production. Raw or synthetic.
  • the arginine deiminase gene can be cloned from the mycoplasma genome, or chemically synthesized; the arginine deiminase gene can be a natural sequence, or an optimized sequence generated by genetic engineering mutation; A sequence of mycoplasma sequences or a plurality of mycoplasma optimized combinations.
  • the arginine deiminase uses a sequence derived from arginine mycoplasma, Mycoplasma hominis, and arthritic mycoplasma. More preferably, the arginine deiminase uses a sequence derived from arginine mycoplasma.
  • a particularly preferred ADI sequence is listed in Figure 2.
  • the average molecular weight of the polyethylene glycol is from 1,000 to 20,000; preferably from 2,000 to 12,000; more preferably from 3,000 to 8,000, still more preferably from 4,000 to 6,000; most preferably about 5,000 Da.
  • PEG is attached to ADI via an activating group.
  • the activating group used is itself a leaving group in which the activating group is shed and PEG is directly attached to the protein.
  • an organic sulfonyl group including toluenesulfonyl group, trifluoromethylsulfonyl group, trifluoroethylsulfonyl group), halogen (F, Cl, 1).
  • An exception is acetaldehyde-activated PEG.
  • Acetaldehyde as an activating group does not fall off during the reaction, but the ethoxy group formed after the reaction cannot be distinguished from PEG.
  • a preferred activating group of the invention is trifluoroethyl sulfsyl or Cl.
  • the linkerless polyethylene glycol modification technology used in the present invention is a kind of
  • the invention also provides a method of preparing a modified arginine deiminase, which generally comprises the steps of:
  • mPEG is monomethoxypolyethylene glycol and X is a leaving group.
  • preferred X is an organic sulfonyl chloride (including trifluoromethylsulfonyl chloride, trifluoroethylsulfonyl chloride, phenylmethylsulfonyl chloride) or a halogen (including F, Cl, 1).
  • Particularly preferred leaving groups are trisethyl sulphonyl chloride (tresyl) and chlorine (Cl).
  • the separation step may be a conventional method such as column chromatography or affinity chromatography.
  • a preferred method is to first remove the unreacted PEG by dialysis and then obtain the compound of formula (I) by column chromatography or affinity chromatography.
  • the degree of modification can be controlled by adjusting the molar ratio of PEG to ADI and controlling the reaction time.
  • the primary amino group on the arginine deiminase is 30% to 70% modified by PEG, more preferably 40% to 60% of the primary amino group is modified by PEG.
  • PEG-modified proteins are usually heterogeneous, protein molecules can be linked to varying amounts of PEG molecules, and sometimes a more uniform fraction needs to be isolated and purified.
  • a preferred component is a complex which has an average of about 9 to 12 PEG molecules per ADI subunit.
  • the ADI product is modified with mPEG-tresyl, mPEG-Cl, and then the fraction with a molecular weight of about 200 000 Da is separated by column chromatography, and the composite of about 9-12 PEG molecules can be connected to each ADI subunit on average. Things.
  • the therapeutically effective dose of the compound of the present invention is a dose effective to inhibit tumor growth. Generally, the treatment is started in a small dose so as to continuously increase the dose until the optimum is optimal.
  • PEG-ADI Prior to injection, PEG-ADI can be mixed with phosphate buffer or any other suitable solution known to those skilled in the art.
  • the PEG-ADI formulation can be administered as a solid (freeze-dried) or liquid formulation as desired.
  • administration of the compound PEG-ADI of the present invention can be accomplished by intraorbital, intranasal, intraperitoneal, parenteral, intravenous, intralymphatic, intratumoral, intramuscular, interstitial, intraarterial, subcutaneous, ocular. Internal, synovial cavity, transepithelial, transdermal administration.
  • a pharmaceutical composition is also passed which typically contains a safe and effective amount of a PEG-ADI of the invention together with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to: I: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical formulation should be compatible with the mode of administration.
  • the pharmaceutical compositions of the present invention can be formulated into injections. The form is prepared, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by a conventional method. Pharmaceutical compositions such as injections, solutions, tablets The capsules are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, from about 1 microgram per kilogram of body weight to about 10 milligrams per kilogram of body weight per day.
  • the PEG-ADI of the present invention may be combined with other treatments. Use together with the agent.
  • a safe and effective amount of PEG-ADI is administered to the mammal, wherein
  • the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 10 milligrams per kilogram of body weight, preferably the dosage is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the PEG-ADI modification obtained by the linkerless PEG modification technique PEG is directly connected to ADI, and the formed C-N bond is very stable.
  • the existing modified ADI with a linking group it has superior comprehensive properties, namely excellent antitumor effect, more stable chemical properties and lower immunogenicity.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually prepared according to the conditions described in the conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer.
  • mPEG-tresyl (tresyl refers to trifluoroethylsulfonyl chloride) was prepared according to the method disclosed in the literature W09506058; mPEG-Cl was prepared according to the method disclosed in the literature W09832466. 'Example 1
  • the gene for arginine deiminase is synthesized according to the sequence of arginine mycoplasma published by T. Ohno et al., Infect. Immun., 58: 3788-3795 (1990), and the open reading frame includes 1230 base pairs (Fig. 1 and SEQ ID NO: 1), wherein 5 codons encoding the tryptophan TGA are unintentional codons of E. coli and changed to TGG.
  • SEQ ID NO: 1 encodes a 410 amino acid arginine deiminase (as shown in SEQ ID NO: 2 and Figure 2).
  • the expression and renaturation in E. coli were carried out as follows: Specifically, the synthesized ADI gene was inserted into the pET32 vector (Novagen) Spel, BamHI site, and the expression plasmid pET32-ADI was constructed. pET32-ADI was transformed into a conventional E. coli strain BL21, and the transformant was cultured in 500 ml of LB medium, and expression was induced with IPTG of 1.0 M for 4 hours. The bacteria were disrupted by sonication and the inclusion bodies were collected by centrifugation.
  • the inclusion bodies were denatured with buffer (50 mM Tris-Cl, pH 8.5, 6 M guanidine hydrochloride 10 mM dithiothreitol) at 37 ° C for 1 hour.
  • the dissolved supernatant was renatured in 10 volumes of pH 7.5, 10 mM phosphate buffer, and incubated at 15 ° C for 90 hours. Time. Purified by DEAE-Sepharose and arginine affinity chromatography.
  • the purified product was 45 kD in denaturing SDS-PAGE and 90 kD in non-denaturing electrophoresis, indicating that the arginine deiminase was present as a dimer. N-terminal sequencing indicated that the methionine encoded by the translation initiation codon was removed.
  • the Km value of ADI in combination with arginine is about 0.3 mM, and the optimal enzyme reaction condition is 41 ° C, pH 6.4.
  • the stability test showed that the enzyme activity remained above 50% under physiological conditions for 5 days.
  • the ADI prepared in Example 1 and the TMPEG having an average molecular weight of 5000 Da were used in this example.
  • the crosslinking reaction of PEG with ADI was carried out at pH 7.5, 50 mM phosphate buffer containing 0.125 M sodium chloride, and the mass ratio of PEG to ADI was 30:1, and stirred at room temperature for 2 hours. After the completion of the reaction, the unbound PEG in the mixture was dialyzed off by an ultrafiltration membrane, and the degree of modification and enzyme activity were measured. The following experiment was used to determine the degree of modification of arginine deiminase:
  • BUN assay buffer 10 ⁇ l sample was placed in a 96-well microtiter plate, 40 ⁇ l of BRN assay buffer was added to 0.5 mM arginine, covered with a culture plate cap, and warmed at 37 ° C. Foster for 15 minutes. 20ul of complete BUN reagent (Sigma Diagnostics) was added and the plates were incubated for 10 minutes at 100 C. The plates were then cooled to 22 and analyzed at 490 nm using a microplate reader (Biored). The amount of enzyme that can convert the amount of lu mol L-arginine to L-citrulline per minute is 1.0 IU. The enzyme activity before and after the modification was measured by this method. The protein content was determined by Coomassie Brilliant Blue method, and the specific activity of the enzyme preparation before and after the modification was calculated.
  • the total degree of modification of the conjugate was 51%, and the residual enzyme activity after modification accounted for 57% of the initial enzyme activity.
  • the conjugate was further purified by Sephacryl S-300HR (Pharmacia) gel chromatography to collect components having a molecular weight of about 200,000 Da (note: in the composite, 9-12 PEG was attached per ADI in the complex. Molecular), used in animal experiments.
  • Sephacryl S-300HR Pulharmacia gel chromatography
  • Example 1 The ADI prepared in Example 1 and the mPEG-Cl having an average molecular weight of 5000 Da were used in this example.
  • crosslinking was carried out by a different method, and the same linkerless PEGylated ADI as in Example 2 was also obtained.
  • the cross-linking reaction of raPEG-Cl with ADI was carried out in a phosphate buffer of pH 7.0, 20 mM, and the molar ratio of PEG to ADI was about 40:1, and the mixture was stirred at room temperature for 2 hours. After the completion of the reaction, the unbound PEG in the mixture was dialyzed off by an ultrafiltration membrane, and the degree of modification and enzyme activity were measured.
  • the conjugate was further purified by Sephacryl S-300HR (Pharmacia) gel chromatography, and components having a molecular weight of about 200, OOO Da were collected for animal experiments.
  • Example 2 The ADI prepared in Example 1 and the mPEG having an average molecular weight of 12,000 Da were used in this example.
  • the cross-linking reaction of mPEG-Cl with ADI was carried out in ⁇ 7 ⁇ 0, 20 mM phosphate buffer, and the molar ratio of PEG to ADI was about 40:1, and the mixture was stirred at room temperature for 2 hours.
  • the unbound PEG in the mixture was dialyzed off by an ultrafiltration membrane, and the degree of modification and enzyme activity were measured. It was purified by Sephacryl S-300HR (Pharmacia) gel chromatography to collect a component having a molecular weight of about 300, OOO Da.
  • the total modification of the conjugate was 41%, and the residual enzyme activity after modification accounted for 38% of the initial enzyme activity.
  • the molecular weight of 200, OOO Da PEG-ADI conjugate prepared in Example 2 was used for the determination of circulating half-life.
  • the circulating half-life is measured by the decrease in serum arginine concentration and the increase in citrulline concentration after a single administration.
  • male BDFi Rats were administered intravenously in a single dose of 5 IU/mouse.
  • blood was taken from the tail vein.
  • Serum was prepared by conventional methods and stored at -70 ⁇ . The samples were treated with NDB-F fluorescent reagent during the measurement, and the contents of arginine and citrulline were determined by HPLC on a u-bondapack C 18 column.
  • the immunogenicity of the native ADI, the molecular weight of 200, OOODa PEG- ADI conjugate prepared in Example 2 was determined as follows: 0.5 IU of natural or modified ADI was administered intravenously for 12 weeks, once a week, Immunize BalbC mice. At 4, 8, and 12 weeks after the start of the experiment, blood was taken from the eye of the animal, and the serum was separated and stored at -70C, and the antibody against ADI was measured by ELISA. The antibody titer was defined as the highest dilution of serum that was 2 times higher than the background absorbance 0D value. Table II Antibody titers of ADI, PEG-ADI immunized mice
  • PEG5000-ADI Example 2
  • mice in each group were injected with 10 6 SK-Hepl human hepatoma cells. After 2 weeks of tumor growth, intravenous injection was performed with 5. OIU of PEG5000-ADI once a week. The mortality of the control group and the treatment group was counted weekly.

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Abstract

La présente invention concerne une arginine déiminase modifiée n’ayant aucun groupe lieur, c’est-à-dire un composé de formule (I) : ADI-(PEG)n. Dans ladite formule, PEG représentant un polyéthylène glycol ayant un poids moléculaire moyen se situant dans un intervalle de 1 000 à 2 0000 Dalton. ; ADI représentant l’arginine déiminase ; le symbole « - » représentant une liaison covalente entre PEG et ADI ; et n étant un nombre entier se situant dans un intervalle de 2 à 30. L’invention propose également un procédé de production de l’arginine déiminase modifiée et d’une composition pharmaceutique. Le composé de l’invention présente de bien meilleures performances totales, par exemple d’excellents effets anti-tumoraux, une bonne stabilité chimique et une faible immunogénicité.
PCT/CN2004/000933 2004-08-11 2004-08-11 Arginine déiminase modifiée WO2006015512A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI784982B (zh) * 2016-11-02 2022-12-01 開曼群島商北極星藥業集團股份有限公司 聚乙二醇化精胺酸脫亞胺酶之調配物
EP4153167A4 (fr) * 2020-06-29 2024-02-28 Vision Global Holdings Limited Méthode de traitement de sous-types de lma à l'aide d'agents de déplétion de l'arginine

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WO1998032466A1 (fr) * 1997-01-29 1998-07-30 Polymasc Pharmaceuticals Plc Procede de p.e.g.ylation
WO2002044360A2 (fr) * 2000-11-28 2002-06-06 Phoenix Pharmacologics, Inc. Arginine deiminase modifiee
WO2004046309A2 (fr) * 2002-11-18 2004-06-03 Phoenix Pharmacologics, Inc. Methodes d'inhibition d'une replication virale in vivo

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* Cited by examiner, † Cited by third party
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TWI784982B (zh) * 2016-11-02 2022-12-01 開曼群島商北極星藥業集團股份有限公司 聚乙二醇化精胺酸脫亞胺酶之調配物
EP4153167A4 (fr) * 2020-06-29 2024-02-28 Vision Global Holdings Limited Méthode de traitement de sous-types de lma à l'aide d'agents de déplétion de l'arginine

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