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WO2012137991A1 - Composition pharmaceutique pour la prévention ou le traitement de la mucolipidose ii, comprenant une enzyme lysosomale comme principe actif - Google Patents

Composition pharmaceutique pour la prévention ou le traitement de la mucolipidose ii, comprenant une enzyme lysosomale comme principe actif Download PDF

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Publication number
WO2012137991A1
WO2012137991A1 PCT/KR2011/002317 KR2011002317W WO2012137991A1 WO 2012137991 A1 WO2012137991 A1 WO 2012137991A1 KR 2011002317 W KR2011002317 W KR 2011002317W WO 2012137991 A1 WO2012137991 A1 WO 2012137991A1
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WO
WIPO (PCT)
Prior art keywords
mucofatosis
lysosomal enzyme
treatment
pharmaceutical composition
prevention
Prior art date
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Ceased
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PCT/KR2011/002317
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English (en)
Korean (ko)
Inventor
이성열
박성익
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Medigenebio Corp
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Medigenebio Corp
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Priority to PCT/KR2011/002317 priority Critical patent/WO2012137991A1/fr
Priority to KR1020137026846A priority patent/KR101619815B1/ko
Publication of WO2012137991A1 publication Critical patent/WO2012137991A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating mucodiosis I I comprising a lysosomal enzyme as an active ingredient.
  • Mucolipidosis I l Onucol ipidosis I I; Mycolipidosis I I; Mycolipidosis I I is also called I cell disease and is a metabolic disease inherited by autosomal recessive. Characteristic symptoms are 1) unique facial features, 2) skeletal abnormalities, and 3) mental retardation. Symptoms of Mucofatosis I I are similar to Hurler Syndrome, but appear much more seriously. Symptoms associated with the disease are usually pronounced in infancy, with various lesions on the skull and face and slow growth.
  • Lysosomes are substances in the cytoplasm and contain various enzymes and play an important role in the intracellular digestion of extracellular subtypes by phagocytosis as well as metabolism of intracellular components. Lack of these enzymes results in the accumulation of mucoids in tissue cells.
  • Mucofatosis II is caused by a mutation in the GNPTA gene located on chromosome 4 (4q21-q23). GNPTA gene mutations result in a deficiency of an enzyme called UDP—N-acetylglucosamine-1-phosphortransferase, which is involved in the synthesis of mannose-6-phosphate. Lack of this enzyme causes cells with lysosomal enzyme levels to It falls within and rises in blood and body fluids. Symptoms of Mucofatosis II are caused by a lack of lysosomal enzymes, which result in the accumulation of certain fatty substances and carbohydrate complexes in the tissues and cells of the body.
  • Mucofatosis II can be diagnosed as a decrease in the activity of UDP-N-acetylglucosamine-1-phosphortransferase on prenatal amniotic fluid and chorionic membrane tests. And special laboratory test findings.
  • the activity of UDP-N-acetylglucosamine-1-phosphorase enzyme in leukocytes or fibroblasts can be measured. Levels of lysosomal enzymes are particularly elevated in serum and decreased in fibroblasts.
  • supportive therapy such as nutrition, treatment of recurrent respiratory tract infections, etc. is best, and no specific treatment has been developed.
  • the present invention provides a pharmaceutical composition for preventing or treating Mucofatosis II, comprising a lysosomal enzyme as an active ingredient. Since the pharmaceutical composition comprising the lysosomal enzyme according to the present invention improves the clinical findings of mucofatosis II, it can be usefully used for the prevention or treatment of mucofatosis II.
  • 1 is a schematic diagram of a targeting vector for the production of GNPTA knockout mice. In the figure, the black square bars represent exons and the other parts represent introns. 2 is a detailed view for the construction of a targeting vector for the production of GNPTA knockout mice.
  • FIG. 3 shows the results of Southern blotting of embryonic stem cell clones identified as having the GNPTA knockout gene.
  • WT represents the wild type GNPTA gene and K0 represents the GNPTA knockout gene.
  • FIG. 5 is a photograph comparing normal mice and mucofatosis II mice at 5 weeks of age.
  • the upper mouse is Mucofatosis II mouse and the lower mouse is normal mouse.
  • 6A and 6B show the results of bone characteristics using normal-energy X-ray absorpt iometry (DXA) in normal mice and mucofatosis I I model mice, respectively.
  • Figure 7 shows the results of measuring the amount of lysosomal enzymes in the serum according to the culture time in normal mice (wi ld-type) and mucofatosis II mice (mutant).
  • lysosomal enzyme refers to a lysosomal enzyme obtained through lysosomal fractionation and purification from normal tissue isolated from a mammal, preferably a human.
  • the "lysosomal enzyme” used in the present invention is a complex lysosomal enzyme including two or more enzymes among about 50 known lysosomal enzymes, except for a single lysosomal enzyme. That is, the "lysosomal enzyme” used in the present invention may be used interchangeably with the “complex lysosomal enzyme", the "lysosomal enzyme complex” or the "lysosomal rich fract ion". Lysosomal enzymes of the present invention can be obtained in any tissue in vivo, including placenta, liver and the like.
  • GNPTA gene refers to GNPTA
  • GNPTA knockout gene means that a part of the gene involved in the synthesis of GNPTA is deleted and is not normally expressed.
  • the present invention refers to a gene characterized in that the exon 20 part of the axon 12 in the GNPTA gene is deleted.
  • GNPTA Knockout Gene "Modified GNPTA Gene”
  • GNPTA Mutation Gene “nests can be used interchangeably.
  • Mucofatosis II mouse refers to a mouse model showing symptoms of mucofatosis II.
  • the mouse model is used to search for substances that have an effect on Mucofatosis II.
  • the most important symptom of mucopyridosis II is a condition in which weight is not greatly increased. Therefore, the prophylactic or therapeutic effect of mucofatosis II can be confirmed by the rapid weight gain of the mouse model.
  • the present invention provides a pharmaceutical composition for preventing or treating mucofatosis II, comprising a lysosomal enzyme as an active ingredient.
  • the lysosomal enzyme used as an active ingredient in the pharmaceutical composition according to the present invention can be separated from the human placenta through conventional fractionation and purification procedures known in the art.
  • the lysosomal enzyme is isolated from the human placenta, but can be obtained in the same way from mammals in addition to humans, and obtained from other tissues in vivo, including the liver, which is known to contain lysosomal enzymes in addition to the placenta You may.
  • Lysosomal enzymes used in pharmaceutical compositions according to the invention are described in C. ALQUIER et al. , Biochem. J. (1985) 232, 529-537. Specifically, the method may include the following steps:
  • the lysosomal enzyme comprises two or more of the 50 known lysosomal enzymes.
  • the lysosomal enzymes of the present invention include arylsulfatase A, arylsulfatase B, alpha-galactosidase, beta-galactosidase, beta-glucosidase, nucleososaminidase A, and the like. can do.
  • Arylsulfatase A, arylsulfatase B, alpha-galactosidase, beta-galactosidase and nucleosamidase A are each present in an amount of 0.01 to 1.0 X 10 "3 units per mg of lysosomal enzyme of the present invention. It may be included as, beta-glucosidase may be included in an amount of 5 to 20 X 10 "3 units, but is not limited to the above content.
  • the effect of lysosomal enzyme isolated according to the present invention on mucolipidosis can be confirmed through in vivo experiments using Mucolipidosis II mouse model.
  • the mucofatosis ⁇ mouse model may be a knock-out mouse obtained by removing a part of the exon of the GNPTA gene of a wild-type mouse.
  • the lysosomal enzyme according to the present invention has an effect of improving mucofatosis. In particular, it prevents weight gain failure, the most important indicator of mucofatosis II, and shows a steady weight gain effect.
  • the compositions of the present invention can be prepared in pharmaceutical formulations according to conventional methods. In the preparation of the formulation, it is preferred that the lysosomal enzyme is mixed or diluted with the carrier or enclosed in a carrier in the form of a container.
  • the carrier when used as a diluent, it may be a solid, semi-solid or liquid substance which acts as a carrier, excipient or medium for the antibody.
  • the formulation may be in the form of tablets, pills, powders, sasei, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectables, sterile powders and the like.
  • Suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, solbi, manni, stag silicate salose, methyl salose, microcrystalline salose, polyvinylpyridone, water, methyl Hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • the formulation may further comprise a layering agent, an antifoaming agent, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.
  • composition of the present invention It can be formulated using methods well known in the art to provide rapid, sustained or delayed release after administration to a mammal. Accordingly, the present invention provides the use of lysosomal enzymes for the manufacture of a medicament for the prevention or treatment of mucofatosis II. Also provided is a method of preventing or treating mucofatosis ⁇ in a mammal, comprising administering a lysosomal enzyme to a mammal in need of prevention or treatment of mucofatosis II.
  • the composition of the present invention can prevent or treat mucofatosis I I by administering to a mammal including a human.
  • the dosage of the composition depends on the subject to be treated, the severity of the disease or condition, the rate of administration and the judgment of the prescribing physician.
  • the lysosomal enzyme is administered via a parenteral route in mammals in an amount of 0.001 to 10 mg / kg (weight) per day, preferably 0.005 to 1 mg / kg (body weight), once a day or divided into mammals. Can be.
  • GCJBP Human placenta sections (GCJBP, South Korea) were passed through a metal sieve (pore size 0.3 mm 3) and then suspended in 1 L TS buffer (10 mM-Tris / HCl, 0.25 M sucrose, pH 7.4). The tissue suspension was centrifuged at 770 Xg for 20 minutes to remove supernatant (SO). It was removed and the precipitate was recovered. The recovered precipitate was resuspended with TS complete layer solution, washed and centrifuged at 770Xg for 20 minutes to recover the precipitate. The obtained precipitate was resuspended in 50 mL of TS complete solution, which was named 'open follicle fraction'.
  • the pellet (P2) was resuspended in 40 mL TS complete solution, re-homogenized and centrifuged under the same conditions.
  • the supernatant obtained in the above process was recovered and centrifuged at 4,000 ⁇ g for 20 minutes to obtain a supernatant (S3) and pellets (P3).
  • the supernatant (S3) was centrifuged at 26,000Xg for 20 minutes to recover the pellet (P4) and the supernatant (S4).
  • the resulting pellets (P4) were resuspended in 15 mL of TS complete solution.
  • Example 2 Analysis of Lysosomal Enzymes Representative components of the lysosomal enzyme obtained in Example 1 were analyzed according to the conventional analytical method as follows. Arylsulfatase A and B were measured spectrophotometrically to measure the enzyme activity by measuring the sulfate released when P-nitrocatechol sulfate was degraded by arylsulfatase A or B. Alpha-galactosidase was measured by measuring the NuF released when MuF-galactoside was hydrolyzed by alpha-galactosidase using fluorescence photometry (f luorometry). Similarly, beta-galactosidase
  • the enzyme activity was measured by measuring the MuF released when MuF-beta-galactopyranoside was hydrolyzed by beta-galactosidase.
  • Beta-glucosidase measured enzyme activity by measuring MuF released when MuF-beta-glucoside was hydrolyzed by beta-glucosidase.
  • Nucleosaminidase A measured the enzyme activity by measuring the MuF released when MuF-glucosamine was hydrolyzed by nucleosaminedase A.
  • the lysosomal enzyme assay showed the highest amount of beta-glucosidase, and arylsulfatase A, arylsulfatase B, alpha-galactosidase and nucleosamynidase A. Was found to be present in trace amounts. Repeated experiments showed that arylsulfatase A, arylsulfatase B, alpha-galactosidase, beta-galactosidase, and nucleosaminidase A were each 0.01 to 1.0 X 10 "3 units per mg of lysosomal enzyme.
  • Example 3 Preparation of Mucofatosis II Mouse Model A mouse model showing Mucofatosis II for verifying the efficacy of the lysosomal enzyme isolated by Example 1 was prepared according to the following method.
  • a targeting vector for deleting axon 20 (about 8.5 kb) from exon 12 of the GNPTA gene was prepared as shown in FIG. 1. Consisting of left arm (10105 bp), MCI promoter and neomycin resistance gene (MCl-neo; -1300 bp), a positive selection marker, and right arm (-5800 bp) Targeting vectors were prepared by ligation of the gene cassette with pOsdupdel vector (Gene Targeting Laboratory).
  • the targeting vector does not express normal GNPTA genes because homologous recombinat ions occur in the complementary fragments, resulting in deletion of the GNTPA gene exon 12 through exon 20, resulting in the loss of a portion of the gene (see FIGS. 1 and 2).
  • the targeting vector was linearized by cleavage with Not I, and then cultured by electroporation into J1 embryonic 21 cells of 129 / SvJ mice. After the incubation, 336 clones that were resistant to G418 and ganciclovir were selected.
  • Example ⁇ 3-3> Preparation of Mucofatosis II Mouse Model
  • the four clones screened in Example ⁇ 3-2> were injected into the 3.5-day pc blastocysts of C57BL6 mice 9-9: L0 cells. Specifically, 3.5 days after male and female mating The females were regenerated by cervical dislocation and the uterus was extracted and 1 ml syringe was used to perfusion 1 ml of injection solution containing 20 mM HEPES, 10% fetal bovine serum, 0.1 mM 2-mercaptoethanol and DMEM.
  • Embryos were isolated from uterine tissue using microglass tubes under a dissecting microscope, and the isolated embryonic embryos were transferred onto 35 mL Petri dishes and the embryonic stem cell clones selected above were introduced into blastocysts of the blastocysts.
  • the clone-implanted blastocysts were transplanted into gestational surrogate mouse uterus to induce the development of chimeric mice, a type of heterotypic hybrids formed from embryonic stem cell clones and C57BL6 mice.
  • the embryonic stem cell-injected blastocyst was transplanted into the surrogate mouse uterus and cultured for about 19 days, thereby transforming the embryonic stem cell-derived transformed cells and the mouse blastocyst-derived cells to GNPTA +/- genotype.
  • Genie produced chimeric mice. Thereafter, the chimeric mice obtained above were crossed with C57BL6 mice six times or more, thereby preparing GNPTA + / + and GNPTA-/-mice in the F1 step, and finally preparing GNPTA-/-nick out mice in which all GNPTA genes were knocked out. It was.
  • Example 4 Analysis of Mucofatosis Pathology in Mucofatosis II Mouse Model Mucofatosis pathology was analyzed while mucofatosis II mice prepared according to Example 3 and normal mice were bred under the same conditions.
  • Body weights of Mucofatosis II mice and normal mice were measured weekly, and the results are shown in FIG. 4.
  • normal mice controls 1 to 4
  • mucofatosis II mouse models homo 1 and 2 of the present invention show that growth is delayed compared to normal mice. appear.
  • Body weight is the most important characteristic that represents Mucofatosis II, the subject with Mucofatosis II does not increase the body weight at all compared to the normal control group based on this can determine whether Mucofatosis II. 4 of the above
  • the results show that the GNPTA knockout mice of the present invention are mouse models representing Mucofatosis II.
  • FIG. 6A shows the DXA results of normal mice
  • FIG. 6B shows the DXA results of the mucodilipidemic II mice prepared in the present invention.
  • the amount of lysosomal enzyme in serum was measured in normal mice and mucofatosis II mice prepared in the present invention. The measurement results are shown in FIG. 7. Degree As shown in Fig. 7, mucofatosis II mice (mutant) were measured in serum compared to normal mice (wi ld-type)
  • Beta galactosidase
  • Example 5 Mucofatosis Pathology Analysis of Mucofatosis II Mouse Model Following Lysosomal Enzyme Treatment Mucofatosis II prepared in Example 3 to confirm the effect of lysosomal enzyme on mucofatosis II
  • the mouse model was examined for weight change. Weight loss is the most important indicator of Mucofatosis II. Since patients with Mucofatosis II rarely gain weight despite a normal diet, a substance that rapidly increases weight by administration is effective for Mucofatosis II. It can be determined that there is.
  • the Mucofatosis II mouse model prepared in Example 3 was divided into 10 experimental groups and 10 control groups, the experimental group was administered with the lysosomal enzyme of the present invention prepared in Example 1, and the control group was administered normal saline. Test substances were administered intravenously in the tail vein for a total of 4 weeks, twice a week, 2 mg per kg body weight of the mouse.

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Abstract

La présente invention concerne une composition pharmaceutique pour la prévention ou le traitement de la mucolipidose II, comprenant un complexe d'enzyme lysosomale. La composition améliore les découvertes cliniques de la mucolipidose II, et permet ainsi d'être utilisée à profit dans la prévention ou le traitement de la mucolipidose II.
PCT/KR2011/002317 2011-04-04 2011-04-04 Composition pharmaceutique pour la prévention ou le traitement de la mucolipidose ii, comprenant une enzyme lysosomale comme principe actif Ceased WO2012137991A1 (fr)

Priority Applications (2)

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PCT/KR2011/002317 WO2012137991A1 (fr) 2011-04-04 2011-04-04 Composition pharmaceutique pour la prévention ou le traitement de la mucolipidose ii, comprenant une enzyme lysosomale comme principe actif
KR1020137026846A KR101619815B1 (ko) 2011-04-04 2011-04-04 리소좀 효소를 유효성분으로 포함하는 뮤코지방증 ii의 예방 또는 치료용 약학 조성물

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PCT/KR2011/002317 WO2012137991A1 (fr) 2011-04-04 2011-04-04 Composition pharmaceutique pour la prévention ou le traitement de la mucolipidose ii, comprenant une enzyme lysosomale comme principe actif

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060057114A1 (en) * 2002-08-13 2006-03-16 Whitley Chester B Methods of using vectors to treat metabolic disorders
US20060083718A1 (en) * 2004-06-16 2006-04-20 University Of Massachusetts Novel therapy for lysosomal enzyme deficiencies

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2004296848A1 (en) * 2003-12-04 2005-06-23 Regents Of The University Of Minnesota Compositions and methods for the treatment of lysosomal storage disorders

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060057114A1 (en) * 2002-08-13 2006-03-16 Whitley Chester B Methods of using vectors to treat metabolic disorders
US20060083718A1 (en) * 2004-06-16 2006-04-20 University Of Massachusetts Novel therapy for lysosomal enzyme deficiencies

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GREWAL, S. ET AL.: "Continued neurocognitive development and prevention of cardiopulmonary complications after successful BMT for I-cell disease: a long-term follow-up report", BONE MARROW TRANSPLANTATION, vol. 32, 2003, pages 957 - 960 *
VOGEL, P. ET AL.: "Comparative Pathology of Murine Mucolipidosis Types II and IIIC", VET PATHOL, vol. 46, 2009, pages 313 - 324 *

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KR20140048105A (ko) 2014-04-23

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