WO2019111791A1 - Oligonucléotide antisens apte à annuler la rétention d'introns dans le gène de la dystrophine - Google Patents

Oligonucléotide antisens apte à annuler la rétention d'introns dans le gène de la dystrophine Download PDF

Info

Publication number
WO2019111791A1
WO2019111791A1 PCT/JP2018/043864 JP2018043864W WO2019111791A1 WO 2019111791 A1 WO2019111791 A1 WO 2019111791A1 JP 2018043864 W JP2018043864 W JP 2018043864W WO 2019111791 A1 WO2019111791 A1 WO 2019111791A1
Authority
WO
WIPO (PCT)
Prior art keywords
antisense oligonucleotide
dystrophin gene
pharmaceutically acceptable
seq
sequence
Prior art date
Application number
PCT/JP2018/043864
Other languages
English (en)
Japanese (ja)
Inventor
雅文 松尾
小泉　誠
Original Assignee
第一三共株式会社
学校法人神戸学院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 第一三共株式会社, 学校法人神戸学院 filed Critical 第一三共株式会社
Publication of WO2019111791A1 publication Critical patent/WO2019111791A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to antisense oligonucleotides that eliminate intron retention of the dystrophin gene.
  • Treatment of solid tumors in children generally involves chemotherapy in addition to surgical removal of the tumor. Although there is a case that the tumor can be resolved successfully in response to such treatment, there are not a few cases where the enlargement of the tumor is seen despite the administration of various chemotherapeutic agents.
  • Rhabdomyosarcoma (Rhabdomyosarcoma: RMS) is one of the typical malignancies that develop in children. Improvements in therapy have resulted in tumor regression in many cases, but the rest are resistant to current therapies and have high mortality rates.
  • Neuroblastoma is also one of the typical malignancies of children of sympathetic origin. Chemotherapy is performed in addition to surgical resection as well as RMS. However, there are examples showing treatment resistance, and the mortality rate is high. For such treatment-resistant solid tumors, development of novel and novel therapeutic methods is urgently required.
  • the dystrophin gene is the largest human gene of 4200 kb and encodes a 14 kb transcript consisting of 79 exons. There are at least eight promoters within the gene, each of which produces a tissue specific dystrophin isoform.
  • Dystrophin Dp71 is the smallest isoform produced from the promoter in intron 62 and is expressed in many tissues. Some of the 78 introns of the dystrophin gene are as large as several hundred kb, but the 11 introns are smaller than 1000 bp and smaller than the other introns.
  • the dystrophin gene is expressed in skeletal muscle and produces dystrophin. This dystrophin gene abnormality results in the onset of muscular dystrophy. Skeletal muscle dystrophin mRNA has been considered to be only mature mRNA for expressing dystrophin. However, analysis of splicing of 11 small introns for skeletal muscle dystrophy mRNA revealed that immature mRNA was unexpectedly present in skeletal muscle (Nishida A, Minegishi M, Takeuchi A, Niba ET, Awano H, Lee T, Iijima K, Takeshima Y, Matsuo M. Tissue- and case-specific retention of intron 40 in mature dystrophin mRNA. J Hum Genet.
  • Non-patent Document 1 2015 Jun; 60 (6) : 323-33 (Non-patent Document 1)). That is, when primers for the region of intron 40 are designed on the exons on both sides and RT-PCR is performed, the retained mRNA left behind as it is without being cut off is a part of the amplification product. was detected. In this sequence, a stop codon is generated within the amino acid open reading frame of dystrophin mRNA, and this mRNA was mRNA without dystrophin-producing ability.
  • SH-SY5Y cells are neuroblastoma-derived cells and are widely used as a model of neural cells.
  • the intron 40 region of dystrophin mRNA was analyzed by RT-PCR in this cell, and three products were obtained (Nishida A, Minegishi M, Takeuchi A, Awano H, Niba ET, Matsuo M.
  • Neuronal SH-SY5Y cells use of C-dystrophin promoter coupled with exon 78 skipping and display multiple patterns of alternative splicing involving two intronic insertion events. Hum Genet. 2015 Sep; 134 (9): 993-1001 (non-patent document 2)).
  • An object of the present invention is to provide a compound capable of eliminating intron retention of the dystrophin gene and promoting the expression of dystrophin.
  • the present inventors assumed that by using this antisense oligonucleotide, the expression of dystrophin can be promoted if this intron retention is eliminated.
  • LESE large exon splicing enhancer
  • the gist of the present invention is as follows.
  • a therapeutic agent for a disease having intron retention of a dystrophin gene which comprises an antisense oligonucleotide capable of eliminating intron retention of a dystrophin gene, a pharmaceutically acceptable salt or solvate thereof.
  • the therapeutic agent according to (1), wherein the intron is intron 40.
  • the antisense oligonucleotide which can eliminate intron retention of the dystrophin gene is described in (1) or (2) which contains a sequence complementary to all or part of the following sequences present in the exon 41e of the dystrophin gene Therapeutic agent.
  • the sequence of the antisense oligonucleotide includes all or part of 3 'tccctgttgtc 5' (SEQ ID NO: 8) (however, t in the sequence may be u) of (1) to (4)
  • the therapeutic agent according to any one of (7) to (9), wherein the modification of the phosphodiester bond is phosphorothioate.
  • the disease having intron retention of dystrophin gene is at least one selected from the group consisting of neuroblastoma, rhabdomyosarcoma, mesothelioma, gastric cancer and brain tumor (1) to (10) Therapeutic agent described in.
  • Antisense oligonucleotide capable of eliminating intron retention of dystrophin gene, pharmaceutically acceptable salt or solvate thereof.
  • the sequence of the antisense oligonucleotide contains all or part of 3 'tccctgttgtc 5' (SEQ ID NO: 8) (provided that t in the sequence may be u) (12) to (14) The antisense oligonucleotide, pharmaceutically acceptable salt or solvate thereof according to any of the foregoing.
  • a pharmaceutical composition comprising the antisense oligonucleotide according to any of (12) to (16), a pharmaceutically acceptable salt or solvate thereof.
  • a preparation for oral or parenteral administration which comprises an antisense oligonucleotide capable of eliminating intron retention of a dystrophin gene, a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides an antisense oligonucleotide that eliminates intron retention of the dystrophin gene.
  • the present specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2017-235359, which is the basis of the priority of the present application.
  • the result of the cell transfer experiment of AO41E-LESE is shown.
  • the cell introduction experiment result of AO88 is shown.
  • transduction to CRL2061 cell is shown.
  • transduced cell is shown.
  • the result of a scratch test (test example 4) is shown.
  • the results of cell migration and invasion assay (Test Example 5) are shown.
  • the result of a soft agar colony formation assay (Test Example 6) is shown.
  • the results of the plate colony formation assay (Test Example 7) are shown.
  • the cell-introduction experiment result of AO41 E- LESE, 3, 4, and 5 is shown (cancellation of intron 40 retention).
  • the present invention provides a therapeutic agent for a disease having intron retention of a dystrophin gene, which comprises an antisense oligonucleotide capable of eliminating intron retention of the dystrophin gene, a pharmaceutically acceptable salt or solvate thereof.
  • intron retention refers to the fact that all or part of an intron sequence remains in mature mRNA without being cut off during splicing.
  • Intron retention of the dystrophin gene was reported to occur in intron 40 (Non-patent Document 1). It is then clarified that the intron 31 or 70 is also present.
  • Such retention of intron 40 is also detected in Rhabdomyosarcoma-derived cells (Niba ETE, Yamanaka R, Rani AQM, Awano H, Matsumoto M, Nishio H, et al: DMD transcripts in CRL-2061 rhabdomyosarcoma cells Cancer cell Int 2017, 17: 58.).
  • Diseases having intron retention of the dystrophin gene include neuroblastoma, rhabdomyosarcoma, mesothelioma, stomach cancer, brain tumor and the like, and the present invention can treat these diseases, but is limited thereto. It does not mean that
  • the therapeutic agent of the present invention comprises, as an active ingredient, an antisense oligonucleotide capable of eliminating intron retention of the dystrophin gene, a pharmaceutically acceptable salt or solvate thereof.
  • an antisense oligonucleotide capable of eliminating intron retention of the dystrophin gene one comprising a sequence complementary to all or part of the following sequences present in the exon 41 e of the dystrophin gene can be exemplified.
  • 5 'ggatgacaata aagg gacaaca gcctttgaaattttgagag 3' The nucleotide sequence of SEQ ID NO: 1 is deduced to be a sequence comprising LESE (large exon splicing enhancer).
  • a large exon splicing enhancer is considered to be a sequence that aids in the recognition of very large size exons.
  • Antisense oligonucleotides capable of eliminating intron retention of the dystrophin gene are the second g to the 34th (counting from the 3 'end 8th) counting from the 5' end of the sequence of SEQ ID NO: 1
  • a sequence complementary to a sequence consisting of a plurality of consecutive nucleotides in the nucleotide sequence up to t may be included.
  • the number of bases of the antisense oligonucleotide is suitably 15 to 30, preferably 15 to 21, and more preferably 16 to 20.
  • a part of the sequence is usually 80% or more of the whole sequence, preferably 85%, more preferably 90%, most preferably 94%. It is.
  • SEQ ID Nos: 2-5 are the nucleotide sequences of 41E-LESE-1, 41E-LESE-3, 41E-LESE-4 and 41E-LESE-5 (see Examples below), respectively.
  • the nucleotides constituting the antisense oligonucleotide may be either naturally occurring DNA, naturally occurring RNA, or modified forms thereof, but it is preferable that at least one is a modified nucleotide.
  • Modified nucleotides include those in which sugar is modified (for example, those in which D-ribofuranose is 2'-O-alkylated, those in which D-ribofuranose is 2'-O, 4'-C-alkylenated) And phosphodiester bond modified (eg, thioated), base modified, and combinations thereof can be exemplified.
  • At least one D-ribofuranose constituting an antisense oligonucleotide that is 2'-O-alkylated or 2'-O, 4'-C-alkylenated has high avidity to RNA
  • higher therapeutic effects can be expected than natural nucleotides (ie, oligo DNAs, oligo RNAs).
  • those in which at least one phosphodiester bond constituting an oligonucleotide is thioated are expected to have a higher therapeutic effect than natural nucleotides (ie, oligo DNAs, oligo RNAs) because they are highly resistant to nucleases. it can.
  • Oligonucleotides containing both the modified sugar and the modified phosphate as described above can be expected to have a higher therapeutic effect because they are more resistant to nucleases.
  • examples of sugar modifications include modification of the hydroxyl group at the 2'-position, eg, 2'-O-alkylation of D-ribofuranose (eg, 2'-O-methylation, 2'- O-aminoethylated, 2'-O-propylated, 2'-O-allylated, 2'-O-methoxyethylated, 2'-O-butylated, 2'-O-pentylated, 2'- O-propargylation, etc.), 2′-O, 4′-C-alkylenation of D-ribofuranose (eg, 2′-O, 4′-C-ethylenation, 2′-O, 4′-C- Methyleneation, 2'-O, 4'-C-Propylation, 2'-O, 4'-C-Tetramethylenation, 2'-O, 4'-C-Pentamethyleneation etc.), D-ribof
  • examples of modification of phosphodiester bond can include phosphorothioate bond, methylphosphonate bond, methylthiophosphonate bond, phosphorodithioate bond, phosphoroamidate bond and the like.
  • Examples of base modification include 5-methylation, 5-fluorination, 5-bromination, 5-iodination, N4-methylation, 5-demethylation of thymine (uracil), 5-fluorination, 5-bromination, 5-iodination, N6-methylation of adenine, 8-bromination, N2-methylation of guanine, 8-bromination and the like can be mentioned.
  • Antisense oligonucleotides can be prepared according to the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)) using a commercially available synthesizer (for example, model 392 according to the phosphoramidite method of Perkin-Elmer). Can be synthesized.
  • the phosphoroamidite reagent used in that case includes naturally occurring nucleosides and 2′-O-methyl nucleosides (ie, 2′-O-methyl guanosine, 2′-O-methyl adenosine, 2′-O-methyl cytidine, For 2′-O-methyluridine), commercially available reagents can be used.
  • the 2'-O-alkyl guanosine having 2 to 6 carbon atoms in the alkyl group, adenosine, cytidine and uridine are as follows.
  • 2'-O-aminoethyl guanosine, adenosine, cytidine, uridine can be synthesized according to the literature (Blommers et al. Biochemistry (1998), 37, 17714-17725.).
  • 2'-O-propyl guanosine, adenosine, cytidine, uridine can be synthesized according to the literature (Lesnik, E. A. et al. Biochemistry (1993), 32, 7832-7838.).
  • 2′-O-methoxyethyl guanosine, adenosine, cytidine, uridine can be synthesized according to the patent (US Pat. No. 6,261,840) or the literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504.).
  • 2'-O-butyl guanosine, adenosine, cytidine, uridine can be synthesized according to the literature (Lesnik, E. A. et al. Biochemistry (1993), 32, 7832-7838.).
  • 2'-O-pentyl guanosine, adenosine, cytidine, uridine can be synthesized according to the literature (Lesnik, E. A. et al. Biochemistry (1993), 32, 7832-7838.).
  • 2'-O 4'-C-methylene guanosine, adenosine, cytidine, 5-methyl cytidine and thymidine, 2'-O having 2 to 5 carbon atoms in the alkylene group according to the method described in WO 99/14226.
  • the 4, 4′-C-alkylene anosine, adenosine, cytidine, 5-methyl cytidine and thymidine can be prepared according to the method described in WO 00/47599.
  • a 2'-deoxy-2'-C, 4'-C-methyleneoxymethyleneated nucleoside of D-ribofuranose is synthesized according to the literature (Wang, G. et al. Tetrahedron (1999), 55, 7707-7724 it can.
  • S-cEt (constrained ethyl) can be synthesized according to the literature (Seth, P.P. et al. J. Org. Chem (2010), 75, 1569-1581.).
  • AmNA can be synthesized according to the literature (Yahara, A. et al. ChemBioChem (2012), 13, 2513-2516.) Or WO 2014/109384.
  • uracil (U) and thymine (T) are compatible. Both uracil (U) and thymine (T) can be used for base pairing with the complementary strand adenine (A).
  • Antisense having a phosphorothioate bond by coupling a phosphoroamidite reagent and then reacting a reagent such as sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthan hydride or the like Oligonucleotides can be synthesized (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / WO 98/54198).
  • CPG controlled pore glass
  • 2′-O-methyl nucleosides are linked
  • 2'-O, 4'-C-methylene guanosine, adenosine, 5-methyl cytidine and thymidine 2'-O having 2 to 5 carbon atoms in the alkylene group according to the method described in WO 99/14226.
  • nucleosides prepared according to the method described in WO 00/47599 are described according to the literature (Oligonucleotide Synthesis, Edited by MJ Gait, Oxford University Press, 1984), It can bind to CPG.
  • modified CPG described in Example 12b of JP-A-7-87982
  • Antisense oligonucleotides may be used in the form of pharmaceutically acceptable salts.
  • “Pharmaceutically acceptable salt” refers to a salt of an antisense oligonucleotide, such as sodium salt, potassium salt, alkali metal salt such as lithium salt, calcium salt, magnesium salt and the like Alkaline earth metal salts, aluminum salts, iron salts, zinc salts, copper salts, nickel salts, metal salts such as cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts Glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt,
  • the antisense oligonucleotide and its pharmaceutically acceptable salt may also exist as a solvate (eg, hydrate), and may be such a solvate.
  • Antisense oligonucleotide, pharmaceutically acceptable salt or solvate thereof is mixed with itself or a suitable pharmaceutically acceptable excipient, diluent, etc., and is tablet, capsule, granule, powder Alternatively, they can be administered orally by syrup or the like, or parenterally by injection, suppository, patch or external preparation.
  • excipients for example, sugar derivatives such as lactose, sucrose, sucrose, mannitol and sorbitol; starch derivatives such as corn starch, potato starch, alpha starch and dextrin; and cellulose derivatives such as crystalline cellulose Gum arabic; dextran; organic excipients such as pullulan; light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, silicate derivatives such as magnesium metasilicate aluminium; phosphate such as calcium hydrogen phosphate; calcium carbonate Carbonates; inorganic excipients such as sulfates such as calcium sulfate), lubricants (eg stearic acid; calcium stearate, metal salts of stearic acid such as magnesium stearate; talc; colloidal silica Bead wax, wax like gay wax Abasic acid, adipic acid, sulfuric acid salt such as sodium sulfate, glycol, fumaric acid, sodium
  • Anionic surfactants cationic surfactants such as benzalkonium chloride; polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, nonionic surfactants such as sucrose fatty acid esters, etc.), stabilizers (P-hydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosers ; Dehydroacetic acid; and sorbic acid), corrigents (e.g., sweeteners commonly used, acidulant, flavor, etc.), are prepared in a known manner by using additives such as diluents.
  • cationic surfactants such as benzalkonium chloride
  • polyoxyethylene alkyl ethers polyoxyethylene sorbitan fatty acid esters
  • nonionic surfactants such as sucrose
  • the therapeutic agent of the present invention may contain 0.1 to 250 ⁇ moles / ml of antisense oligonucleotide, a pharmaceutically acceptable salt or solvate thereof, preferably 0.2 to 150 ⁇ moles / ml of antisense.
  • An oligonucleotide, or a pharmaceutically acceptable salt or solvate thereof more preferably an antisense oligonucleotide of 0.5 to 100 ⁇ moles / ml, a pharmaceutically acceptable salt or solvate thereof, Even more preferably, 1 to 50 ⁇ moles / ml of an antisense oligonucleotide, a pharmaceutically acceptable salt or solvate thereof, 0.02 to 10% w / v carbohydrate or polyhydric alcohol and 0.01 to 0.4% w
  • a pharmaceutically acceptable surfactant of / v may be included.
  • carbohydrate monosaccharides and / or disaccharides are particularly preferable.
  • these carbohydrates and polyhydric alcohols include glucose, galactose, mannose, lactose, maltose, mannitol and sorbitol. These may be used alone or in combination.
  • Preferred examples of the surfactant include polyoxyethylene sorbitan mono- to tri-ester, alkylphenyl polyoxyethylene, sodium taurocholate, sodium cholate, and polyhydric alcohol ester.
  • polyoxyethylene sorbitan mono- to tri-esters are particularly preferred, and as ester here, oleate, laurate, stearate and palmitate are particularly preferred. These may be used alone or in combination.
  • the therapeutic agent of the present invention may further preferably contain 0.03 to 0.09 M of a pharmaceutically acceptable neutral salt such as sodium chloride, potassium chloride and / or calcium chloride.
  • the therapeutic agent of the present invention can more preferably contain 0.002 to 0.05 M of a pharmaceutically acceptable buffer.
  • a pharmaceutically acceptable buffer examples include sodium citrate, sodium glycinate, sodium phosphate, tris (hydroxymethyl) aminomethane. These buffers may be used alone or in combination.
  • the above therapeutic agents may be supplied in solution.
  • it is usually preferable to freeze it for a certain period of time, etc. It may be used as reconstituted (ie, in distilled water for injection), that is, in a liquid state to be administered. Therefore, the therapeutic agent of the present invention also includes those in a lyophilised state for reconstitution with a solution so that each component is in a predetermined concentration range.
  • amino acids such as albumin and glycine may be further contained.
  • the antisense oligonucleotide, or a pharmaceutically acceptable salt or solvate thereof is administered to humans, for example, about 0.01 to 100 mg / kg of body weight per adult, preferably 0.1 to 100 mg / day.
  • the dose of 20 mg / kg (body weight) may be injected once or divided into subcutaneous injection, intravenous drip infusion, or intravenous injection, but the dosage and frequency of administration may depend on the type of disease, symptoms, age, It may be changed as appropriate depending on the administration method and the like.
  • Administration of the antisense oligonucleotide, a pharmaceutically acceptable salt or solvate thereof to a patient can be performed, for example, as follows. That is, an antisense oligonucleotide, or a pharmaceutically acceptable salt or solvate thereof is produced by a method well known to those skilled in the art and sterilized by a conventional method to prepare, for example, a 125 mg / ml solution for injection. . This solution is instilled, for example in the form of an infusion, in a patient vein such that the dose of antisense oligonucleotide is, for example, 10 mg / kg of body weight. Administration is performed, for example, at intervals of one week, and thereafter this treatment is repeated as appropriate while confirming the therapeutic effect. Other than intravenous administration, direct injection into a tumor or injection into an artery flowing into a tumor may be used.
  • the therapeutic agent of the present invention may be used in combination with other therapeutic agents.
  • the present invention also provides an antisense oligonucleotide, pharmaceutically acceptable salt or solvate thereof which can eliminate intron retention of the dystrophin gene.
  • the antisense oligonucleotides of the present invention, pharmaceutically acceptable salts or solvates thereof are useful because they can be used as medicaments such as therapeutic agents for diseases with intron retention of the dystrophin gene.
  • the present invention also provides a pharmaceutical composition comprising an antisense oligonucleotide capable of eliminating intron retention of a dystrophin gene, a pharmaceutically acceptable salt or solvate thereof.
  • Example 1 HO-T e2s -U m1s -T e2s -C m1s -A e2s -A m1s -A e2s -G m1s -G e2s -C m1s -T e2s -G m1s -T e2s -U m1s -G e2s -U m1s - C e2s -C m1s -C e2s -U m1t -H
  • a 200 nmol RNA program was performed using an automatic nucleic acid synthesizer (MerMade 192X manufactured by BioAutomation).
  • the concentration of solvent, reagent and phosphoramidite in each synthesis cycle is the same as in the case of natural oligonucleotide synthesis, and the solvent, reagent and phosphoramidite of 2'-O-Me nucleoside (adenosine product product No. ANP-5751, The cytidine body product No. ANP-5752, the guanosine body product No. ANP-5753, the uridine body product No. ANP-5754) was from ChemGenes.
  • Example 14 The non-natural type phosphoroamidite is disclosed in Example 14 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-6-N-benzoyladenosine-3'-O- of JP-A-2000-297097).
  • Example 27 (2-cyanoethyl N, N-diisopropyl) phosphoroamidite),
  • Example 27 (5′-O-dimethoxytrityl-2′-O, 4′-C-ethylene-N-isobutyryl guanosine-3′-O -(2-Cyanoethyl N, N-diisopropyl) phosphoroamidite)
  • Example 22 (5'-O-Dimethoxytrityl-2'-O, 4'-C-ethylene-4-N-benzoyl-5-methylcytidine -3'-O- (2-cyanoethyl N, N-diisopropyl) phosphoroamidite)
  • Example 9 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylene-5-methyluridine-
  • the compound of 3'-O- (2-cyanoethyl N, N-diisopropyl) phosphoroamidite) was used
  • the protecting group cyanoethyl group on the phosphorus atom and the protecting group on the nucleic acid base were removed.
  • the mixed solution of oligomers was mixed with 300 uL of Clarity QSP DNA Loading Buffer (manufactured by Phenomenex), and charged on a Clarity SPE 96 well plate (manufactured by Phenomenex).
  • TEAB triethylammonium bicarbonate
  • DCA dichloroacetic acid
  • the compound was identified by negative ion ESI mass spectrometry (calculated: 7066.7733, found: 7066.7759)
  • the nucleotide sequence of the present compound is a sequence complementary to nucleotide number 1002224-1002243 of Homo sapiens dystrophin (DMD) gene (NCBI-GenBank accession No. NG_012232).
  • Example 2 HO-T e2s -U m1s -U m1s -C e2s -A m1s -A m1s -A e2s -G m1s -G m1s -C e2s -U m1s -G m1s -T e2s -U m1s -G m1s -T e2s - C m1s -C m1s -C e2s -U m1t -H
  • the title compound was synthesized in the same manner as in Example 1.
  • the protecting group cyanoethyl group on the phosphorus atom and the protecting group on the nucleic acid base were removed.
  • the mixed solution of oligomers was mixed with 300 uL of Clarity QSP DNA Loading Buffer (manufactured by Phenomenex), and charged on a Clarity SPE 96 well plate (manufactured by Phenomenex).
  • TEAB triethylammonium bicarbonate
  • DCA dichloroacetic acid
  • the compound was identified by negative ion ESI mass spectrometry (calculated: 7030.7733, found: 7030.7767)
  • the nucleotide sequence of the present compound is a sequence complementary to nucleotide number 1002224-1002243 of Homo sapiens dystrophin (DMD) gene (NCBI-GenBank accession No. NG_012232).
  • Example 3 HO-C e2s -A m1s -A e2s -A m1s -G e2s -G m1s -C e2s -U m1s -G e2s -U m1s -T e2s -G m1s -T e2s -C m1s -C 2s -C m1s - Te 2s -U m1s -Te 2s -A m1t -H
  • the title compound was synthesized in the same manner as in Example 1.
  • the compound was identified by negative ion ESI mass spectrometry (calculated: 7103.8161, found: 7103.8156)
  • the base sequence of the present compound is a sequence complementary to nucleotide number 1002221-1002240 of Homo sapiens dystrophin (DMD) gene (NCBI-GenBank accession No. NG_012232).
  • Example 4 HO-A e2s -G m1s -G e2s -C m1s -T e2s -G m1s -T e2s -U m1s -G e2s -U m1s -C e2s -C m1s -C e2s -U m1s -T e2s -U m1s - A e2s -U m1s -T e2s -G m1t -H
  • the title compound was synthesized in the same manner as in Example 1.
  • the compound was identified by negative ion ESI mass spectrometry (calculated: 7083.7522, found: 7083.748)
  • the base sequence of the present compound is a sequence complementary to nucleotide numbers 1002218-1002237 of Homo sapiens dystrophin (DMD) gene (NCBI-GenBank accession No. NG_012232).
  • Example 5 HO-C e2s -U m1s -G e2s -U m1s -T e2s -G m1s -T e2s -C m1s -C 2s -C m1s -T e2s -U m1s -T e2s -A m1s -T e2s -U m1s- G e2s -U m1s -C e2s -A m1t -H
  • the title compound was synthesized in the same manner as in Example 1.
  • the compound was identified by negative ion ESI mass spectrometry (calculated: 7032.7552, found: 7032.7396)
  • the base sequence of the present compound is a sequence complementary to nucleotide number 1002215-1002234 of Homo sapiens dystrophin (DMD) gene (NCBI-GenBank accession No. NG_012232).
  • Test Example 1 Intron 40 Retention Release of Dystrophin Gene in SH-SY5Y Cells by Example Compounds SH-SY5Y cells are purchased from ATCC, and the cells are cultured in 5% CO 2 in air at 37 ° C. Used for the test.
  • the compound AO41E-LESE prepared in the example was transfected into SH-SY5Y cells as follows. 1. Compounds prepared in the example (10 ⁇ g in milli Q) in 100 ⁇ l of Opti-MEM (GIBCO-BRL) 200 pmol was dissolved. 6 ⁇ l plus reagent (GIBCO-BRL) was added to the solution of 2.1 and left at room temperature for 15 minutes. 3. In another tube, 8 ⁇ l Lipofectamine (GIBCO-BRL) was dissolved in 100 ⁇ l Opti-MEM. After the treatment of 4.2, 3 was added to the treatment solution and left at room temperature for 15 minutes. 5.
  • Opti-MEM Opti-MEM
  • the myoblasts on day 4 after induction of differentiation were washed once with PBS, and then 800 ⁇ l of Opti-MEM was added. After the treatment of 6.4, the treatment solution was added to 5. After culturing the cells of 7.6 at 37 ° C. in 5% CO 2 in air for 3 hours, 500 ⁇ l of DMEM (containing 6% HS) was added to each well. 8. The culture was further continued.
  • RNA extraction RNA extraction was performed as follows. 1. The cells transfected with the compounds prepared in the examples were cultured for 1 day, then washed once with PBS, and 500 ⁇ l of ISOGEN (Nippon Gene) was added to the cells. After standing at room temperature for 2.5 minutes, the ISOGEN in the wells was collected in a tube. 3. RNA was extracted according to the ISOGEN (Nippon Gene) protocol. 4. Finally, RNA was dissolved in 20 ⁇ l of DEPW.
  • Reverse transcription reaction was performed as follows. 1. To 2 ⁇ g of RNA, DEPW (sterile water treated with diethyl pyrocarbonate) was added to make 6 ⁇ l. To the solution of 2.1, 2 ⁇ l of random hexamer (20 ⁇ l diluted with 3 ⁇ g / ⁇ l of Invitrogen) was added. Heated at 3.65 ° C. for 10 minutes. 4. Cooled on ice for 2 minutes. 5.
  • DEPW sterile water treated with diethyl pyrocarbonate
  • PCR reaction was performed as follows. 1. After mixing the following components, the mixture was heated at 94 ° C. for 4 minutes. Reverse transcription reaction product 3 ⁇ l, forward primer (10 pmol / ⁇ l) 1 ⁇ l, reverse primer (10 pmol / ⁇ l) 1 ⁇ l, dNTP (attached to TAKARA Ex Taq) 2 ⁇ l, buffer (attached to TAKARA Ex Taq) 2 ⁇ l, Ex Taq (TAKARA) 0.1 ⁇ l, 11 ⁇ l of sterile water. After treatment at 2.94 ° C. for 4 minutes, 35 cycles of treatment at 94 ° C. for 1 minute, 60 ° C. for 1 minute and 72 ° C. for 3 minutes were performed.
  • the base sequences of the forward and reverse primers used in the PCR reaction for detecting the cancellation of the retention of intron 40 are as follows.
  • Forward primer Dys-ex40f 5'- CTGAGCCCAGAGTGAAAAGG-3 '(exon 40) (SEQ ID NO: 6)
  • Reverse primer c41r 5'-TGCGGCCCCCATCCTC AGACAA-3 '(exon 41) (SEQ ID NO: 7) 4.
  • Analysis of PCR reactions was performed using an Agilent Bioanalyzer. After electrophoretic separation, the amount of each band was quantified.
  • Sequence amplification products of the reaction products of the PCR reaction were analyzed by 2% agarose gel electrophoresis. The band of the amplification product is excised from the electrophoresed gel, and the PCR product is subcloned into pT7 Blue-T vector (Novagen), and Thermo SequenqseTM II dye terminator cyc ABI PRISM 310 Genet using le sequencing kit (Amersham Pharmacia Biotec) The sequencing reaction was performed by ic analyzer (Applied Biosystems) to confirm the nucleotide sequence. The reaction procedure followed the attached manual.
  • CCL-136 cells were purchased from ATCC. CCL-136 cells were seeded on a 60 mm culture plate (manufactured by Iwaki) so as to be 2 ⁇ 10 5 cells, and cultured at 37 ° C. under 5.0% carbon dioxide gas for 24 hours (Panasonic Health Care). The compounds of Examples were transfected in the same manner as in Test Example 1, and after 1, 3, 5 and 7 days, the number of cultured cells was manually counted. Trypan blue was used to remove dead cells and cell counts were counted using a hemocytometer.
  • CCL-136 cells were purchased from ATCC. CCL-136 cells were seeded at 2 ⁇ 10 5 on a 6 mm culture plate (manufactured by Iwaki) and cultured at 37 ° C. under 5.0% carbon dioxide gas for 24 hours (Panasonic Health Care). After 24 hours, the example compounds were transfected in the same manner as in Test Example 1.
  • the 6 mm culture plate was cultured in an incubation chamber at 37 ° C. under 5.0% carbon dioxide gas under a time-lapse microscope (cellSens, manufactured by Olympus). Images were taken every 30 minutes for 5 days at 100 ⁇ magnification and analyzed using cellSens software (Olympus) to observe temporal changes in cell morphology.
  • AO41E-LESE of Example 1 was introduced into SH-SY5Y cells, and 24 hours later, mRNA was analyzed by RT-PCR. Before the introduction, two intron 40 retention products were detected as amplification products of the intron 40 region of dystrophin, in addition to the normal splicing product band. That is, a full-length retention band and a band corresponding to exon 40e.
  • FIG. 1a A schematic diagram of the relationship between intron 40 and AO41E-LESE is shown in FIG. 1a.
  • Introduction of AO41E-LESE (50 ⁇ M) of Example 1 caused a large change in RT-PCR products, and the concentration of the bands of the two products containing the intron 40 sequence was reduced.
  • CRL-2061 and CCL-136 were purchased from ATCC as two types of cells derived from RMS, and cultured using the same culture method as in Test Example 1.
  • the AO41E-LESE of Example 1 was introduced into these cultured cells in the same manner as in Test Example 1.
  • AO was introduced into both cells at a concentration of 50 ⁇ M, and dystrophin mRNA produced after 24 hours was analyzed.
  • retention of intron 40 and a band corresponding to exon 41e were amplified in cells not introduced.
  • such intron retention bands disappeared, and only bands corresponding to normal mRNAs (FIG. 4).
  • AO88 the compound of Example 30 described in International Patent No.
  • WO 2004/048570 complementary to the sequence of exon 45 of the dystrophin gene was introduced into CCL-136 cells, and the same analysis was performed. With the introduction of AO88, intron 40 retention and elimination of exon 41e were not observed (FIG. 4). These results indicate sequence specific intron retention of antisense oligonucleotides.
  • Test Example 3 Inhibition of Cell Growth in Rhabdomyosarcoma (RMS) by Example Compounds
  • the AO 41 E-LESE of Example 1 was introduced into CCL-1361 cells in the same manner as in Test Example 2, and the number of cells was measured over 7 days. Change was observed. As a result, the AO41E-LESE non-transfected cells of Example 1 proliferated, and the number of cells linearly increased (FIG. 5). On the other hand, the proliferation of the AO41E-LESE-introduced cells of Example 1 was suppressed and the number of cells was hardly increased.
  • Test Example 5 Cell Migration and Invasion Assay (FIG. 7) Migration / invasion ability was analyzed using CytoSelect 24-well migration and invasion assay kit. The cells migrated into the wells were measured at an absorbance of 570 mm. Both migration ability and infiltration ability decreased in AO41E-LESE transfected cells. In the colony formation reaction, the number of colonies increased to 300 or more with non-administration and AO88 administration, but remained at 80 with AO41E-LESR administration, and a significant colony growth inhibitory effect was observed. Also, in the infiltration analysis, there were many infiltrating cells in the non-administered cases and in the AO88-administered cases. However, significant suppression was observed in patients treated with AO41E-LESE. There were significant differences between the two groups.
  • the present invention can be used to treat diseases having intron retention of the dystrophin gene.
  • ⁇ SEQ ID NO: 1> A sequence presumed to be LESE (large exon splicing enhancer) present in exon 41e of dystrophin gene. 5 'ggatgacaata aagg gacaaca gcc ttt gaaatttt gagag 3' ⁇ SEQ ID NO: 2> This shows the nucleotide sequence of the antisense oligonucleotide (41E-LESE-1) produced in Examples 1 and 2. 3 'tccctgttgtcggaaacttt 5' ⁇ SEQ ID NO: 3> This shows the nucleotide sequence of the antisense oligonucleotide (41E-LESE-3) produced in Example 3.
  • LESE large exon splicing enhancer
  • 3'atttccctgttgtcggaac 5 ' ⁇ SEQ ID NO: 4> This shows the nucleotide sequence of the antisense oligonucleotide (41E-LESE-4) produced in Example 4.
  • 3'gttatttccctgttgtcgga 5 ' ⁇ SEQ ID NO: 5> This shows the nucleotide sequence of the antisense oligonucleotide (41E-LESE-5) produced in Example 5.
  • 3 'actgttatttccctgttgtc 5' ⁇ SEQ ID NO: 6> This shows the nucleotide sequence of forward primer Dys-ex40f used in Test Example 1.
  • nucleotides constituting the antisense oligonucleotides of SEQ ID NOs: 2 to 5 and 8 may be any of natural DNA, natural RNA, DNA / RNA chimera, and modified forms thereof, but at least one of them is a modified nucleotide. Is preferred.

Abstract

L'invention concerne un composé pouvant annuler la rétention d'introns dans le gène de la dystrophine pour favoriser l'expression de la dystrophine. L'invention concerne un oligonucléotide antisens apte à annuler la rétention d'introns dans le gène de la dystrophine, ou un sel ou un solvate pharmaceutiquement acceptable de celui-ci. L'invention concerne également une composition pharmaceutique contenant l'oligonucléotide antisens ou un sel ou solvate pharmaceutiquement acceptable de celui-ci. L'invention concerne encore un agent thérapeutique contre une maladie caractérisée par la rétention d'introns dans le gène de la dystrophine, ledit agent comprenant l'oligonucléotide antisens ou un sel ou solvate pharmaceutiquement acceptable de celui-ci.
PCT/JP2018/043864 2017-12-07 2018-11-28 Oligonucléotide antisens apte à annuler la rétention d'introns dans le gène de la dystrophine WO2019111791A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2017235359 2017-12-07
JP2017-235359 2017-12-07

Publications (1)

Publication Number Publication Date
WO2019111791A1 true WO2019111791A1 (fr) 2019-06-13

Family

ID=66750234

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2018/043864 WO2019111791A1 (fr) 2017-12-07 2018-11-28 Oligonucléotide antisens apte à annuler la rétention d'introns dans le gène de la dystrophine

Country Status (1)

Country Link
WO (1) WO2019111791A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000297097A (ja) * 1999-02-12 2000-10-24 Sankyo Co Ltd 新規ヌクレオシド及びオリゴヌクレオチド類縁体
JP2002501534A (ja) * 1997-05-30 2002-01-15 ハイブリドン・インコーポレイテッド オリゴヌクレオチド合成用新規硫黄転移試薬
JP2002521310A (ja) * 1997-09-12 2002-07-16 エクシコン エ／エス オリゴヌクレオチド類似体
CN101775401A (zh) * 2009-01-14 2010-07-14 北京联合大学生物化学工程学院 中国林蛙功能基因Rd-RNase3序列、构建方法及其氨基酸序列和用途
JP2011032236A (ja) * 2009-08-04 2011-02-17 Dainippon Sumitomo Pharma Co Ltd 腫瘍血管新生阻害剤
JP2017519766A (ja) * 2014-06-16 2017-07-20 ユニバーシティ・オブ・サザンプトン イントロン保持の減少
JP2017522010A (ja) * 2014-06-10 2017-08-10 エラスムス ユニバーシティ メディカルセンター ロッテルダムＥｒａｓｍｕｓ Ｕｎｉｖｅｒｓｉｔｙ Ｍｅｄｉｃａｌ Ｃｅｎｔｅｒ Ｒｏｔｔｅｒｄａｍ ポンペ病の治療に有用なアンチセンスオリゴヌクレオチド

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002501534A (ja) * 1997-05-30 2002-01-15 ハイブリドン・インコーポレイテッド オリゴヌクレオチド合成用新規硫黄転移試薬
JP2002521310A (ja) * 1997-09-12 2002-07-16 エクシコン エ／エス オリゴヌクレオチド類似体
JP2000297097A (ja) * 1999-02-12 2000-10-24 Sankyo Co Ltd 新規ヌクレオシド及びオリゴヌクレオチド類縁体
CN101775401A (zh) * 2009-01-14 2010-07-14 北京联合大学生物化学工程学院 中国林蛙功能基因Rd-RNase3序列、构建方法及其氨基酸序列和用途
JP2011032236A (ja) * 2009-08-04 2011-02-17 Dainippon Sumitomo Pharma Co Ltd 腫瘍血管新生阻害剤
JP2017522010A (ja) * 2014-06-10 2017-08-10 エラスムス ユニバーシティ メディカルセンター ロッテルダムＥｒａｓｍｕｓ Ｕｎｉｖｅｒｓｉｔｙ Ｍｅｄｉｃａｌ Ｃｅｎｔｅｒ Ｒｏｔｔｅｒｄａｍ ポンペ病の治療に有用なアンチセンスオリゴヌクレオチド
JP2017519766A (ja) * 2014-06-16 2017-07-20 ユニバーシティ・オブ・サザンプトン イントロン保持の減少

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 15 September 2017 (2017-09-15), HERSHBERGER, R.E. ET AL.: "Homo sapiens dystrophin (DMD), RefSeqGene (LRG_199) on chromosome X.", XP055616965, retrieved from NCBI Database accession no. NG_012232.1 *
NIBA ET AL.: "DMD transcripts in CRL-2061 rhabdomyosarcoma cells show high levels of intron retention by intron-specific PCR amplification", CANCER CELL INTERNATIONAL, vol. 17, no. 58, 23 May 2017 (2017-05-23), pages 1 - 10, XP055616958, DOI: 10.1186/s12935-017-0428-4 *
NIBAETE ET AL.: "Cryptic splice activation but not exon skipping is observed in minigene assays of dystrophin c.9361+lG>A mutation identified by NGS", JOURNAL OF HUMAN GENETICS, vol. 62, no. 5, April 2017 (2017-04-01), pages 531 - 537, XP055616967, DOI: 10.1038/jhg.2016.162 *
NISHIDA ET AL.: "Neuronal SH-SY5Y cells use the C-dystrophin promoter coupled with exon 78 skipping and display multiple patterns of alternative splicing including two intronic insertion events", HUMAN GENETICS, vol. 134, no. 9, 2015, pages 993 - 1001, XP035526831, DOI: 10.1007/s00439-015-1581-2 *
NISHIDA ET AL.: "Tissue- and case-specific retention of intron 40 in mature dystrophin mRNA", JOURNAL OF HUMAN GENETICS, vol. 60, no. 6, 2015, pages 327 - 333, XP055616962, ISSN: 1434-5161, DOI: 10.1038/jhg.2015.24 *
SAKABE NJ ET AL.: "Sequence features responsible for intron retention in human", BMC GENOMICS, vol. 8, no. 59, 2007, pages 1 - 14, XP021022365, DOI: 10.1186/1471-2164-8-59 *

Similar Documents

Publication Publication Date Title
EP2386636B1 (fr) Médicaments à base d'acide nucléique ena modifiant l'épissage dans un précurseur d'arnm
KR100809166B1 (ko) Ｓｔａｔ3 발현의 안티센스 올리고뉴클레오티드 조절
JP5763539B2 (ja) ５’及び２’ビス置換ヌクレオシド及びそれから製造されるオリゴマー化合物
KR100750788B1 (ko) ｅＩＦ4Ｅ 발현의 조정
AU2004270741B2 (en) Gapped oligomeric compounds having linked bicyclic sugar moieties at the termini
US20040014957A1 (en) Oligonucleotides having modified nucleoside units
US20040014108A1 (en) Oligonucleotides having modified nucleoside units
JP2008501693A (ja) 遺伝子調節で使用するための個別に調節された鎖を有する二本鎖組成物
JP2002508944A (ja) ヒトｍｄｍ２発現のアンチセンス修飾
JP2002541784A (ja) Ｓｔａｔ３発現のアンチセンスオリゴヌクレオチド調節
US20040171566A1 (en) Antisense modulation of p38 mitogen activated protein kinase expression
CN111655851A (zh) 靶向srebp1的反义寡核苷酸
JP2007104971A (ja) ミオスタチンのスプライシング制御アンチセンスオリゴヌクレオチド
WO2019111791A1 (fr) Oligonucléotide antisens apte à annuler la rétention d'introns dans le gène de la dystrophine
WO2001074136A2 (fr) Polynucleotides inhibiteurs de la telomerase
EP3998108A1 (fr) Oligonucléotide antisens permettant de modifier l'épissage pré-arnm de dux4
WO2019240164A1 (fr) Agent thérapeutique contre le dysfonctionnement myocardique
US20040171564A1 (en) Antisense oligonucleotide modulation of human serine/threonine protein phosphatase gene expression
JP6536911B2 (ja) ＣＤ４４遺伝子のバリアントエクソンのスキッピングを誘導し、正常型ＣＤ４４ｍＲＮＡの発現を増加させる核酸医薬
JP7072748B2 (ja) アルポート症候群治療薬
US20040102393A1 (en) Modulation of heat shock protein 90-alpha expression
EP1631235A2 (fr) Compositions et methodes de modulation de l'expression de la proteine b7
MXPA06008869A (en) Antisense oligonucleotide modulation of stat3 expression

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18886757

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18886757

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP