US20100329993A1 - Method of fixing and expressing physiologically active substance - Google Patents

Method of fixing and expressing physiologically active substance Download PDF

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US20100329993A1
US20100329993A1 US12/666,983 US66698308A US2010329993A1 US 20100329993 A1 US20100329993 A1 US 20100329993A1 US 66698308 A US66698308 A US 66698308A US 2010329993 A1 US2010329993 A1 US 2010329993A1
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physiologically active
sirna
active substances
submucous
tissue
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Hiroyuki Yoneyama
Kenji Suzuki
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Niigata University NUC
Stelic Institute and Co Inc
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Publication of US20100329993A1 publication Critical patent/US20100329993A1/en
Priority to US14/158,607 priority Critical patent/US20140135379A1/en
Priority to US15/241,830 priority patent/US20160355818A1/en
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    • 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
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/396—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having three-membered rings, e.g. aziridine
    • 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
    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to methods for retaining and expressing physiologically active substances in a target tissue-specific manner, in which the physiologically active substances are administered to the target submucous tissue.
  • nucleic acid pharmaceutical agents using such a gene or an siRNA (generally termed “nucleic acid”)
  • the nucleic acid administered to the living body needs to continuously produce its effect and be retained over a long period.
  • DDS drug delivery system
  • nucleic acids when nucleic acids are administered to the body as is, they are rapidly degraded and thus fail to work. Accordingly, such nucleic acids are usually administered by using a carrier such as a viral vector, liposome, or atelocollagen as a DDS.
  • a carrier such as a viral vector, liposome, or atelocollagen as a DDS.
  • nucleic acid pharmaceutical agents have a serious disadvantage in that the carrier itself may induce an adverse immune response or such in the body and thus not only the nucleic acid but also carrier must be assessed for its influence on the body.
  • atelocollagen when used as a carrier, induces a hypersensitive immune reaction to calf dermis derived collagen; the instruction manual (Non-patent Document 1) attached to the product named Koken Atelocollagen implant (syringe type) describes that adverse effects were clinically found in 24 of a total of 1,192 patients.
  • Another problem is that, even when a carrier is used, normally, the introduced nucleic acid can only be retained for about one week.
  • Patent Documents 1 to 7 listed below.
  • all of these inventions use carriers.
  • the above-described problems still remain unsolved.
  • Patent Document 1 Japanese Patent Application Kohyo Publication No. (JP-A) 2003-516365 (unexamined Japanese national phase publication corresponding to a non-Japanese international publication)
  • Patent Document 2 JP-A (Kohyo) 2005-538943
  • Patent Document 4 Japanese Patent Application Saikohyo Publication No. (JP-A) WO01-093856 (unexamined Japanese national phase publication corresponding to a Japanese international publication
  • Patent Document 6 Japanese Patent Application Kokai Publication No. (JP-A) 2007-119498 (unexamined, published Japanese patent application)
  • Patent Document 7 (Granted/Registered) Japanese Patent No. 4054352
  • Non-patent Document 1 Instruction manual attached to the product named Koken Atelocollagen implant (syringe type)
  • the present invention provides methods for locally retaining and expressing physiologically active substances at the administration site. Specifically, the present invention provides methods for retaining and expressing physiologically active substances in a target tissue-specific manner, in which the physiologically active substances are administered to target submucous tissue.
  • the present inventors conducted dedicated studies to solve the above-described issues. Specifically, as a method of solving the above problems, the present inventors evaluated establishment of a novel method that produces effects of physiologically active substances without using carriers.
  • the present inventors conducted an experiment of injecting nucleic acids, which are an embodiment of the physiologically active substances, into the submucous tissue of the large intestine in experimental animals such as rats, and evaluated the effect. Common knowledge envisaged that the solution would be rapidly lost due to diffusion or degradation. However, the present inventors obtained an unpredictable result. When the present inventors actually injected solutions containing nucleic acids into submucous tissues, the injected solutions were retained at the administration site without diffusing and produced effects acting as a reservoir. This suggests that the submucous microenvironment is very special and the living body functions like a carrier by utilizing its own environment.
  • the submucous tissue of the intestinal tract has been shown to be much more effective than other matrices, for example, when pancreatic Langerhans islets are cultured in vitro (Tian X H et al. Hepatobiliary Pancreat Dis Int. 4: 524, 2005). Even this shows that submucous tissues have a very special microenvironment. In particular, the submucous pH, sugar chains, and cell composition are assumed to create an environment that is very suitable for maintaining nucleic acids.
  • the present inventors injected iopamidol, India ink, or siRNA, each of which is an embodiment of the physiologically active substances, into the submucous tissue of the large intestine in rats and mice.
  • the result showed that every substance was selectively retained at the injection sites in the submucous tissue of the large intestine.
  • the present inventors discovered that, when siRNA was administered into the submucous tissue of colitis model mice, the increase in expression of the GalNAc4S-6ST gene in the large intestine was significantly suppressed while it had no influence on other normal organs. In addition, it was also revealed that the suppression of GalNAc4S-6ST gene expression in the large intestine resulted in suppression of inflammatory activity and thus strong suppression of intestinal fibrous degeneration. Furthermore, the histological therapeutic effect was also demonstrated based on the finding that the administration significantly suppresses epithelial disruption, infiltration of inflammatory cells into lamina limba and submucosa, and thickening of muscular layer in the large intestinal tissue.
  • the present invention demonstrated that, when nucleic acids are administered endoscopically into the submucous tissues of the large intestine of subjects, the nucleic acids were specifically retained at the administration site over a long period and could continuously produce their effect.
  • the present invention also enables physiologically active substances to produce their effects without assistance of a carrier, and thus has solved the previous problem associated with the use of carriers.
  • the present invention relates to methods for retaining and expressing physiologically active substances in a tissue-specific manner, in which the physiologically active substances are administered to the submucous tissue, and more specifically,
  • the physiologically active substance is selected from nucleic acids, proteins, carbohydrates, lipids, or low-molecular-weight compounds;
  • nucleic acid is an siRNA
  • the present invention also provides:
  • a method for treating or preventing a disease which comprises the step of retaining and expressing a physiologically active substance in a submucous tissue-specific manner, by administering the physiologically active substance to the diseased submucous tissue.
  • the present invention provides a Drug Delivery System (DDS) for maintaining a physiologically active substance (for example, a nucleic acid, protein, carbohydrate, lipid, low-molecular-weight compound, etc.) in a target submucous tissue over a long period, for continuously producing a physiologically active substance useful to the living body, or for continuously removing a physiologically active substance that is harmful to the living body.
  • DDS Drug Delivery System
  • the present invention enables safe and effective retention of physiologically active substances at the administration sites over long periods without assistance of a carrier.
  • physiologically active substances can be retained without considering side effects of carriers as before.
  • physiologically active substances can be retained without considering side effects of carriers as before.
  • physiologically active substances for example, nucleic acid pharmaceutical agents.
  • the above-described pharmaceutical agents have very little systemic side effects, because they specifically produce their effects at the injection sites. Thus, the pharmaceutical agents are much safer than conventional methods.
  • the present invention has the advantage in that physiologically active substances (for example, nucleic acids) can be injected (for treatment) at the same time as diagnosis.
  • physiologically active substances for example, nucleic acids
  • the present invention is also expected to be applicable to the submucous tissues of the nose, subconjunctival tissue, or such, and thus enables actual clinical use of pharmaceuticals for cranial nerve diseases or ophthalmic diseases, in which physiologically active substances have been difficult to deliver in the past.
  • the methods of the present invention have a superior effect than the conventional local administration methods, specifically, enemas, nasal drips, and ocular instillations.
  • FIG. 1 show photographs of histological images 24 hours after injection of FITC-labeled siRNA into the submucous tissues of rat large intestines. The images show retention of the siRNA in the submucous tissues.
  • A an image of the submucous tissue of the large intestine
  • B an image observed under a fluorescence microscope after FITC staining
  • C an image obtained by superimposing A with B.
  • FIG. 2 photographs showing retention of a substance administered into the submucous tissue of the large intestine in normal rats.
  • An X-ray finding showed that iopamidol (white; within circle) was retained at a specific site within the large intestine (left).
  • the upper panels show lateral view images, and the bottom panel shows a magnified image.
  • a CT finding (right) showed that iopamidol was retained at the injection site on the ventral side of large intestine (upper panel) but it was undetectable on the craniad slice (lower panel).
  • FIG. 3 is a photograph showing retention of a substance administered into the submucous tissue of the large intestine in a normal mouse. Carbon particles (black) were retained within the submucous tissue of the large intestine.
  • FIG. 4 show photographs depicting retention of a nucleic acid administered into the submucous tissue of the large intestine in a normal mouse.
  • FITC-labeled siRNA green was retained at a specific site in the large intestine. Images obtained with a fluorescence stereoscopic microscope; the magnification is 8.6-, 39-, or 102-fold from left.
  • FIG. 5 is a graph showing the kinetics of a nucleic acid administered into the submucous tissue of the large intestine in a normal mouse.
  • the graph shows the concentrations of siRNA retained in the large intestine 0.5, 3, 6, and 24 hours after injection into the submucous tissue of the large intestine.
  • the rightmost bar (black) is a positive control, indicating the siRNA concentration before injection.
  • FIG. 6 shows a result of agarose gel electrophoresis after stability test at 37° C.
  • the upper panel shows the pattern of GalNAc4S-6ST siRNA alone; the middle panel shows the pattern of GalNAc4S-6ST siRNA treated with ribonuclease; the bottom panel shows atelocollagen-embedded GalNAc4S-6ST siRNA treated with ribonuclease.
  • the time shows the duration of reaction at 37° C.
  • FIG. 7 show graphs depicting the results on the effect of a nucleic acid administered into the submucous tissue of the large intestine in dextran sulfate sodium (DSS)-induced colitis model mice.
  • DSS dextran sulfate sodium
  • FIG. 8 show graphs depicting an inflammatory activity-suppressing effect of a nucleic acid administered into the submucous tissue of the large intestine in DSS colitis model mice.
  • the left panel shows stool consistency, while the right panel shows fecal occult blood.
  • the administration of siRNA into the submucous tissue of the large intestine resulted in suppression of disease activity.
  • FIG. 9 is a graph showing the colonic length-improving effect of a nucleic acid administered into the submucous tissue of the large intestine in DSS colitis model mice.
  • the administration of siRNA into the submucous tissue of the large intestine significantly suppressed the contraction of large intestine.
  • FIG. 10 show photographs depicting the tissue-improving effect of a nucleic acid administered into the submucous tissue of the large intestine in DSS colitis model mice.
  • the administration of siRNA into the submucous tissue of large intestine significantly suppressed the epithelial disruption, inflammation, and fibrosis associated with colitis.
  • HE stain 100-fold magnification.
  • the present inventors discovered that by directly administering physiologically active substances into submucous tissues, these physiologically active substances are retained at the administration sites over a long period without loss or diffusion, and exert effects acting as a reservoir.
  • the present invention provides methods for retaining and expressing physiologically active substances in a target tissue-specific manner, in which the physiologically active substances are administered to the submucous tissue.
  • mucous more specifically refers to the macroscopic membrane covering the surface of the lumen of hollow organs such as the gastrointestinal tract, respiratory and urogenital systems, including membranes covering the surface of auditory tubes, the middle ear cavity connecting to the airway, and bulbar and palpebral conjunctivae.
  • epithelium is the outermost layer, and the underlayer is lamina intestinal of the mucous membrane.
  • submucous tissue refers to the tissue under the lamina basement membrane that supports the epithelium. In actual clinical practice, however, a “submucous tissue” may be distinguished depending on a physician's “feeling”, and thus the subepithelial tissue may be taken as “intramucosal tissue” and deeper tissue between the mucosa and muscle may be identified as “submucous tissue”.
  • the “submucous tissue” is not particularly limited, as long as its properties (pH, sugar chains, cellular composition, etc.) are appropriate to maintain the physiologically active substances of the present invention.
  • Such submucous tissues include, for example, those of the intestinal tract, eye, ear, nose, uterus, urinary bladder, or oral cavity, subcutaneous tissues, or such, but are not limited to these examples.
  • submucous tissues of the intestinal tract have been demonstrated to be much more effective than other matrices when pancreatic Langerhans islets are cultured in vitro (Tian X H et al. Hepatobiliary Pancreat Dis Int. 4: 524, 2005).
  • This also suggests that the submucous tissues of the intestinal tract may have a very special microenvironment. More specifically, in a preferred embodiment of the present invention, submucous tissues include those of the intestinal tract.
  • submucous tissues of the intestinal tract include, for example, those of intestinal tracts such as the esophagus, stomach, duodenum, small intestine, appendix, large intestine, and rectum.
  • submucous tissues of the intestinal tract include submucous tissue of the large intestine.
  • physiologically active substance is a general name for chemical substances that exert various biological effects in living organisms, and is not particularly limited.
  • a physiologically active substance is preferably selected, for example, from nucleic acids, proteins, carbohydrates, lipids, or low-molecular-weight compounds.
  • Nucleic acids used in the present invention refer to both RNAs and DNAs. Chemically synthesized nucleic acid analogs, such as so-called “PNAs” (peptide nucleic acids) or Morpholino antisense oligos, are also included in the nucleic acids of the present invention. PNAs are nucleic acids in which the fundamental backbone structure of nucleic acids, the pentose-phosphate backbone, is replaced by a polyamide backbone with glycine units, and Morpholino antisense oligos are nucleic acids in which the pentose-phosphate backbone is replaced by a morpholino backbone. PNAs and morpholino antisense oligos have a three-dimensional structure quite similar to that of nucleic acids.
  • nucleic acids of the present invention include, for example, antisense nucleic acids against transcripts of target genes or portions thereof, nucleic acids with the ribozyme activity of specifically cleaving transcripts of target genes, and nucleic acids with the activity of using RNAi effect to inhibit the expression of target genes.
  • antisense nucleic acids inhibit the expression of target genes by inhibiting various processes, such as transcription, splicing, and translation.
  • the antisense nucleic acids may inhibit the expression and/or function of target genes, based on any of the actions described above.
  • antisense sequences designed to be complementary to an untranslated region adjacent to the 5′ end of an mRNA for a target gene may be effective for inhibiting translation of the gene.
  • Sequences complementary to a coding region or 3′-untranslated region can also be used.
  • the antisense nucleic acids to be used in the present invention include not only nucleic acids comprising sequences antisense to the coding regions, but also nucleic acids comprising sequences antisense to untranslated regions of target genes.
  • Such antisense nucleic acids to be used are linked downstream of adequate promoters and are preferably linked with transcription termination signals on the 3′ side.
  • Nucleic acids thus prepared can be introduced into desired animals (cells) using known methods.
  • the sequences of the antisense nucleic acids are preferably complementary to a target gene or portion thereof that is endogenous to the animals (cells) to be transformed with them.
  • the sequences need not be perfectly complementary, as long as the antisense nucleic acids can effectively suppress expression of a gene.
  • the transcribed RNAs preferably have 90% or higher, and most preferably 95% or higher complementarity to target gene.
  • the antisense nucleic acids are preferably at least 15 nucleotides long, and less than 25 nucleotides long.
  • the lengths of the antisense nucleic acids of the present invention are not necessarily limited to the lengths mentioned above, and they may be 100 nucleotides or more, or 500 nucleotides or more.
  • Ribozymes refer to RNA molecules with catalytic activity. There are various ribozymes with different activities. Among others, studies that focused on ribozymes functioning as RNA-cleaving enzymes have enabled the design of ribozymes that cleave RNAs in a site-specific manner Some ribozymes have 400 or more nucleotides, such as group I intron type ribozymes and M1 RNA, which is comprised by RNase P, but others, called hammerhead and hairpin ribozymes, have a catalytic domain of about 40 nucleotides (Koizumi, M. and Otsuka, E., Tanpakushitsu Kakusan Koso (Protein, Nucleic Acid, and Enzyme) 1990, 35, 2191).
  • the autocatalytic domain of a hammerhead ribozyme cleaves the sequence G13U14C15 at the 3′ side of C15.
  • Base pairing between U14 and A9 has been shown to be essential for this activity, and the sequence can be cleaved when C15 is substituted with A15 or U15 (Koizumi, M. et al., FEBS Lett. 1988, 239, 285; Koizumi, M. and Otsuka, E., Tanpakushitsu Kakusan Koso (Protein, Nucleic Acid, and Enzyme) 1990, 35, 2191; and Koizumi, M. et al., Nucl. Acids Res. 1989, 17, 7059).
  • hairpin ribozymes are also useful. Such ribozymes are found in, for example, the minus strand of satellite RNAs of tobacco ringspot viruses (Buzayan, J. M., Nature 1986, 323, 349). It has been shown that target-specific RNA-cleaving ribozymes can also be created from hairpin ribozymes (Kikuchi, Y. and Sasaki, N., Nucl Acids Res. 1991, 19, 6751; and Kikuchi, Y. Kagaku to Seibutsu (Chemistry and Biology) 1992, 30, 112). Thus, the expression of the above-described target genes can be inhibited by using ribozymes to specifically cleave the gene transcripts.
  • nucleic acids of the present invention include nucleic acids with the inhibition activity of using RNAi effect (siRNA).
  • RNA interference RNA interference
  • target genes can be suppressed by RNA interference (hereinafter abbreviated as “RNAi”), using double-stranded RNAs comprising a sequence the same as or similar to a target gene sequence.
  • RNAi is a phenomenon where an mRNA comprising a base sequence complementary to a double-stranded RNA is degraded. RNAi is a method based on this phenomenon, in which the expression of an arbitrary gene is suppressed by artificially introducing a 21- to 23-mer double-stranded RNA (small interfering RNA; siRNA).
  • siRNA small interfering RNA
  • Fire et al. discovered using C. elegans that double-stranded RNA silences genes in a sequence-specific manner (Fire, A. et al., Nature 1998, 391, 806-811). After elucidating the underlying mechanism of mRNA cleavage by 21- to 23-mer processed double-stranded RNA (Zamore P D.
  • RNA-induced silencing complex RISC
  • RISC RNA-induced silencing complex
  • Dicer cloning Dicer
  • RNAi is a phenomenon in which, when cells or such are introduced with short double-stranded RNAs (hereinafter abbreviated as “dsRNAs”) comprising sense RNAs that comprise sequences homologous to the mRNAs of a target gene, and antisense RNAs that comprise sequences homologous a sequence complementary thereto, the dsRNAs bind specifically and selectively to the target gene mRNAs, induce their disruption, and cleave the gene transcript, thereby effectively inhibiting (suppressing) target gene expression.
  • dsRNAs short double-stranded RNAs
  • RNAi can suppress the expression of target genes, and is thus drawing attention as a method applicable to gene therapy, or as a simple gene knockout method replacing conventional methods of gene disruption, which are based on complicated and inefficient homologous recombination.
  • the RNAs to be used in RNAi are not necessarily perfectly identical to the target genes or portions thereof; however, the RNAs are preferably perfectly homologous to the genes or portions thereof.
  • the targets of the siRNAs to be designed are not particularly limited, as long as they are regions of target genes. Any region of the gene can be a candidate for a target.
  • shRNAs are RNA molecules with a stem-loop structure, since a portion of the single strand constitutes a strand complementary to another portion. Thus, molecules capable of forming an intramolecular double-stranded RNA are also included in the siRNAs.
  • RNAs of the present invention even double-stranded RNAs with a structure having a deletion or addition of one or a small number of bases are included in the siRNAs of the present invention, as long as they have the function of suppressing the expression of target genes by RNAi effect.
  • RNAi mechanism still remain poorly understood, but it is known that an enzyme called “DICER” (a member of the RNase III nuclease family) binds to a double-stranded RNA and degrades it in to small fragments, called “siRNAs”.
  • the double-stranded RNAs of the present invention that have RNAi effect include such double-stranded RNAs prior to being degraded by DICER.
  • the length of the double-stranded RNAs of the present invention is not particularly limited.
  • long double-stranded RNAs covering the full-length or near full-length mRNA of a target gene can be pre-digested, for example, by DICER, and then the degradation products can also be used.
  • These degradation products are expected to contain double-stranded RNA molecules with RNAi effect.
  • siRNAs of the present invention are not necessarily single pairs of double-stranded RNAs directed to target sequences, but may be mixtures of multiple double-stranded RNAs directed to regions that cover the target sequence.
  • the siRNAs of the present invention include so-called “siRNA cocktails”.
  • RNAs ribonucleotides
  • one or more of the ribonucleotides constituting the siRNAs of the present invention may be replaced with corresponding deoxyribonucleotides.
  • corresponding means that although the sugar moieties are structurally differently, the nucleotide residues (adenine, thymine (uracil), guanine, or cytosine) are the same.
  • deoxyribonucleotides corresponding to ribonucleotides with adenine refer to deoxyribonucleotides with adenine.
  • the term “or more” described above is not particularly limited, but preferably refers to a small number of about two to five ribonucleotides.
  • siRNAs of the present invention include, for example, the siRNAs of SEQ ID NOs: 1 and/or 2.
  • nucleic acids aiming at suppressing the expression of a target gene includes microRNAs (miRNAs), aptamers, and locked nucleic acids (LNAs) generated by modifying oligonucleotides.
  • miRNAs microRNAs
  • LNAs locked nucleic acids
  • nucleic acids are not limited to the examples described above, and it is possible to use nucleic acids that are appropriate for the purposes.
  • protein of the present invention refers to a polymer comprising several amino acids, and includes not only polypeptides but also oligopeptides.
  • polypeptides include both naturally-occurring polypeptides without modification and modified polypeptides.
  • Such modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavin, covalent bonding of heme moiety/moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, ⁇ -carboxylation, glycosylation, formation of GPI anchors, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • proteins of the present invention include, for example, a target protein variants, which are dominant negative for the target protein, and antibodies that bind to the target protein.
  • a target protein variant that is dominant negative for a target protein refers to a protein that, when expressed a gene encoding the protein, has the function of reducing or eliminating the activity of the endogenous wild type protein.
  • antibodies that bind to the target protein can be prepared by methods known to those skilled in the art.
  • Polyclonal antibodies can be obtained, for example, by the following procedure: small animals such as rabbits are immunized with a natural target protein or a recombinant target protein expressed in microorganisms such as E. coli as a fusion protein with GST, or a partial peptide thereof.
  • Sera are obtained from these animals and purified by, for example, ammonium sulfate precipitation, Protein A or G columns, DEAE ion exchange chromatography, affinity columns coupled with the target protein or a synthetic peptide to prepare antibodies.
  • Monoclonal antibodies can be obtained, for example, by the following procedure: small animals such as mice are immunized with a target protein or a partial peptide thereof. Spleens are removed from the mice and crushed to isolate cells. The cells are fused with mouse myeloma cells using a reagent such as polyethylene glycol. Clones producing antibodies that bind to a target protein is selected from among the resulting fused cells (hybridomas). The obtained hybridomas are then transplanted in the peritoneal cavities of mice, and ascites collected. The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, Protein A or G columns, DEAE ion exchange chromatography, affinity columns coupled with the target protein or a synthetic peptide.
  • the forms of above-described antibodies are not particularly limited as long as they bind to a target protein.
  • the antibodies of the present invention may include human antibodies, humanized antibodies created by gene recombination, fragments or modified products of such antibodies, in addition to the polyclonal and monoclonal antibodies described above.
  • the target proteins used as sensitizing antigens to prepare antibodies are not limited in terms of the animal species from which the proteins are derived. However, the proteins are preferably derived from mammals, for example, mice and humans Human-derived proteins are particularly preferred.
  • the proteins to be used as sensitizing antigens may be whole proteins or partial peptides thereof. Such partial peptides of the proteins include, for example, amino (N)-terminal fragments and carboxyl (C)-terminal fragments of the proteins.
  • antibodies refer to antibodies that react with a full-length protein or fragment thereof.
  • human lymphocytes for example, EB virus-infected human lymphocytes
  • the sensitized lymphocytes can be fused with human-derived myeloma cells with the ability to divide permanently, for example, U266, to obtain hybridomas that produce desired human antibodies with binding activity to the proteins.
  • the antibodies are preferably human or humanized antibodies in order to reduce immunogenicity.
  • proteins include, for example, enzymes; and their forms are not particularly limited.
  • carbohydrates include all of monosaccharides, oligosaccharides, and polysaccharides.
  • the carbohydrates of the present invention also include complex carbohydrates in which the above saccharides are covalently linked to proteins or lipids, and glycosides in which the reducing groups of monosaccharides or oligosaccharides are linked to an aglycon such as alcohol, phenol, saponin, or a pigment.
  • lipids include all of simple lipids, complex lipids, and derived lipids.
  • low-molecular-weight compounds include chemically synthesized substances with a low molecular weight, typically ranging from about several hundreds to thousands.
  • a target protein For inhibiting (suppressing) the function of a target protein, it is possible to use, for example, low-molecular-weight substances that bind to the target protein.
  • Such low-molecular-weight substances that bind to a target protein may be natural or artificial compounds.
  • the compounds can be obtained or produced by methods known to those skilled in the art.
  • the physiologically active substances are not limited, and include, for example, single compounds such as natural compounds, organic compounds, inorganic compounds; and compound libraries, expression products of gene libraries, cell extracts, cell culture supernatant, products of fermenting microorganisms, extracts of marine organisms, and plant extracts.
  • single compounds such as natural compounds, organic compounds, inorganic compounds
  • compound libraries expression products of gene libraries, cell extracts, cell culture supernatant, products of fermenting microorganisms, extracts of marine organisms, and plant extracts.
  • Each of the above-described physiologically active substances may be used alone or in combination with other physiologically active substances.
  • the physiologically active substances are preferably used (administered) in an unmodified, non-labeled form (occasionally herein referred to as “naked form”).
  • the physiologically active substances of the present invention can be used after labeling, if needed.
  • labels include, for example, radioisotope labels and fluorescent labels.
  • the labels are not particularly limited, and include alkaline phosphatase labels, peroxidase labels, biotin-labeled/streptavidin-conjugated enzymes (alkaline phosphatase, peroxidase, and such), and fluorescein isothiocyanate (FITC).
  • physiologically active substances of the present invention can be used in combination with pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carriers include, for example, vectors that are typically used as gene therapy vectors.
  • the above-described vectors include, for example, viral vectors such as retroviral vectors, adenoviral vectors, and adeno-associated viral vectors, and non-viral vectors such as liposomes and atelocollagen.
  • viral vectors such as retroviral vectors, adenoviral vectors, and adeno-associated viral vectors
  • non-viral vectors such as liposomes and atelocollagen.
  • the carriers of the present invention include, but are not limited to, for example, water, physiological saline, phosphate buffered saline, polyvinyl alcohol, polyvinylpyrrolidone, carboxylvinyl polymer, sodium alginate, water-soluble dextran, pectin, xanthan gum, gum Arabic, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, and lactose.
  • the physiologically active substances of the present invention may further comprise additives such as preservatives.
  • the physiologically active substances of the present invention may further comprise other pharmacological ingredients.
  • physiologically active substances of the present invention are preferably parenteral preparations; liquid preparations such as solutions and suspensions are preferred, including, for example, injections.
  • liquid preparations such as solutions and suspensions are preferred, including, for example, injections.
  • Other preferred dosage forms include, for example, liniments and coating agents which are applied onto or coat the surface of indwelling devices such as balloons and stents.
  • physiologically active substances of the present invention can be administered to subjects (patients or such) by methods known to those skilled in the art, such as using injection into target submucous tissues, or application or coating onto the surface of indwelling devices such as balloons and stents. If needed, devices such as endoscopes may be used to administer the physiologically active substances.
  • the applied dose of a physiologically active substance of the present invention varies depending on the body weight and age of the subject (patient or such), administration method, and the like; however, the optimum dose can be appropriately selected by those skilled in the art.
  • the present invention also provides methods for treating or preventing diseases, which comprise the step of retaining and expressing the physiologically active substances in a target tissue-specific manner affected with the diseases, by administering the physiologically active substances to the submucous tissues with diseases.
  • diseases include those associated with mucosa (specifically, inflammatory bowel disease and Crohn's disease), fibrotic diseases, arthritis (osteoarthritis and rheumatoid arthritis), Alzheimer's disease, organ transplant toxicity and rejection, cachexia, allergy, cancer (for example, solid tumors/cancers including colon, breast, prostate, and brain, and malignant hematopoietic tumors including leukemia and lymphoma), tissue ulceration, restenosis, periodontal diseases, bullous epidermolysis, osteoporosis, loosening of artificial joint implants, atherosclerosis (including divulsion of atherosclerosis lesion), aortic aneurysm (including abdominal aneurysm and cerebral aneurysm), congestive heart failure, myocardial infarct, seizure, cerebral ischemia, head injury, spinal cord injury, neurodegenerative disorders (acute and chronic), autoimmune diseases, Huntington's chorea, Parkinson's disease,
  • Preferred subjects to be administered with the physiologically active substances of the present invention are mammals including humans, and domestic animals, pets, and experimental animals.
  • mammals (patients) with a disease described above are preferred subjects in the present invention.
  • DSS enteritis was induced by allowing 12-week-old male Wistar rats to freely drink water containing 3% dextran sulfate sodium (DSS) (molecular weight; 50,000) (Okayasu I, Hatakeyama S, Ohkusa T, Inagaki Y, Nakaya R. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 1990, 98: 694-702). An ultrathin endoscope for humans was inserted into the large intestines of rats anesthetized with Nembutal on day 0 and 3 after the start of feeding with DSS water.
  • DSS dextran sulfate sodium
  • siRNA was injected into the submucous tissue at equally spaced four sites in the left colon.
  • the ultrathin endoscope used was a prototype model (outer diameter of the scope, 5.6 mm) having a working forceps channel (channel diameter, 2 mm) which had been developed as an upper gastrointestinal endoscope for humans by OLYMPUS. Untreated rats were used as a control.
  • the therapeutic effect was evaluated based on: (1) clinical disease activity index (CDAI) determined in terms of the three items: weight loss, diarrhea, and melena; (2) intestinal length; and (3) pathohistological analysis of HE stained samples prepared using section of large intestine, in the treated and control groups.
  • CDAI clinical disease activity index
  • Iopamidol refers to the compound named N,N′-Bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5-[(2S)-2-hydroxypropanoylamino]-2,4,6-triiodoisophthalamide (C17H22I3N308; molecular weight, 777.09) represented by formula (I):
  • the results are shown in FIG. 2 .
  • the X-ray images showed that the injected iopamidol was retained in the submucous tissue of the colon without intraperitoneal leakage and transfer to other sections.
  • the CT images also showed that the injected iopamidol was retained specifically at the injection sites and did not spread to other slices.
  • the clinical evaluation method also demonstrated that the methods of the present invention could retain physiologically active substances very specifically within target submucous tissues.
  • mice which are more common experimental animals.
  • Eight-week-old female C57BL/6J mice were anesthetized with Nembutal and laparotomized to expose the lower part of the large intestines.
  • 20 ⁇ l of carbon particles (India ink), corresponding to a physiologically active substance of the present invention, alone was macroscopically injected to the submucous tissues. Then, the abdomen was closed.
  • mice Five days after, the mice were sacrificed to prepare tissue sections of the large intestine. The sections were observed under a light microscope. The result is shown in FIG. 3 .
  • the injected carbon particles were found to be retained within the submucous tissue without physical diffusion and leakage to other portions.
  • FITC-labeled siRNA 20 nM of BLOCK-iT Fluorescent Oligo (Invitrogen) was injected into the submucous tissue of mouse large intestine by the same method as described in Examples 1 and 3. The retention of the injected siRNA was assessed under a fluorescent stereomicroscope (Leica) 24 hours after injection.
  • FIG. 4 The results are shown in FIG. 4 .
  • the FITC-labeled siRNA was confined at the injection site in the large intestine.
  • FIG. 1 of Example 1 is a histological image obtained by injecting the same FITC-labeled siRNA into the submucous tissue of rat large intestine. Based on the findings described above, the same result is predicted to be obtained when the labeled siRNA is injected into the submucous tissue of mouse large intestine.
  • the concentration of siRNA in the large intestine was determined by using the ELISA method for direct quantitation of siRNA according to a published report (Rosie Z et al. Analytical Biochem. 304: 19-25, 2002).
  • the nucleotide sequences of GalNac 4S-6ST siRNA used in this Example are shown below, but the sequences are not limited to the example shown herein.
  • GalNAc4S-6ST siRNA (0.3 ⁇ g/10 ⁇ l) was injected at three sites to the submucous tissue of large intestine in eight-week-old female C57BL/6J mice by the same method described in Example 3. The mice were sacrificed 0.5, 3, 6, or 24 hours after injection to prepare histological samples of the large intestine. The siRNA concentrations were determined by the ELISA methods.
  • siRNA 1 ⁇ g/ml siRNA before injection was used as a positive control without any additional treatment. At the time point after 0.5 hour, the siRNA was retained at a very high concentration, while the concentration was decreased by half after three hours. However, the result shows that siRNA continually remained retained 24 hours later.
  • GalNAc4S-6ST siRNA the same substance used in Example 5, was assessed for its stability.
  • test solutions were prepared by adding 1 ⁇ g of GalNAc4S-6ST siRNA to 200 ⁇ l of sterile phosphate buffer or 0.1% atelocollagen and stirring the mixtures at 4° C. for 20 minutes.
  • 0.1% atelocollagen was prepared by combining 1% atelocollagen (Koken) with ten volumes of sterile phosphate buffer and stirring the mixture at 4° C. for 16 hours.
  • RNA iso (Takara Bio) was added to the test solutions after reaction. The mixtures were incubated on ice for five minutes, and centrifuged at 14,000 rpm for 15 minutes. The resulting supernatants were collected, and 500 ⁇ l of isopropanol and 1 ⁇ l of glycogen (Invitrogen) were added thereto.
  • the gel was shaken for 20 minutes in a stain solution prepared by 10,000 times diluting Ethidium Bromide (Invitrogen) with 1 ⁇ LoTE (composition: 3 mM Tris-HCl (pH 7.5) (Invitrogen), 0.2 mM EDTA (pH 7.5) (Sigma Aldrich Japan)). The gel was photographed with Fluourchem (Innotech) and analyzed.
  • GalNAc4S-6ST siRNA in the absence of ribonuclease, GalNAc4S-6ST siRNA was relatively stable even after 60 minutes. However, in the presence of ribonuclease, siRNA degradation was observed at the time point after 15 minutes and the band disappeared after 30 minutes. On the other hand, when GalNAc4S-6ST siRNA was embedded in atelocollagen, the band was faint but detectable even after 30 minutes. Short-chain RNA (siRNA) embedded in atelocollagen has been reported to be resistant to ribonuclease. An equivalent result was obtained in this Example. Furthermore, the present invention also suggests that the same result is obtained not only when siRNA is embedded in atelocollagen but also in other biological substances having the same characteristics.
  • a colitis model mice was prepared by allowing C57BL/6J mice (female, six weeks old; CLEA Japan Inc.) to freely drink high-concentration chlorine water containing 3% dextran sulfate sodium (DSS; Wako Pure Chemical Industries Inc.) for five days.
  • This DSS-induced colitis model has excellent reproducibility, and is thus used widely as a standard experimental model for inflammatory bowel diseases such as mouse ulcerative colitis and Crohn's disease (Sasaki N, J. Inflamm. 2005 2: 13; as a review, Pucilowska J B et al. Am J Physiol Gastroenterol Liver Physiol. 279: G653-G659, 2000).
  • the same GalNAc4S-6ST siRNA (0.3 ⁇ g/head) as used in Example 5 was injected to the submucous tissue of mouse large intestine, while the mice were allowed to drink water containing 3% DSS.
  • the control groups used were: a group injected with scramble siRNA (“BLOCK-iT Fluorescent Oligo (Invitrogen)” in Example 4) and an untreated group in which mice were allowed to drink DSS water only.
  • the body weight and disease activity index (DAI) score (Kihara M, Gut. 2003 52: 713-9) were recorded for five days while the mice were allowed to drink water containing 3% DDS. Then, the mice were sacrificed on the fifth day.
  • RNA iso (Takara Bio) was added to 50 mg each of excised organs (large intestine and kidney). The organs were crushed using an electrical homogenizer (DIGITAL HOMOGENIZER; AS ONE), then, 200 ⁇ l of chloroform (Sigma-Aldrich Japan) was added to the resulting suspension. The mixture was gently mixed and then cooled on ice for about five minutes, and centrifuged in a centrifuge (Centrifuge 5417R; Eppendorf) at 12,000 rpm and 4° C. for 15 minutes.
  • DIGITAL HOMOGENIZER an electrical homogenizer
  • chloroform Sigma-Aldrich Japan
  • RNA precipitate obtained after washing three times with 1,000 ⁇ l of 75% ethanol (Sigma-Aldrich Japan) was air-dried for 30 minutes to one hour, and then dissolved in Otsuka distilled water (Otsuka Pharmaceutical Co., Ltd). The solution was 100 times diluted with Otsuka distilled water.
  • the RNA concentrations of extracted samples in UV plates were determined using a plate reader (POWER Wave XS; BIO-TEK).
  • RNA synthesis reverse transcription reaction
  • concentrations of the obtained RNA samples were adjusted to 500 ng/20 ⁇ l.
  • the samples were heated at 68° C. for three minutes in a BLOCK INCUBATOR (ASTEC), and cooled on ice for ten minutes.
  • RT PreMix solution composition: 18.64 ⁇ l of 25 mM MgC12 (Invitrogen), 20 ⁇ l of 5 ⁇ Buffer (Invitrogen), 6.6 ⁇ l of 0.1 M DTT (Invitrogen), 10 ⁇ l of 10 mM dNTP mix (Invitrogen), 2 ⁇ l of RNase Inhibitor (Invitrogen), 1.2 ⁇ l of MMLV Reverse Transcriptase (Invitrogen), 2 ⁇ l of Random primer (Invitrogen), and 19.56 ⁇ l of sterile distilled water (Otsuka distilled water; Otsuka Pharmaceutical Co., Ltd.)), which had been prepared in advance, was added to the samples.
  • RT PreMix solution composition: 18.64 ⁇ l of 25 mM MgC12 (Invitrogen), 20 ⁇ l of 5 ⁇ Buffer (Invitrogen), 6.6 ⁇ l of 0.1 M DTT (Invitrogen), 10 ⁇ l of 10 mM d
  • the GalNAc4S-6ST siRNA was demonstrated to significantly suppress the increase in the expression of GalNAc4S-6ST in the large intestine.
  • the result described above suggests that the present invention is very effective for the retention and function of the injected physiologically active substances at the administration sites, and produces no side effect on other normal organs.
  • DAI colitis activity index
  • the first day (day 0) of feeding with DSS water is defined as 1, and the stool consistency and fecal occult blood in each mouse were recorded.
  • the results are shown in FIG. 8 .
  • the score was lower in the group administered with GalNAc4S-6ST siRNA. This result suggests that the GalNAc4S-6ST siRNA injected into the submucous tissue of large intestine suppresses the expression of GalNAc4S-6ST gene in a colon-specific manner and exerted the effect of suppressing the inflammatory activity.
  • the length of the large intestine was measured after sacrificing mice on day five. Colonic contraction was significantly suppressed in the group administered with GalNAc4S-6ST siRNA (p ⁇ 0.05; t test) ( FIG. 9 ).
  • the length of the large intestine is a crucial indicator that reflects the intestinal fibrosis.
  • the GalNAc4S-6ST siRNA injected into the submucous tissue of large intestine was demonstrated to suppress the expression of GalNAc4S-6ST gene in a colon-specific manner.
  • the siRNA was demonstrated to strongly suppress fibrotic degeneration of the intestine.
  • tissue sections were prepared from collected large intestines and stained with hematoxylin-eosin.
  • the resulting histological images were analyzed ( FIG. 10 ).
  • the epithelial disruption, infiltration of inflammatory cells into lamina intestinal and submucosa, and thickening of muscular layer were markedly suppressed in the group administered with GalNAc4S-6ST siRNA.
  • the GalNAc4S-6ST siRNA injected into the submucous tissue of large intestine was demonstrated to suppress the expression of GalNAc4S-6ST gene in a colon-specific manner.
  • the siRNA was also histologically demonstrated to produce a therapeutic effect.

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150337313A1 (en) * 2012-07-17 2015-11-26 Stelic Institute & Co. Mucosal healing promoter
US10646579B2 (en) 2015-11-04 2020-05-12 Tme Therapeutics Inc. Complex comprising RNAi molecule and N-acetylated chitosan

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3673910B1 (de) * 2007-12-27 2023-11-22 Stelic Institute & Co., Inc. Mit zuckerkette assoziiertes gen und verwendung davon
US8834423B2 (en) 2009-10-23 2014-09-16 University of Pittsburgh—of the Commonwealth System of Higher Education Dissolvable microneedle arrays for transdermal delivery to human skin
EP4112112A1 (de) 2012-05-01 2023-01-04 University of Pittsburgh - Of the Commonwealth System of Higher Education Spitzenbelastete mikronadelanordnungen zum transdermalen einführen
WO2016149673A1 (en) 2015-03-18 2016-09-22 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Bioactive components conjugated to substrates of microneedle arrays
US11684763B2 (en) 2015-10-16 2023-06-27 University of Pittsburgh—of the Commonwealth System of Higher Education Multi-component bio-active drug delivery and controlled release to the skin by microneedle array devices
US11744889B2 (en) 2016-01-05 2023-09-05 University of Pittsburgh—of the Commonwealth System of Higher Education Skin microenvironment targeted delivery for promoting immune and other responses
WO2018105708A1 (ja) 2016-12-07 2018-06-14 株式会社ステリック再生医科学研究所 慢性疾患の治療及び予防用医薬組成物
US20180206726A1 (en) 2016-12-07 2018-07-26 Progenity Inc. Gastrointestinal tract detection methods, devices and systems
EP3600249A1 (de) 2017-03-30 2020-02-05 Progenity Inc. Behandlung von erkrankungen des magen-darm-traktes mit einem chst15-inhibitor

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4054352A (en) * 1975-08-25 1977-10-18 Rudin Marvin B Electrical power take-off unit for cigarette lighter socket of vehicle
US6464998B1 (en) * 1993-11-23 2002-10-15 Aventis Pharma S.A. Composition for the in vivo production of therapeutic products
US20030105061A1 (en) * 2001-11-05 2003-06-05 Seikagaku Corporation Medical composition for protuberance of epithelium
US20030191070A1 (en) * 2000-06-06 2003-10-09 Kinuko Oku Medical composition kit for treating lesioned abnormal tissue
US20040120970A1 (en) * 1999-01-26 2004-06-24 Jacobs Antonius Arnoldus Christiaan Use of bacterium for manufacture of a vaccine
US20040241843A1 (en) * 2001-05-07 2004-12-02 Baker Brenda F. Antisense modulation of interleukin 12 p40 subunit expression
US20050013854A1 (en) * 2003-04-09 2005-01-20 Mannino Raphael J. Novel encochleation methods, cochleates and methods of use
US20050164964A1 (en) * 2002-05-03 2005-07-28 Fmc Biopolymer As Non-viral gene delivery system
US20060128659A1 (en) * 2002-07-10 2006-06-15 Osami Habuchi Sulfotransferase inhibitors
US20060286073A1 (en) * 2002-07-24 2006-12-21 Tolentino Michael J COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF ANGIOGENESIS
US20090060892A1 (en) * 2005-10-27 2009-03-05 Stelic Institute of Regenerative Medicine, Stelic Institute & Company Agents for suppressing hepatic fibrosis
US20090182136A1 (en) * 2006-03-23 2009-07-16 Jesper Wengel Small Internally Segmented Interfering RNA
US20090202515A1 (en) * 2006-09-08 2009-08-13 Stelic Institute Of Regenerative Medicine, Stelic Institute & Co. Agents for suppressing neural fibrotic degeneration

Family Cites Families (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59130074A (ja) 1983-01-18 1984-07-26 Sanyo Electric Co Ltd 非水電解液二次電池用負極の製造方法
US5695998A (en) 1995-02-10 1997-12-09 Purdue Research Foundation Submucosa as a growth substrate for islet cells
FR2797274B1 (fr) 1999-08-02 2003-09-05 Agronomique Inst Nat Rech Gene mutant de la famille gras, et plantes a developpement reduit comprenant ledit gene
AU782370B2 (en) 1999-12-08 2005-07-21 Merck Patent Gmbh Nucleic acid delivery system
CZ302719B6 (cs) * 2000-12-01 2011-09-21 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Izolovaná molekula dvouretezcové RNA, zpusob její výroby a její použití
EP1386004A4 (de) * 2001-04-05 2005-02-16 Ribozyme Pharm Inc Modulation der mit der entzündungsausbreitung und dem neuritenauswuchs assoziierten genexpression unter verwendung von technologien auf nukleinsäurebasis
CA2450070C (en) 2001-06-14 2010-03-02 Cook Incorporated Endovascular filter
JP2007119498A (ja) 2001-11-05 2007-05-17 Seikagaku Kogyo Co Ltd 上皮膨隆高の維持用組成物
US20050106729A1 (en) * 2002-05-03 2005-05-19 Mark Irvin Rosenblatt Gene transfer methods and compositions
PT1581812E (pt) * 2003-01-06 2008-09-22 Wyeth Corp Composições e métodos para diagnóstico e tratamento de cancros do cólon
US7825235B2 (en) * 2003-08-18 2010-11-02 Isis Pharmaceuticals, Inc. Modulation of diacylglycerol acyltransferase 2 expression
WO2005063293A1 (ja) * 2003-12-26 2005-07-14 Masatoshi Hagiwara Sr蛋白質のリン酸化制御方法、および、sr蛋白質の活性制御剤を有効成分とする抗ウイルス剤
WO2005074633A2 (en) * 2004-02-03 2005-08-18 The Regents Of The University Of Michigan Compositions and methods for characterizing, regulating, diagnosing, and treating cancer
US20050272682A1 (en) * 2004-03-22 2005-12-08 Evers Bernard M SiRNA targeting PI3K signal transduction pathway and siRNA-based therapy
US7972295B2 (en) * 2005-03-11 2011-07-05 Boston Scientific Scimed, Inc. Apparatus and methods for delivering a bolus of therapeutic material
WO2007072469A2 (en) * 2005-12-23 2007-06-28 Vysera Biomedical Limited A medical device suitable for treating reflux from a stomach to an oesophagus
US20100028350A1 (en) * 2006-03-03 2010-02-04 London Health Sciences Centre Research Inc. Preventing il-2 mediated inflammation in epithelial cells
KR20080102196A (ko) 2006-03-15 2008-11-24 도요다 지도샤 가부시끼가이샤 복수개의 육각셀로 구성된 허니콤 구조체
TW200806789A (en) * 2006-03-27 2008-02-01 Globeimmune Inc RAS mutation and compositions and methods related thereto
WO2007116395A2 (en) * 2006-04-10 2007-10-18 Ester Neurosciences Ltd. Antisense oligonucleotides against acetylcholinesterase for treating inflammatory diseases
US20070258952A1 (en) * 2006-05-04 2007-11-08 Baylor Research Institute Anti-Tumor Activity of an Oncolytic Adenovirus-Delivered Oncogene siRNA
EP3673910B1 (de) * 2007-12-27 2023-11-22 Stelic Institute & Co., Inc. Mit zuckerkette assoziiertes gen und verwendung davon
WO2010086849A1 (en) * 2009-01-27 2010-08-05 Silenseed Ltd Method and system for implant delivery

Patent Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4054352A (en) * 1975-08-25 1977-10-18 Rudin Marvin B Electrical power take-off unit for cigarette lighter socket of vehicle
US6464998B1 (en) * 1993-11-23 2002-10-15 Aventis Pharma S.A. Composition for the in vivo production of therapeutic products
US20040120970A1 (en) * 1999-01-26 2004-06-24 Jacobs Antonius Arnoldus Christiaan Use of bacterium for manufacture of a vaccine
US7381419B2 (en) * 2000-06-06 2008-06-03 Lequio Pharma Co., Ltd. Medical composition kit for treating lesioned abnormal tissue
US20030191070A1 (en) * 2000-06-06 2003-10-09 Kinuko Oku Medical composition kit for treating lesioned abnormal tissue
US20040241843A1 (en) * 2001-05-07 2004-12-02 Baker Brenda F. Antisense modulation of interleukin 12 p40 subunit expression
US7008626B2 (en) * 2001-11-05 2006-03-07 Seikagaku Corporation Medical composition for protuberance of epithelium
US20060105991A1 (en) * 2001-11-05 2006-05-18 Seikagaku Corporation Medical composition for protuberance of epithelium
US20030105061A1 (en) * 2001-11-05 2003-06-05 Seikagaku Corporation Medical composition for protuberance of epithelium
US20090325899A1 (en) * 2001-11-05 2009-12-31 Seikagaku Corporation Medical composition for protuberance of epithelium
US20050164964A1 (en) * 2002-05-03 2005-07-28 Fmc Biopolymer As Non-viral gene delivery system
US20080085242A1 (en) * 2002-05-03 2008-04-10 Fmc Biopolymer As Non-viral gene delivery system
US20060128659A1 (en) * 2002-07-10 2006-06-15 Osami Habuchi Sulfotransferase inhibitors
US20060286073A1 (en) * 2002-07-24 2006-12-21 Tolentino Michael J COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF ANGIOGENESIS
US20050013854A1 (en) * 2003-04-09 2005-01-20 Mannino Raphael J. Novel encochleation methods, cochleates and methods of use
US20090060892A1 (en) * 2005-10-27 2009-03-05 Stelic Institute of Regenerative Medicine, Stelic Institute & Company Agents for suppressing hepatic fibrosis
US20090182136A1 (en) * 2006-03-23 2009-07-16 Jesper Wengel Small Internally Segmented Interfering RNA
US20090202515A1 (en) * 2006-09-08 2009-08-13 Stelic Institute Of Regenerative Medicine, Stelic Institute & Co. Agents for suppressing neural fibrotic degeneration

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Bass (2001, Nature, v.411:428-9). *
Bennett et al. (Investigative Opthal. Visual Sci 1994: 2535-2542). *
Stedman's Medical Dictionary http://www.medilexicon.com/medicaldictionary.php?t=58990 07/16/2013 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150337313A1 (en) * 2012-07-17 2015-11-26 Stelic Institute & Co. Mucosal healing promoter
US20160348118A1 (en) * 2012-07-17 2016-12-01 Stelic Institute & Co. Mucosal healing promoter
TWI639433B (zh) * 2012-07-17 2018-11-01 史特利克再生醫科學研究所股份有限公司 黏膜治癒促進劑
US20190376063A1 (en) * 2012-07-17 2019-12-12 Stelic Institute & Co. Mucosal healing promoter
US10646579B2 (en) 2015-11-04 2020-05-12 Tme Therapeutics Inc. Complex comprising RNAi molecule and N-acetylated chitosan

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