WO2015147575A1 - Composition pharmaceutique permettant le traitement d'une dysérection, contenant un inhibiteur du trpc4 en tant que principe actif - Google Patents

Composition pharmaceutique permettant le traitement d'une dysérection, contenant un inhibiteur du trpc4 en tant que principe actif Download PDF

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WO2015147575A1
WO2015147575A1 PCT/KR2015/002993 KR2015002993W WO2015147575A1 WO 2015147575 A1 WO2015147575 A1 WO 2015147575A1 KR 2015002993 W KR2015002993 W KR 2015002993W WO 2015147575 A1 WO2015147575 A1 WO 2015147575A1
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trpc4
activity
erectile dysfunction
inhibitor
protein
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Korean (ko)
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이성원
성현환
채미리
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Samsung Life Public Welfare Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • 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
    • C12N15/1138—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 against receptors or cell surface proteins
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/344—Disorders of the penis and the scrotum and erectile dysfuncrion

Definitions

  • composition for treating erectile dysfunction comprising an inhibitor of TRPC4 as an active ingredient
  • the present invention is supported by the Ministry of Health and Welfare
  • HI11C1489 (A111535), which is a research and management institution of the above-mentioned project, is the Korea Health Industry Development Institute.
  • the research project name is "Disease Overcoming Technology / Disease-Centered Intermediate-Based Research of Genitourinary System Disease," Development of safe erectile dysfunction treatment method through ion channel gene regulation ", Host institution is Samsung Seoul Hospital, The research period is from November 01, 2011 to October 31, 2014.
  • the present invention relates to a pharmaceutical composition for treating or preventing erectile dysfunction comprising an inhibitor of TRPC4 (transient receptor potential cation channel, subfamily C, member 4) as an active ingredient.
  • TRPC4 transient receptor potential cation channel, subfamily C, member 4
  • Erectile dysfunction is a disease mainly affecting men over 40 years old and it is known that 10 to 30 million men in the United States have this disease (2, 3). The annual incidence of the disease increases with age, with 12.4, 29.8, and 46.4 cases reported per 1,000 men, 40-49 years, 50-59 years, and 60-69 years, respectively. (4).
  • An erection is the result of a balance between contraction and relaxation of the penile corpus smooth muscle.
  • Nitric oxide (NO) and cyclic guanyl monophosphate (cGMP) pathways play an important role in the relaxation of penile smooth muscle.
  • PDE5 inhibitors based on the NO-cGMP pathway have been introduced to treat erectile dysfunction, and over 50 million patients Regardless of the cause, it was successfully treated with a PDE5 inhibitor (5). However, 30-40% of patients with erectile dysfunction no longer respond to PDE5 inhibitors and require additional invasive treatment (6, 7). Other pathophysiological pathways, such as the RhoA / ROCK pathway, the maxi-K channel, and the TRP channel, are also known to be involved in erectile dysfunction, which may lead to potential new compounds for the treatment of patients who did not obtain satisfactory results from PDE5 inhibitors. Research is ongoing (8).
  • TRP Transient receptor potential
  • TRPC canonical
  • TRPV vallinoid
  • TRPM mesattat in
  • TRPA ankyrin
  • TRPP polycystin
  • TRPML mucolipin
  • TRPC is a non-selective Ca 2+ -permeable cation channel and has seven channels (TRPC1-TRPC7).
  • TRPC channels are expressed in smooth muscle cells of the uterus and gastrointestinal tract as well as blood vessels of various sizes (9). Recently, TRPC channels are known to play an important role in the regulation of smooth muscle function (10).
  • TRPC4 channels are known to play an important role in regulating endothelial function, which is critical for the development and progression of erectile dysfunction (14). It has been reported that the expression of TRPC4 channels in diabetes increased the levels of mRNA and protein (15, 16). However, there are no known changes in the expression of TRPC4 channels in diabetic erectile dysfunction penile cavernous smooth muscle.
  • mRNA and protein 15, 16
  • TRPC4 channels there are no known changes in the expression of TRPC4 channels in diabetic erectile dysfunction penile cavernous smooth muscle.
  • TRPC4 transient receptor..potential cation channel, subfamily C
  • TRPC4 activity in the cavernous tissue By inhibiting experimentally confirmed that the therapeutic effect of erectile dysfunction is achieved to complete the present invention.
  • an object of the present invention to provide a pharmaceutical composition for treating erectile dysfunction comprising an inhibitor of TRPC4 as an active ingredient.
  • Another object of the present invention is to provide a dominant negative mutant protein of TRPC4 and a polynucleotide encoding the same.
  • Another object of the present invention to provide a pharmaceutical composition for treating erectile dysfunction comprising the dominant negative mutant protein of TRPC4 and a polynucleotide encoding the same as an active ingredient.
  • the present invention provides a method for screening an erectile dysfunction drug candidate comprising the following steps:
  • TRPC4 transient receptor potential cation channel subfamily C, member 4
  • TRPC4 transient receptor potential cation channel subfamily C, member 4
  • the term "subject to inhibit the activity of TRPC4" as a substance that is expected to inhibit the activity of TRPC4 means a substance used in the screening in anticipation of a therapeutic effect of erectile dysfunction.
  • Treatment of the activity inhibitory candidate of TRPC4" as used herein includes not only contact of the activity inhibitory candidate of TRPC4 with a cell, but also introducing a candidate into a cell.
  • candidates for inhibiting the activity of TRPC4 include not only substances which inhibit the activity of TRPC4 protein, but also substances which decrease the activity of TRPC4 protein by down-regulating the expression level of TRPC4 gene.
  • candidates for inhibiting activity of TRPC4 of the present invention are substances that down-regulate the expression level of the TRPC4 gene, and include RNAi (RNA interference) molecules, antisense oligonucleotides, siRNA, shR A, or miRNA, Oligopeptides, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, peptide nucleic acids (PNAs), aptamers, small molecule or high molecular compounds, and dominant substances that bind and inhibit their activity Negative mutants.
  • RNAi RNA interference
  • antisense oligonucleotides siRNA, shR A, or miRNA
  • Oligopeptides Oligopeptides
  • monoclonal antibodies polyclonal antibodies
  • chimeric antibodies humanized antibodies
  • PNAs peptide nucleic acids
  • aptamers small molecule or high molecular compounds
  • Compounds that can be used as candidates in the present invention can be obtained from a library of synthetic compounds or natural product compounds, the synthetic compound library is Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA) commercially available. Libraries of natural compounds are commercially available from Pan Laboratories (USA) and MycoSearch (USA). Available for purchase.
  • the compounds may be obtained by a variety of combinatorial library methods known in the art, such as biological libraries, spatial addressable parallel solid or liquid libraries (spat ia Uy addressable parallel sol id phaseor solution phase libraries), deconvolution required By a synthetic library method, a "1-bead 1-compound” library method, and a synthetic library method using affinity chromatography screening.
  • Methods of synthesizing molecular libraries are described in DeWitt et al. , Proc. Natl. Acad. Sci. USA 90, 6909, 1993; Erb et al. , Proc. Natl. Acad. Sci. USA 91, 11422, 1994; Zuckermann et al., J. Med. Chem.
  • Recombinant vectors comprising polynucleotides encoding TRPC4 in the present invention can be prepared by cloning a nucleic acid molecule encoding T PC4 into the vector, wherein the vector is typically used to express nucleic acid in prokaryotic or eukaryotic host cells.
  • the vector of the present invention includes a signal sequence required for efficient polyadenylation sequence, transcription termination, ribosomal binding site, or translation termination in addition to a promoter operably linked with a polynucleotide sequence encoding TRPC4, Additional elements may include enhancer elements, heterologous splicing signals or nuclear localization signals (NLS), and the like.
  • the method for transforming cells with the recombinant vector includes liposome mediated delivery, electroporation, chemicals that increase free DNA uptake, direct injection of DNA into plants, particle total stratification, virus or pollen transformation. Conversion and microprojection, calcium / polyethylene glycol method for plasma (Krenset et al, 1982; Negrutiu et al, 1987), plasma electroporation (Shillito et al, 1985), microinjection (Crossway et al, 1986), DNA or RNA coated particle bombardment (Klein et al, 1987), and methods by viral infection.
  • the cells used in the screening method of the present invention include prokaryotic and eukaryotic cells, and include bacteria, yeast, nasal cells, and animal cells, preferably animal cells.
  • Determination of whether TRPC4 is inhibited in the screening method of the present invention It is carried out using a method capable of measuring the activeol of the ion channel, preferably
  • the patch clamp method is used in cells expressing TRPC4.
  • a pharmaceutical composition for treating erectile dysfunction which comprises an inhibitor of TRPC4 (transient receptor potential cat ion channel, subfamily C, member 4) as an active ingredient.
  • the term "Erectile dysfunct ion” means a disease in a state in which there is not enough erection or maintenance for sexual life.
  • the erectile dysfunction in the present invention includes diabetic erectile dysfunction.
  • the inhibitor of TRPC4 is an expression inhibitor of TRPC4 gene.
  • the expression inhibitor of the TRPC4 gene is an RNAi (RNA interference) molecule that specifically binds to the TRPC4 gene.
  • RNAi RNA interference molecules that bind to the gene as TRPC4, specifically is an antisense oligonucleotide, siRNA, shRNA, miRNA, or having a sequence complementary to the gene sequence TRPC4.
  • anti-sense oligonucleotide refers to a DNA or RNA or derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, and binds to the complementary sequence in the mRNA to
  • the antisense sequence of the present invention refers to a DNA or RNA sequence complementary to the TRPC4 gene sequence and capable of binding to TRPC4 mRNA, and the translation of TRPC4 mRNA into the cytoplasm. May inhibit the essential activity for translocation, maturation or any other overall biological function.
  • To 100 bases preferably 8 to 60 bases, and more preferably 10 to 40 bases.
  • siRNA small interfering RNA
  • siRNA refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (W0 00/44895, W0 01/36646, W0 99/32619, W0 01/29058, W0 99/07409 and W0 00/44914).
  • siRNA is provided as an efficient gene knockdown method or gene therapy method because it can inhibit the expression of the target gene.
  • the siRNA molecule of the present invention may have a structure in which the sense strand (the sequence corresponding to the mRNA sequence of the TRPC4 gene) and the antisense strand (the sequence complementary to the mRNA sequence of the TRPC4 gene) are positioned opposite to each other to form a double strand.
  • siRNA molecules of the present invention may have a single chain structure with self-complementary sense and antisense strands.
  • siRNAs are not limited to pairing of complementary RNAs completely, and may include portions that are not paired by mismatches (bases that are not complementary) or bulges (there is no base corresponding to one chain). have.
  • the total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases.
  • the siRNA terminal structure can be either blunt or cohesive, as long as the expression of the TRPC4 gene can be suppressed by the RNAi effect.
  • the cohesive end structure is possible for both 3'-end protrusion structures and 5'-end protrusion structures.
  • the siRNA molecules of the present invention may have a form in which a short nucleotide sequence (eg, about 5-15 nt) is inserted between a self-complementary sense and an antisense strand, in which case by expression of the nucleotide sequence
  • the siRNA molecules formed form a hairpin structure by intramolecular shaking, and form a stem-and-loop structure as a whole. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule that can mediate RNAi.
  • shRNA small hairpin RNA Ehsms short hairpin RNA
  • shRNA refers to an RNA nucleotide sequence that creates a robust hairpin turn structure that is used to silence expression of target genes through RAN inhibition (RNA interference). Or molecule.
  • shRNA is a single-stranded RNA with a length of 45 to 70 nucleotides
  • Oligonucleotide DNA is synthesized by linking 3-10 base linkers between the sense sequence of the gene sequence and the antisense sequence complementary thereto. Expression of shRNA in cells is accomplished using plasmid vectors or vectors for virus or bacteria and the promoter selects suitable ones for robust shRNA expression.
  • Polymerase ⁇ promoters such as U6 and HI may be used, and polymerase ⁇ promoters may be used to regulate the expression of shRNA.
  • Various viral vectors can be used for the expression of shRNAs, such as adenoviruses, adeno-associated viruses or lentiviruses. Inserting and expressing a shRNA into a target cell to express it in a vector produces a small hairpin RNA (shRNA) with a looped 'hairpin' structure, which is converted into an siRNA by Dicer in the cell, and the RNAi effect. Indicates.
  • shRNA small hairpin RNA
  • Expression promoters / vectors including shRNAs can be prepared by methods known in the art, for example, US Patent Publications 20040106567 and 20040086884 include viral vectors including shRNAs. Information regarding expression constructs / vectors is disclosed.
  • miRNA (microRNA) ' is a single-stranded NA molecule of 21-25 nucleotides and is a modulator that controls gene expression in eukaryotes through inhibition of the disruption or translation of target mRNAs. It consists of two stages of processing: the first miRNA transcript (primary miRNA) is made into a 70-90 base stem-loop structure, or pre-miRNA, by the RNase type enzyme Drosha in the nucleus and then migrates into the cytoplasm. It is cleaved by an enzyme called Dicer into a mature miRNA of 21-25 bases, which binds complementarily to the target mRNA and acts as a post-transcript ional gene suppressor. Induces translation inhibition and m RNA destabilization.
  • primary miRNA is made into a 70-90 base stem-loop structure, or pre-miRNA, by the RNase type enzyme Drosha in the nucleus and then migrates into the cytoplasm. It is cleaved by an enzyme called Dicer
  • the inhibitor of TRPC4 is an activity inhibitor of TRPC4 protein.
  • the activity inhibitor of TRPC4 is a substance that binds to TRPC4 protein and inhibits its activity,
  • oligopeptide monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, PNA (Peptide nucleic acid), aptamer, small molecule or high molecular compound, and dominant negative mutant ).
  • antibody refers to a specific protein molecule directed against an antigenic site.
  • antibody refers to an antibody that specifically binds to TRPC4 protein, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies.
  • the antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.
  • Antibodies can be prepared by methods conventionally practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4 ⁇ 816,56). Or) phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol., 222: 58, 1-597 (1991)). have. General procedures for antibody preparation are described in Harlow, E.
  • Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis salt precipitation, ion exchange chromatography, affinity chromatography, and the like. Polyclonal antibodies inject the protein antigen into a suitable animal and the antisera from the animal. After collection, the antibody can be obtained by separating the antibody from the antiserum using known affinity techniques.
  • antibodies of the invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains.
  • a functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.
  • the term “chimeric antibody” refers to an antibody produced by combining a source other than human, such as a mouse derived antibody site with a human derived antibody site.
  • humanized antibody refers to an antibody that has increased the similarity to antibody variants generated in humans by modifying antibodies having amino acid sequences from sources other than humans, as described in Riechmann L et al. . , (1988). "Resha ing human antibodies for therapy”. Nature 332 (6162): 332-323; Queen C et al., "A humanized antibody that binds to the inter leukin 2 receptor.”. Proc Natl Acad Sci U S A. 86 (24): 10029-33.
  • aptamer refers to an oligonucleic acid or peptide molecule, the general content of which is described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide ap tamers: powerful new tools for molecular medicine”. J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent. "An artificial cell- cycle inhibitor isolated from a combinatorial library”. Proc Nat 1 Acad Sci USA. 95 (24): 142727 (1998).
  • PNA protein nucleic acid
  • PNA protein nucleic acid
  • N- (2-aminoethyl-glycine) linked by peptide bonds is composed of oils, and purine and pyrimidine bases are linked to the backbone by methylene bridges (-CH2-).
  • -CH2- methylene bridges
  • the invention provides a TRPC4 comprising a amino acid sequence of SEQ ID 29 It provides a dominant negative mutant protein of the transient receptor potential cation channel, subfamily C, member 4.
  • the present invention provides a polynucleotide encoding the dominant negative mutant protein.
  • the present invention provides an erectile dysfunction treatment comprising a recombinant vector comprising the dominant negative mutant protein or a polynucleotide encoding the protein in an expressible form as an active ingredient.
  • the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation of lactose, dextrose, sucrose, sorbbi, manny, starch, acacia rubber, calcium phosphate, alginate. Gelatin, Calcium Silicate, Microcrystalline Cellulose, Polyvinylpyrrolidone, Sealolose, Water, Syrup, Methyl Cellulose, Methylhydroxybenzoate, Propylhydroxybenzoate, Talc, Magnesium Stearate and Minerals Including but not limited to oils, etc.
  • the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. in addition to the above components.
  • Acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
  • the pharmaceutical composition of the present invention can be oral or parenteral administration, parenteral administration is intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration And the like.
  • the pharmaceutical composition of the present invention can be administered by injecting directly into the corpus cavernosum.
  • Suitable dosages of the pharmaceutical compositions of the invention include formulation methods, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, absorption of the active ingredient in the body, inactivation rate, drugs used in combination It can be prescribed in various ways, such as factors such as excretion rate and response sensitivity.
  • the dosage of the pharmaceutical composition of the present invention is 0.0001 ng / kg body weight to 100 mg / kg body weight per day.
  • compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of axes, powders, granules, tablets or accelerators, and may further include a dispersant or stabilizer.
  • the present invention relates to a method for screening a candidate drug for treating erectile dysfunction comprising measuring the inhibition of activity of TRPC4 and a pharmaceutical composition for treating erectile dysfunction comprising an inhibitor of TRPC4 as an active ingredient.
  • the present invention can effectively screen for candidate drugs for treating erectile dysfunction using the method for measuring activity of TRPC4.
  • Figure la and lb are the results of RT-PCR analysis of TRPC mRNA in the corpus cavernosum smooth muscle of human [(a: cell (cel l) and b: tissue (s)] and rat, respectively. .
  • Figure 2 shows the relative levels of TRPC mRNA expression in the corpus cavernosum compared to the normal control.
  • TRPC3 1.4 ⁇ 0.15 times
  • TRPC4 1.8 ⁇ 0.29 times
  • TRPC6 1.5 ⁇ 0.20 times
  • n 7, * : p ⁇ 0.05 vs. Normal control.
  • TRPC1 1.1 times 0.08 times
  • TRPC4 3.2 times 0.24 times
  • TRPC6 2.24 times 0.37 times
  • n 8 *: p ⁇ 0.05 vs. Normal control.
  • FIGS. 4A and 4B show the results of immunohistochemical experiments of the expression of TRPC ⁇ TRPC4 and TRPC6 in rat penile tissue (X 50) and the immunohistochemistry of TRPC4 in veins, arteries and penile sinus. . Increased intensity of TRPC4 was seen in penile sinus as well as veins and arteries (Panel B, x400). Blue, green and red indicate DAP ⁇ TRPC, and ⁇ -actin, respectively: N: normal, DM: diabetes.
  • 5A and 5B show TRPC1, in the penile tissues of rats as compared to the normal control.
  • TRPC4 and TRPC6 were measured, respectively (see Panel A).
  • 6A-6D show the results of in vivo penile nerve stimulation to compare the erectile function of normal control, diabetic control and TRPC4 DN -transfected diabetic rats.
  • Diabetic rats showed a significantly lower ICP / ABP ratio than the normal control, and the rats transfected with TRPC4 DN gene did not differ from the normal control.
  • Figure 7 shows the results of measuring the expression of TRPC4 DN yumche in rat CSM tissue two weeks after transfection.
  • Human tissue was obtained from the corpus cavernosum of patients with organic erectile dysfunction implanted with penile prosthesis. Tissue according to the conventionally known method (17, 18) Human CSM (corpus smooth muscle) cells were cultured using the transplant method. Human CSM cells were incubated at 37 ° C. and 5% CO 2 conditions using 10% FBS-DMEM (fetal bovine serum-Dulbecco's modified Eagle medium) medium, and the medium was changed every 2-3 days. Primary culture cells were used 2-4 passages.
  • FBS-DMEM fetal bovine serum-Dulbecco's modified Eagle medium
  • RT-PCR was used to screen TRPC expression in CSM.
  • Total RNA was extracted from human CSM cells, frozen tissue and rat penis tissue, respectively, cultured using TRIzol (Invitrogen, Carlsbad, CA) method.
  • RNA was synthesized cDNA using a random nucleomer primer of the Superscript II First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA).
  • the expression of TRPC mRNA was confirmed using Taq DNA polymerase (Roche Applied Science, Mannheim, Germany). The combination of primers used in the reaction is shown in Table 1.
  • GAPDH was used as loading control and RNA control internal control.
  • Real-time quantitative PCR was performed using a 7900HT Fast Real-Time PCR system (Applied Biosysterns, Foster City, CA, USA) and a Taqman gene expression master mix (Applied Biosystems).
  • Gene specific Taqman primers and probes for rat TRPC1 (Rn00585625_ml), TRPC3 (Rn00572928_ml), TRPC4 (Rn00584835_ml), TRPC6 (Rn00677559_ml), and ⁇ actin (actin-Rn00667869_ml) were purchased from Applied Biosystems.
  • PCR conditions are as follows The initial denaturation was performed at 95 ° C for 10 minutes, followed by 40 cycles of amplification with 15 seconds of degeneration at 95 ° C and extended reaction at 60 ° C for 1 minute. Samples without the template were designated as negative controls and three replicate measurements were taken for all samples.
  • the standard curve for quantification of genes was obtained by PCR after synthesizing cDNA with RNA obtained from rat brain and sequentially diluting it 10 times, and the relative fold change of genes was the target gene and housekeeping gene. Calculated by the ⁇ -actin ratio. Gene expression differences were analyzed using Student's t-test and statistical significance level was based on 0.05.
  • the frozen tissue was ground and homogenized using mortar and pestle, and lysed solution containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM NaCl, and 0.1% SDS (Pro-prep protein extract ion solution, i tRON, Seoul, Korea) and the protease inhibitor cocktail (Roche, Mannheim, Germany) were added to lyse the cells and centrifuged at 14,000 rpm for 10 minutes at 4 ° C.
  • the membrane was blocked for 1 hour at room temperature with 5% BSA in TBS (Tris buffered saline; 10 mM Tris, 100 mM NaCl) containing 0.1% Tween-20, followed by mouse monoclonal anti-TRPC1 antibody (1: 2000 dilution, SantaCruz, CA, USA), mouse monoclonal anti-TRPC4 antibody (1: 2000 dilution, Neuromab, Davis, CA), rabbit polyclonal anti-TRPC6 antibody (1: 500 dilution, Alomone Laboratories, Jerusalem, Israel), or monoclonal anti- ⁇ -actin (1: 5000 dilution, Abeam, MA, USA) overnight at 4 ° C.
  • TBS Tris buffered saline
  • mouse monoclonal anti-TRPC4 antibody 1: 2000 dilution, Neuromab, Davis, CA
  • rabbit polyclonal anti-TRPC6 antibody (1: 500 dilution, Alomone Laboratories, Jerusalem, Israel
  • the membrane was reacted with anti-rabbit or anti-mouse IgG alkaline phosphatase bound antibody (1: 1000, Santa Cruz) for 1 hour in a blocking supernatant at room temperature. Excess secondary antibody was washed again and bound secondary antibody was detected by ECL (enhanced chemi luminescence; Pierce). In order to quantify, the film is flat After scanning on a scanner and digitizing band and relative intensities, they were analyzed using UN-SCAN-IT software (Silk Scientific, UT, USA). Western blotting was repeated 6 or more times using different rat tissues. The relative intensities of the bands were quantified using the Image J 1.34 image analysis system (National Institutes of Health).
  • Dual immunofluorescence labeling for TRPC and smooth muscle ⁇ -actin was performed in control and diabetic rat tissues.
  • the penile tissues were fixed for 4 hours at room temperature in 4% paraformaldehyde (5, 10, 15, 20% w / v, 5-15% in various concentrations of sucrose solution in PBS). For 15 minutes each, for 20% ⁇ overnight). Samples were placed in a timber cutting temperature compound (OCT, Sakur Finetek, Torrance, Calif., USA) and quickly frozen in liquid nitrogen and then stored at -70 ° C.
  • OCT timber cutting temperature compound
  • mice monoclonal anti -TRPC1 (1:50, Santacruz) and anti -TRPC4 (1:50, Neuromab)
  • rabbit polyclonal anti— TRPC6 (1:50, Abeam)
  • mouse monoclonal Ronald or rabbit polyclonal smooth muscle term- ⁇ —actin (1: 500, Abeam).
  • Negative controls were performed with the exception of the primary antibody. After three washes with PBS-Triton, the sections were ligated with Alexa fluor 488- and 594-conjugated secondary antibodies (1: 1000, Invitrogen, Carlsbad, Calif., USA) for 1 hour at room temperature. Curated.
  • Induction of diabetes mellitus for 4 months in 8-week-old experimental animals at the time of administration was performed by Streptozocin dissolved in citrate buffer (60 mL of 100 mM citric acid and 40 mL of 200 mM disodium phosphate, pH 4.6), according to known methods (19). (STZ) was done via a single intraperitoneal injection at a dose of 60 mg / kg. The control group was injected with vector only. Diabetic rats were identified as diabetic for one week (diabetes when blood glucose levels were higher than 300 mg / dL) and used in the study. Blood glucose levels were monitored at weekly intervals for the first month and then monthly for up to four months thereafter.
  • MTRPC4P in PCDNA3 was determined by Dr. Provided by M. Schaefer. Preparation of the dominant negative form (DN) of TRPC4
  • a total of 30 adult male Spra ague-Daw ley rats were used in the experiment.
  • the experimental animals were divided into the control group and the diabetic experimental group.
  • Ten rats were used as a control group and the remaining 20 rats were diabetic group.
  • Ten rats were used as diabetic controls and 10 rats were used as TRPC4 DN gene transfection groups. All rats used in the experiment were bred under constant temperature and light conditions.
  • Four months after diabetes induction, STZ-induced diabetic rats were intraperitoneally administered with ketamine (50 mg / kg). After the incision and aseptic exposure of the penis, ligation was performed at the base of the penis.
  • the TRPC4 DN gene injection group injected the TRPC4 DN gene into the corpus cavernosum at a dose of 200 ug / 100yL [20% sucrose -phosphate buffered saline (PBS)] and the control group injected only the same amount of solvent. Ligation was removed 2 minutes after injection.
  • PBS sucrose -phosphate buffered saline
  • TRPC4 DN gene or vector was transfected into penile cavernous cells of STZ-induced diabetic rats and then erection response measurements were taken two weeks later. Detailed methods for measuring erection reactions are described in the prior document (19).
  • systemic arterial blood pressure PB was monitored by inserting a polyethylene (PE) -50 tube into the neck artery.
  • PE polyethylene
  • the prostate and bladder were exposed through median abdominal incisions.
  • Major pelvic nodules, pelvic nerves and cavernous nerves were identified from each side to the prostate as the posterior side, and electrical stimulators with stainless steel bipolar hook electrodes were placed around the cavernous nerve for electrical stimulation.
  • ICP intracavernosal pressure
  • 25-gauge cannula was layered with 250 U / mL heparin, connected to a PE-50 tube, and inserted into the right penis angle.
  • Whole body BP and ICP were measured with a transducer connected to a computer system for data collection (PowerLab ADI Instruments, Sydney, Australia).
  • Real time display and recording of blood pressure were done using Chart 5 software (ADI Instruments).
  • the electric field stimulation parameters were as follows: The voltage was 6V and lasted 60 seconds using an electrical stimulator (model PG 721A; Austin Electronic Specialties Inc, Palo Alto, Calif., USA).
  • PCR reactions using a Perkin-Elmer Thermal Cycler were conducted under the following conditions: initial denaturation at 95 ° C. for 5 minutes; 30 cycles of 30 seconds at 95 ° C., 30 seconds at 50 ° C., 30 seconds at 72 ° C .; Final extension of 7 minutes at 72 ° C. GAPDH was used as a control to confirm the same loading and robustness of RNA.
  • PCR products were subjected to 19 agarose gel electrophoresis and DNA bands were visualized under UV light by EtBr (ethidium bromide) staining. 11.
  • EtBr ethidium bromide
  • RT-PCR reactions were performed using specific primers for each TRPC homolog to determine which TRPC homologs were expressed in CSM (Table 1).
  • hTRPG4 415 Reverse 5 '-AAGGTlTCTTCGTGCAAATTOCATTe-3' (SEQ ID NO: 10)
  • hTRPCe 428 Reverse 5 '-ATGACTGGATCTTCACTAGAC-3' (SEQ ID NO: 10
  • TRPCl TRPC3, TRPC4, and TRPC6 niRNAs were expressed in cells cultured with human corpus cavernosum tissue and rat corpus cavernosum tissue, but TRPC5 and TRPC7 isoforms were not expressed (FIG. la and lb).
  • TRPC4 and TRPC6 proteins were significantly increased in diabetic rats compared to the normal control, while no significant change in expression of TRPC1 was measured. The difference in TRPC4 expression between the control and diabetic models was more pronounced in western blotting.
  • TRPC4 3.2 cells 0.24 fold
  • TRPC6 2.24 mm 0.4 times
  • n 8, p ⁇ 0.05 vs. normal control, see FIGS. 3A and 3B.
  • TRPC4 expression was found to be higher in CSM tissue of diabetic rats compared to normal rats.
  • TRPC4 fluorescence was consistent with Q-SMA fluorescence.
  • TRPC6 immunofluorescence was slightly higher in diabetic rats than in normal control, but there was no statistically significant difference between the two groups as in TRPC1.
  • TRPC4 DN TRPC4 dominant negative mutation
  • TRPC4 DN gene expression in CSM tissues was confirmed by RT-PCR 2 weeks after transfection (see FIG. 7). TRPC4 DN gene transfer in diabetic rats induced recovery of erectile function and an increase in ICP / BP, which was maintained for at least 2 weeks. conclusion
  • TRPC4 channels have increased expression in penile CSM cells of rats with diabetes.
  • the transfer of TRPC4 Dlv ⁇ gene into penile tissue restored erectile function in diabetic rats.
  • the TRPC4 channel is an important pathophysiological component of erectile dysfunction and can be an important target for the treatment of erectile dysfunction.

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Abstract

La présente invention se rapporte à un procédé de recherche par criblage d'un médicament candidat pour traiter une dysérection, le procédé comprenant une étape de mesure de l'inhibition de l'activité du TRPC4; et à une composition pharmaceutique permettant le traitement d'une dysérection, contenant un inhibiteur du TRPC4 en tant que principe actif. La présente invention permet de rechercher efficacement par criblage un médicament candidat pour traiter une dysérection à l'aide d'un procédé de mesure de l'activité du TRPC4. L'inhibiteur de l'activité du TRPC4 selon la présente invention est très efficace pour soulager le syndrome de dysérection et peut ainsi être développé pour un médicament pour traiter une dysérection.
PCT/KR2015/002993 2014-03-27 2015-03-26 Composition pharmaceutique permettant le traitement d'une dysérection, contenant un inhibiteur du trpc4 en tant que principe actif Ceased WO2015147575A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20100135619A (ko) * 2009-06-17 2010-12-27 경상대학교산학협력단 알코올 노출에 의한 생식기능장애 질환 치료제의 스크리닝 방법
KR20110016591A (ko) * 2009-08-12 2011-02-18 성균관대학교산학협력단 발기부전 치료용 조성물

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20100135619A (ko) * 2009-06-17 2010-12-27 경상대학교산학협력단 알코올 노출에 의한 생식기능장애 질환 치료제의 스크리닝 방법
KR20110016591A (ko) * 2009-08-12 2011-02-18 성균관대학교산학협력단 발기부전 치료용 조성물

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI GenBank [O] 7 June 2007 (2007-06-07), "transient receptor potential cation channel, subfamily C, member 4, isoform CRA_a [Mus musculus", XP055227258, Database accession no. EDL35253 *
LEE, SEONG WON ET AL.: "Development of Etiology-Specific Gene Therapy by Regulation of TRPC Signaling Module in Erectile Dysfunction", SAMSUNG SEOUL HOSPITAL, FINAL REPORT OF HEALTH & MEDICAL TECHNOLOGY R&D PROGRAM, 18 April 2013 (2013-04-18) *

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