WO2018024878A1 - Procédés et compositions pour réduire l'expression de pcsk9 - Google Patents

Procédés et compositions pour réduire l'expression de pcsk9 Download PDF

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WO2018024878A1
WO2018024878A1 PCT/EP2017/069779 EP2017069779W WO2018024878A1 WO 2018024878 A1 WO2018024878 A1 WO 2018024878A1 EP 2017069779 W EP2017069779 W EP 2017069779W WO 2018024878 A1 WO2018024878 A1 WO 2018024878A1
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pcsk9
expression
subject
malignant
hepatocytes
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PCT/EP2017/069779
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Raphaël SCHARFMANN
Lukas HUIJBREGTS
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paris Descartes
Centre National De La Recherche Scientifique (Cnrs)
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Publication of WO2018024878A1 publication Critical patent/WO2018024878A1/fr

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    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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 invention is in the field of cardiology, oncology and infectiology. Particularly, the present invention relates to compounds which reduce the PCSK9 expression.
  • Proprotein convertase subtilisin/kexin type 9 is an enzyme involved in cholesterol homeostasis. More particularly, PCSK9 downregulates liver LDL receptor (LDLR) proteins which mediate the endocytosis of cholesterol-rich LDL.
  • LDLR liver LDL receptor
  • Low-density lipoprotein (LDL) is one of the five major groups of lipoprotein. LDL particles are responsible of risk of cardiovascular disease when they invade the endothelium and become oxidized, since the oxidized forms are more easily retained by the proteoglycans.
  • High level of LDL particles is correlated with accumulation of atherosclerosis within the walls of arteries over time, resulting in sudden plaque ruptures, decades later, and triggering clots within the artery opening, or a narrowing or closing of the opening, i.e. cardiovascular disease, stroke, and other vascular disease complications. Accordingly, LDL particles are considered as bad cholesterol. In contrast, High-density lipoprotein particles (HDL) are often called good cholesterol or healthy cholesterol because they can remove lipid molecules from macrophages in the wall of arteries.
  • HDL High-density lipoprotein particles
  • PCSK9 has emerged as an attractive therapeutic target for hypercholesterolemia, cardiovascular diseases and cancers.
  • monoclonal antibodies such as Alirocumab (trade name Praluent) and Evolocumab (trade name Repatha) are commercialized by Sanofi and Amgen respectively. More than 20 clinical trials have shown that monoclonal antibodies against PCSK9 can reduce cholesterol, cardiac events and all-cause mortality. Many side effects are observed with monoclonal antibodies treatment and particularly, before the treatment, patients should be administered with corticosteroids, histamine receptor blockers, and acetaminophen. Peptide mimics, gene silencing or vaccination strategies are ongoing in clinical trials.
  • the invention relates to a method for reducing or inhibiting PCSK9 expression in a subject in need thereof comprising a step of administering to said subject a therapeutically amount of a I-BET 151 and/or JQ1.
  • the present invention is defined by the claims.
  • the invention relates to a method for reducing or inhibiting PCSK9 expression in a subject in need thereof comprising a step of administering to said subject a therapeutically amount of a I-BET 151 and/or JQ1.
  • PCSK9 refers to Proprotein convertase subtilisin/kexin type 9, is a protein that in humans is encoded by the PCSK9 gene and involved in cholesterol homeostasis. PCSK9 binds to the receptor for low-density lipoprotein particles (LDL), which transport fat molecules such as cholesterol.
  • LDL low-density lipoprotein particles
  • the naturally occurring human PCSK9 gene has a nucleotide sequence as shown in Genbank Accession number NM_ 174936.3 and the naturally occurring human PCSK9 protein has an aminoacid sequence as shown in Genbank Accession number NP 777596.2.
  • the murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NM_153565.2 and NP_705793.1).
  • the term "reducing or inhibiting” refers to the at least partial suppression of the expression of the PCSK9 gene, as manifested by a reduction of the amount of mRNA transcribed from the PCSK9 gene.
  • LDLRs LDL receptors
  • the inhibition of PCSK9 leads to an increase of LDLRs and thus to a decrease of LDL-particles which contain for example cholesterol in the blood.
  • subject refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human.
  • the method for reducing or inhibiting PCSK9 expression is useful to treat disorders in which the PCSK9 is overexpressed.
  • the term "overexpressed" refers to a high or strong expression of the PCSK9 in a subject suffering from a disorder comparing to a control subject.
  • the overexpression of PCSK9 may be at genomic and/or nucleic and/or protein level.
  • the overexpression PCSK9 may be determined by any technology known by a person skilled in the art.
  • the overexpression of PCSK9 gene is determined by measuring the amount of nucleic acid transcripts of gene.
  • the overexpression of PCSK9 is determined by measuring the amount of gene corresponding protein.
  • the amount of nucleic acid transcripts can be measured by any technology known by a man skilled in the art.
  • the measure may be carried out directly on an extracted messenger RNA (mR A) sample, or on retrotranscribed complementary DNA (cDNA) prepared from extracted mRNA by technologies well-known in the art.
  • mR A messenger RNA
  • cDNA retrotranscribed complementary DNA
  • the amount of nucleic acid transcripts may be measured using any technology known by a man skilled in the art, including nucleic microarrays, quantitative PCR, microfluidic cards, and hybridization with a labelled probe.
  • the overexpression of PCSK9 is determined using quantitative PCR.
  • Quantitative, or real-time, PCR is a well-known and easily available technology for those skilled in the art and does not need a precise description.
  • Methods for determining the quantity of mRNA are well known in the art.
  • the nucleic acid contained in the biological sample is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions.
  • the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR).
  • hybridization e. g., Northern blot analysis
  • RT-PCR e.g., RT-PCR
  • quantitative or semi-quantitative RT-PCR is performed. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.
  • Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • SCC is a 0.15 M NaCl, 0.015 M Na- citrate).
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the method of the invention comprise the steps of providing total RNAs extracted from a biological samples and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi-quantitative RT-PCR.
  • the expression level is determined by DNA chip analysis.
  • Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
  • Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs.
  • a biological sample from a test subject optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling.
  • Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).
  • the method of the invention is suitable for treating a subject suffering from the disorders in which PCSK9 is overexpressed.
  • disorders in which PCSK9 is overexpressed may be cardiovascular diseases, dyslipidemias, cancer or infections.
  • cardiovascular disease is a general term used to classify numerous conditions affecting the heart, heart valves, blood, and vasculature of the body and encompasses any disease affecting the heart or blood vessels, including, but not limited to, Metabolic Syndrome, Syndrome X, atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina pectoris, stroke, diseases of the aorta and its branches (such as aortic stenosis, thrombosis or aortic aneurysm), peripheral artery disease, peripheral vascular disease, cerebrovascular disease, and including, without limitation, any transiently or permanently ischemic arteriovascular event.
  • Metabolic Syndrome Syndrome X
  • atherosclerosis atherosclerosis
  • atherothrombosis coronary artery disease
  • stable and unstable angina pectoris stable and unstable angina pectoris
  • stroke diseases of the aorta and its branches (such as aortic stenosis, thrombosis or a
  • the subject suffers from dyslipidemias.
  • dyslipidemias or “dyslipidemias” refers to an abnormal amount of lipids (e.g., triglycerides, cholesterol and/or fat phospholipids) in the blood.
  • Dyslipidemias diseases correspond to hyperlipidemia (elevated lipid levels) or hypercholesterolemia (elevated cholesterol levels).
  • the subject suffers from a cancer.
  • the term refers herein, the term
  • cancer refers to an abnormal cell growth with the potential to invade or spread to other parts of the body.
  • cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the subject suffers from an infection.
  • the term as used herein, the term
  • infection refers to diseases caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi; the diseases can be spread, directly or indirectly, from one person to another. Infectious diseases including, but not limited to, those caused by hepatitis viruses (e.g. hepatitis B virus, hepatitis C virus), human immunodeficiency virus (HIV), papillomaviruses, herpesviruses, respiratory viruses (e.g.
  • influenza viruses respiratory syncytial virus, rhinovirus, metapneumo virus, parainfluenzavirus, SARS), West Nile virus, tuberculosis, bacterial pneumonia, aspergillosis, histoplasmosis, candidosis, pneumocystosis, leprosy, chlamydia, cryptococcal disease, cryptosporidosis, toxoplasmosis, leishmania, malaria, and trypanosomiasis.
  • the subject suffers from HIV.
  • a “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject.
  • a “therapeutically effective amount” to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • I -BET 151 refers to a bromodomain (BRD) inhibitor and blocks recruitment of bromodomain and extra-terminal (BET) to chromatin.
  • BET 151 has the formula C23H21N503 and the following structure in the art:
  • JQ1 refers to a thienotriazolodiazepine, has the formula C23H25C1N402S and the followin structure in the art:
  • administering refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., inhibitors of PCSK9 JQ1 and IBET 151) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
  • a disease, or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof.
  • administration of the substance typically occurs before the onset of the disease or symptoms thereof.
  • the two inhibitors as described above are administered in combination to the subject.
  • the inhibitors are administered as a combined preparation. More particularly, the inhibitors are administered simultaneously, separately or sequentially.
  • administration simultaneously refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time.
  • administration separately refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes.
  • administration sequentially refers to an administration of 2 active ingredients at different times, the administration route being identical or different.
  • the two inhibitors as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • pharmaceutically acceptable excipients such as a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, a carboxylate, a pharmaceutically acceptable.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Time course effect of BET inhibitors on Pcsk9 mRNA levels in mouse pancreatic buds.
  • Mouse embryonic pancreatic buds were cultured in presence of either DMSO or I-BET 151 (0.5 ⁇ ) or (+)-JQl ( ⁇ . ⁇ ). After 1, 3, 5 or 7 days of culture, total RNA was extracted. Relative expression of Pcsk9 was measured by RT-qPCR normalized with Cyclophylin A expression. Values represent the average of three experiments with standard deviation error bars. ***P ⁇ 0.001
  • FIG. 2 Dose response effect of BET inhibitors on Pcsk9 mRNA levels in mouse primary hepatocytes.
  • Primary hepatocytes were cultured for 24h in presence of 0.1 % DMSO or increasing concentrations of IBET-151 (0.5 ⁇ ; ⁇ ; 2.5 ⁇ ) or (+)-JQl ( ⁇ . ⁇ ; 0.5 ⁇ ; 2 ⁇ ).
  • Total RNA was then extracted and relative expression of Pcsk9 was measured by RT- qPCR normalized with Cyclophylin A expression. Values represent the average of three experiments with standard deviation error bars. ***P ⁇ 0.001
  • A, B Mouse primary hepatocytes were cultured for 48h in presence of 0.1% DMSO or ⁇ of IBET-151 or 0.5 ⁇ of (+)-JQl . Whole cell lysates were prepared in RIPA buffer. LDLR expression was analyzed by Western blot (A) and quantified using image J (B). Values represent the average of two independent experiments with standard deviation error bars. *P ⁇ 0.05 **P ⁇ 0.01
  • LDLR increase is dependent on Pcsk9 inhibition by BET inhibitors A
  • B WT or LDLR-/- mouse primary hepatocytes were cultured for 48h in presence of 0.1% DMSO or ⁇ of IBET-151 or 0.5 ⁇ of (+)-JQl .
  • Whole cell lysates were prepared in RIPA buffer.
  • LDLR expression was analyzed by Western blot (A) and quantified using image J (B).
  • C Primary hepatocytes from WT or LDLR-/- mice were cultured for 24h in presence of 0.1% DMSO or increasing concentrations of IBET-151 (0.5 ⁇ ; ⁇ ; 2.5 ⁇ ) or (+)-JQl (0.1 ⁇ ; 0.5 ⁇ ; 2 ⁇ ).
  • Total RNA was extracted and relative expression of Pcsk9 was measured by RT-qPCR normalized with CyclophylmA expression. Values represent the average of three independent experiments with standard deviation error bars.
  • FIG. 5 Dose response effect of BET inhibitors on Pcsk9 mRNA levels in human primary hepatocytes.
  • Primary human hepatocytes were cultured for 48h in presence of 0.1% DMSO or increasing concentrations of IBET-151 ( ⁇ ; 5 ⁇ ; 10 ⁇ ) or (+)-JQl ( ⁇ ; 2.5 ⁇ ; 5 ⁇ ).
  • Total RNA was extracted and relative expression of Pcsk9 was measured by RT-qPCR normalized with CyclophylmA expression. Values represent the average of three experiments with standard deviation error bars. *P ⁇ 0.05 **P ⁇ 0.01
  • Mouse pancreatic buds were dissected from El 1.5 C57B16/J embryos and placed at the air/medium interface on 0.45 ⁇ filters in culture dishes containing RPMI medium supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, 1% non-essential amino acids and lOmM HEPES. Pancreatic buds were cultured at 37°C with 5% C02 as previously described (Attali et al, 2007; Rachdi et al, 2012).
  • Ibet-151 was obtained from Sigma- Aldrich and (+)/-JQl from Abeam. Stock solutions were prepared in DMSO. Inhibitors subsequent dilutions or DMSO (0.1% final concentration) were directly added to the culture medium which was changed daily.
  • RNA from at least 500,000 hepatocytes or pools of three or more pancreatic buds were extracted using Qiagen RNA extraction microkit and 250ng were reverse transcribed using maxima first strand cDNA synthesis kit from Thermo Fisher.
  • Real-time PCR analysis was performed in IX Sybr Green Powermix in QuantStudio 3 Applied Biosystem system.
  • Relative expression of PCSK9 was calculated using the comparative method of relative quantification (2-AACT) normalized to cyclophilin A expression. Values represent the average of three independent experiments with standard deviation error bars.
  • Statistical analysis was performed using unpaired Student t test (Guillemain et al, 2007; Haumaitre et al., 2008).
  • mice and WT littermates were kindly provided by the Bertrand Cariou's team (INSERM UMR1087, France). Hepatocytes were prepared by the Catherine Postic's team (INSERM U1016, Paris).
  • PCSK9 was described as a protein strongly expressed in hepatocytes, where it catalyzes LDL-R degradation. Inventors therefore analyzed the effects of BET inhibitors on PCSK9 expression in primary cultures of mouse hepatocytes. They observed a strong and significant dose-response effect of both inhibitors on Pcsk9 expression, with approximatively 90%) loss of expression at higher doses (Fig2).
  • Hepatocytes from human origin were purchased from and plated on 24-well culture dishes by Biopredic International. Upon delivery, they were maintained at 37°C, 5% C02 for 48h in culture medium containing either 0.1% DMSO, Ibet-151 or (+)/-JQl at various concentrations.
  • Histone deacetylase inhibitors modify pancreatic cell fate determination and amplify endocrine progenitors. Mol Cell Biol 28, 6373-6383.
  • L-leucine alters pancreatic beta-cell differentiation and function via the mTor signaling pathway. Diabetes 61, 409-417.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Les inventeurs ont travaillé sur des bourgeons pancréatiques embryonnaires de souris et des cultures primaires d'hépatocytes de souris. Ils ont observé que les niveaux d'état stable de l'ARNm de PCSK9 diminuent de manière surprenante de plus de 90 % après 24h du traitement par des inhibiteurs de BET-151 et JQ1. Les inventeurs ont également travaillé avec des hépatocytes primaires humains et ils ont montré qu'il existe une inhibition nette en dose-réponse de l'expression de Pcsk9. Ces résultats montrent que le traitement utilisant des inhibiteurs de BET-151 et (+)/-JQ1 peut représenter une nouvelle manière d'augmenter l'expression de récepteur au LDL (LDLR) et donc de réduire les niveaux circulants des LDL. En conséquence, la présente invention concerne un procédé pour réduire ou inhiber l'expression de PCSK9 chez un sujet en ayant besoin comprenant une étape d'administration audit sujet d'une quantité thérapeutique d'un I-BET 151 ou JQ1.
PCT/EP2017/069779 2016-08-05 2017-08-04 Procédés et compositions pour réduire l'expression de pcsk9 WO2018024878A1 (fr)

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CN109568324A (zh) * 2018-12-25 2019-04-05 南华大学 一种jq1或其衍生物在制备镇痛药物中的应用

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109568324A (zh) * 2018-12-25 2019-04-05 南华大学 一种jq1或其衍生物在制备镇痛药物中的应用

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