WO2009143619A1 - Procédés de traitement ou de prévention de l'obésité et de l'hypertension liée à l'obésité - Google Patents

Procédés de traitement ou de prévention de l'obésité et de l'hypertension liée à l'obésité Download PDF

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Publication number
WO2009143619A1
WO2009143619A1 PCT/CA2009/000731 CA2009000731W WO2009143619A1 WO 2009143619 A1 WO2009143619 A1 WO 2009143619A1 CA 2009000731 W CA2009000731 W CA 2009000731W WO 2009143619 A1 WO2009143619 A1 WO 2009143619A1
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obesity
reduces
inhibitor
level
seq
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PCT/CA2009/000731
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English (en)
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Julie L. Lavoie
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Chum
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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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/488—Aspartic endopeptidases (3.4.23), e.g. pepsin, chymosin, renin, cathepsin E
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/23—Aspartic endopeptidases (3.4.23)
    • C12Y304/23015—Renin (3.4.23.15)
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96472—Aspartic endopeptidases (3.4.23)
    • G01N2333/96475—Aspartic endopeptidases (3.4.23) with definite EC number
    • G01N2333/96483—Renin (3.4.23.15)

Definitions

  • the present invention relates to the prevention and treatment of obesity and obesity-related hypertension.
  • Obese individuals have an increased risk of suffering from other diseases such as hypertension; type 2 diabetes; dyslipidemia; coronary heart disease; stroke; gallbladder disease; osteoarthritis; sleep apnea and other respiratory problems.
  • RAS renin-angiotensin system
  • BP blood pressure
  • ACE angiotensin-converting enzyme
  • Local RASs have been demonstrated in many tissues, for instance, in the brain, kidney, heart and adipose tissue, and have been found to be involved in different pathologies. For example, brain and kidney RASs seem to contribute to the development of hypertension 1 .
  • the RAS contained in its entirety within the adipocyte may provide an important link between a major cardiovascular control system and obesity and obesity- related diseases. Although the association between body weight and blood pressure is closely linked, the assignment of specific mechanisms underlying this relationship has been more difficult to prove. [0008] Thus, in view of the high prevalence and small pharmacopeia available to treat hypertension related to obesity and given that these patients are more difficult to treat 23 , there remains a need for the identification of new therapeutic targets and methods of treatment derived therefrom.
  • Ren-eGFP mice placed on a high-fat diet to induce obesity
  • renin SEQ ID NOs:6 and 8
  • PRR SEQ ID NOs:4 and 5
  • this receptor was implicated in the development of obesity
  • additional groups with the same diets have been administered the (P)RR blocker.
  • Ang-ll can increase fatty acid and triglyceride synthesis and storage in both human and 3T3-L1 adipocytes 15 while decreasing lipolysis 15 .
  • ACEi ACE inhibitors
  • AT-1 receptor blockers have reported significant weight loss in patients 16 as well as in Sprague-Dawley rats 17 ' 18 , spontaneously hypertensive rats 19 , in obese mice 20 and in Zucker rats 21 ' 22 .
  • renin activity is decreased, thereby reducing Ang-ll production which ultimately translates into a decrease in weight gain.
  • (P)RR in adipose tissue is significantly increased by obesity. This is also a novel finding and further demonstrates the implication of the (P)RR in the development of obesity. Furthermore, Applicant has demonstrated the presence of renin mRNA in adipose tissue showing that there is a fully functional RAS in this tissue. Indeed, all other components of the RAS occur in adipose tissue. Furthermore, the presence of the (P)RR now explains why low concentrations of renin are sufficient to produce significant amounts of Ang-I 2 as it has been shown that binding renin to its receptor increases the catalytic efficiency of AGT conversion to Ang-I by 4-fold 9 .
  • a method of treating or preventing obesity and/or obesity-related hypertension comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of (P)RR activity.
  • the (P)RR activity is the binding of renin/prorenin to the receptor.
  • the (P)RR inhibitor of the present invention a) increases the Ejection Fraction (EF) of the heart, b) reduces blood pressure, c) reduces free fatty acid level, d) reduces weight gain; e) reduces insulin level, f) reduces circulating triglyceride level, g) reduces glucose level or h) any combinations thereof in the subject.
  • EF Ejection Fraction
  • said circulating triglyceride level; said free fatty acid level and/or said glucose level are returned to a comparable normal level found in a healthy subject.
  • the (P)RR inhibitor of the present invention improves vascular reactivity and reduces vascular stiffness, thereby reducing cardiovascular risks.
  • the present invention relates to a method of treating or preventing obesity and/or obesity-related hypertension comprising administering to a subject in need thereof a therapeutically effective amount of a (P)RR inhibitor selected from the group consisting of a) a peptide comprising a fragment of the prosegment of prorenin comprising the handle region (SEQ ID NO:7), or fragment thereof (e.g., RIFLKRMPSI (human, SEQ ID NO:1); IPPLKKMPS (mice, SEQ ID NO:2); RILLKKMPSV (rat, SEQ ID NO:3)); b) a functional derivative, analogue, conjugate or prodrug of a); and c) a (P)RR antibody, whereby obesity and/or obesity-related hypertension are treated or prevented.
  • a (P)RR inhibitor selected from the group consisting of a) a peptide comprising a fragment of the prosegment of prorenin comprising the handle region (SEQ ID NO:7), or fragment thereof (e
  • the present invention provides a method of treating or preventing obesity and/or obesity-related hypertension comprising administering to a subject in need thereof a therapeutically effective amount of an agent which inhibits the expression of the (P)RR nucleic acid or encoded polypeptide.
  • the agent is a siRNA or antisense RNA.
  • the siRNA or antisense RNA of the present invention is substantially complementary to a portion of an mRNA encoding the (P)RR.
  • the siRNA or antisense RNA hybridizes to a (P)RR nucleic acid sequence as set forth in SEQ ID NO:4 or to a sequence which encodes a (P)RR polypeptide (e.g., SEQ ID NO:5).
  • the siRNA or antisense molecule is substantially complementary to a portion of an mRNA encoding the (P)RR.
  • the antisense or siRNA is a fragment of at lease 15 nucleotides, preferably between 15 and 50 nucleotides and more preferably between 19 and 30 nucleotides, of a sequence complementary to SEQ ID NO:4.
  • the si RNA or antisense RNA is between 19 and 21 bp.
  • the present invention also concerns the use of an inhibitor of the (P)RR for preventing or treating hypertensive obesity.
  • the present invention provides a use of an inhibitor of the renin/prorenin receptor activity for the preparation of a medicament for preventing or treating obesity and/or obesity-related hypertension.
  • the inhibitor is selected from the group consisting of: a) a peptide comprising the prosegment of prorenin comprising the handle region (SEQ ID NO:7) or fragment thereof ⁇ e.g., RIFLKRMPSI (human, SEQ ID NO:1); IPPLKKMPS (mice, SEQ ID NO:2); RILLKKMPSV (rat, SEQ ID NO:3) and b) a functional derivative, analogue, conjugate or prodrug of a); and c) a (P)RR antibody.
  • RIFLKRMPSI human, SEQ ID NO:1
  • IPPLKKMPS mice, SEQ ID NO:2
  • RILLKKMPSV rat, SEQ ID NO:3
  • a functional derivative, analogue, conjugate or prodrug of a and c) a (P)RR antibody.
  • the present invention concerns a method for identifying a compound for preventing or treating obesity and/or obesity-related hypertension, said method comprising determining whether: a) the level of expression of the renin/prorenin nucleic acid ⁇ e.g., SEQ ID NO:8) or encoded polypeptide (e.g., SEQ ID NO:6); b) the level of renin/prorenin receptor activity; or c) a combination of a) and b), is decreased in the presence of a test compound relative to in the absence of said test compound, wherein said decrease is indicative that said test compound can be used for preventing or treating obesity and/or obesity-related hypertension.
  • the above-mentioned screening methods further comprise determining whether said test compound: a) increases the Ejection Fraction (EF) of the heart, b) reduces blood pressure, c) reduces free fatty acid level, d) reduces weight gain; e) reduces insulin level, f) reduces circulating triglyceride level, g) reduces glucose level or h) any combinations thereof, as compared to in the absence of said inhibitor.
  • EF Ejection Fraction
  • the (P)RR activity that is inhibited is the binding of the receptor to renin or prorenin.
  • the present invention also relates to a method of identifying or characterizing a compound for preventing or treating obesity and/or obesity-related hypertension comprising: a) contacting a test compound with a cell comprising a first nucleic acid comprising a first transcriptionally regulatory element normally associated with a (P)RR gene, operably-linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and b) determining whether the reporter gene expression or reporter activity is decreased in the presence of the test compound; wherein a decrease in the reporter gene expression or reporter gene activity is indicative that the test compound may be used for treating or preventing obesity and/or obesity- related hypertension.
  • the present invention also provides a package for preventing or treating obesity and/or obesity-related hypertension comprising an inhibitor of (P)RR activity together with instructions for preventing or treating obesity and/or obesity-related hypertension.
  • the (P)RR inhibitor is a) a peptide comprising the prosegment of prorenin comprising the handle region (SEQ ID NO:7) or fragment thereof (e.g., RIFLKRMPSI (human, SEQ ID NO:1); IPPLKKMPS (mice, SEQ ID NO:2); RILLKKMPSV (rat, SEQ ID NO:3)); b) a functional derivative, analogue, conjugate or prodrug of a); or c) a (P)RR antibody.
  • RIFLKRMPSI human, SEQ ID NO:1
  • IPPLKKMPS mice, SEQ ID NO:2
  • RILLKKMPSV rat, SEQ ID NO:3
  • a functional derivative, analogue, conjugate or prodrug of a or c) a
  • the present invention concerns a composition for treating or preventing obesity and/or obesity-related hypertension comprising an inhibitor of (P)RR activity together with a pharmaceutical carrier.
  • the inhibitor is a) a peptide comprising the prosegment of prorenin comprising the handle region (SEQ ID NO:7) or fragment thereof (e.g., RIFLKRMPSI (human, SEQ ID NO:1); IPPLKKMPS (mice, SEQ ID NO:2); RILLKKMPSV (rat, SEQ ID NO:3)); b) a functional derivative, analogue, conjugate or prodrug thereof; or c) a (P)RR antibody.
  • RIFLKRMPSI human, SEQ ID NO:1
  • IPPLKKMPS mice, SEQ ID NO:2
  • RILLKKMPSV rat, SEQ ID NO:3
  • a functional derivative, analogue, conjugate or prodrug thereof or c) a (P)RR antibody.
  • Methods, compositions, uses and packages of the present invention are particularly useful for mammals and preferably humans.
  • the subject to which the (P)RR inhibitor of the present invention is administered already suffers from obesity and/or obesity-related hypertension.
  • the subject is at risk of suffering from obesity and/or obesity-related hypertension.
  • the subject has an increased triglyceride level, increased glucose level, increased free fatty acid level, increased insulin level and/or increased blood pressure compared to normotensive subjects who do not suffer from obesity.
  • the subject's food consumption is generally high in fat.
  • the above-mentioned (P)RR blocker of the present invention a) increases the Ejection Fraction (EF) of the heart, b) reduces blood pressure, c) reduces free fatty acid level, d) reduces weight gain; e) reduces insulin level, f) reduces circulating triglyceride level, g) reduces glucose level or h) any combinations thereof.
  • EF Ejection Fraction
  • Figure 1 shows (P)RR mRNA expression in different adipose tissue depots.
  • FIG. 2 shows (P)RR protein levels in different adipose tissue depots.
  • a significant increase in (P)RR protein could only be detected in subcutaneous adipose tissue (subcu.) from Ren-eGFP mice fed a HF/HC diet (black) compared to those receiving normal diet (white). Values are expressed as means ⁇ SE;
  • Figure 3 shows body weight from week 5 to week 10 of treatment.
  • a significant increase in body weight was observed when Ren-eGFP mice were placed on the HF/HC diet (squares) compared to those on a normal diet (circles).
  • mice received a (P)RR blocker white
  • saline black
  • Values are expressed as means ⁇ SE;
  • Figure 5 shows circulating metabolic markers in mice receiving the (P)RR blocker (black) or saline (white) in concomitance with a HF/HC or normal (N) diet.
  • FFA Free Fatty Acids
  • Ren-eGFP mice receiving a (P)RR blocker in concomitance with the HF/HC diet had a normalization of their circulating triglyceride (A), free fatty acid (B) and glucose (C) levels while insulin levels (D) were reduced. Values are expressed as means ⁇ SE.
  • Figure 6 shows daily food and Kcal consumption during week 8 of treatment.
  • food was weighed daily to determine consumption (A).
  • mice fed the normal diet ate more then those on the HF/HC diet.
  • values were expressed as daily Kcal consumption (B)
  • B when values were expressed as daily Kcal consumption (B), a significant difference could be observed between the mice receiving the (P)RR blocker and saline groups on the HF/HC diet.
  • the difference between the HF/HC and normal diet groups receiving saline was absent in (P)RR blocker animals.
  • Values are expressed as means ⁇ SE. * p ⁇ 0.05 and t P ⁇ 0.001 , statistically different from HF/HC diet. ⁇ p ⁇ 0.05, significantly different from the saline treated group;
  • Figure 7 shows the prevention of weight gain in obese mice receiving a
  • FIG. 8 shows fat body mass (%) measured by MRI oin obese mice receiving a HF/HC diet and treated with the (P)RR blocker (black) or with saline (white) after 5 weeks of treatment. While the average weight in each group was the same at the before treatment, mice placed in the (P)RR blocker group had more fat then the animals in the saline group. Interestingly, after 5 weeks of HF/HC diet and treatment, mice in the saline group increased their fat mass while the (P)RR blocker group decreased their fat mass. Hence, the (P)RR blocker seems to promote fat body mass loss ;
  • Figure 9 shows lean body mass (%) measured by MRI in obese mice receiving a HF/HC diet and treated with the (P)RR blocker (black) or with saline (white) after 5 weeks of treatment.
  • mice in this group had a higher lean body mass then those in the saline group.
  • mice receiving saline decreased the lean body mass % while those receiving the blocker maintained their lean body mass. Hence, this could contribute to weight loss in the (P)RR animals as increased lean body mass produces an increase in basal metabolism;
  • Figure 10 shows the anti-hypertensive effects of the (P)RR blocker in mice receiving the HF/HC diet.
  • Mean arterial pressure was measured by telemetry in anesthetized animals at the end of treatment. Mice receiving the blocker (black) tended to have a lower blood pressure than those receiving saline (white) while on the HF/HC diet;
  • Figure 11 shows the improved response to sodium nitroprusside in animals receiving a HF/HC and the (P)RR blocker. Vessel response to sodium nitroprusside in animals receiving the HF/HC diet treated with (P)RR blocker (black) or with saline (white) in vitro are presented;
  • Figure 12 shows the decreased sensitivity to endothelin in animals treated with (P)RR blocker compared to those receiving saline on the HF/HC diet. Vasoconstriction of vessels following endothelin administration (10 '9 to 10 "6 M) in mice treated with (P)RR blocker (black) or saline (white) receiving a HF/HC diet is shown; [0041] Figure 13 shows decreased vascular stiffness in animals treated with the
  • Figure 14 shows the effect of the (P)RR blocker on cardiac function.
  • Ejection Fraction was measured in animals fed a HF/HC or normal (N) diet and treated with saline or the (P)RR blocker.
  • the present invention generally relates to the inhibition of the expression or activity of the (P)RR receptor for the prevention or treatment of obesity and/or obesity-related hypertension.
  • the present invention relates to methods of inhibiting (P)RR biological activity in cells.
  • the present invention concerns the use of (P)RR inhibitors to decrease for example (P)RR signaling pathways, Ang I production, Ang Il production, circulating triglyceride level or the like.
  • the present invention concerns the use of (P)RR inhibitors to inhibit locally, in adipose tissue, the RAS.
  • a method of treating or preventing obesity and/or obesity-related hypertension comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of (P)RR activity.
  • the (P)RR inhibitor is selected from the group consisting of a) a peptide comprising the prosegment of prorenin comprising the handle region (SEQ ID NO:7) or a fragment thereof (e.g., RIFLKRMPSI (human, SEQ ID NO:1); IPPLKKMPS (mice, SEQ ID NO:2); RILLKKMPSV (rat, SEQ ID NO:3); b) a functional derivative, analogue, conjugate or prodrug of a); and c) a (P)RR antibody.
  • suitable inhibitors are peptides competing with the binding of renin and/or prorenin to the receptor such as fragments of renin or prorenin devoid of enzymatic activity which are still able to bind to the receptor.
  • the inhibitors of the present invention reduce or completely abolish (P)RR biological activity.
  • the inhibitors of the present invention compete with natural endogenous (P)RR interacting molecules (e.g., renin/prorenin) for binding to (P)RR.
  • P)RR interacting molecules e.g., renin/prorenin
  • peptides or small molecules mimicking (P)RR interacting domains ⁇ e.g., domain which is responsible for binding to renin or prorenin, domain responsible for the coupling of (P)RR to downstream effectors, etc.
  • P)RR interaction with endogenous proteins can be used to inhibit (P)RR interaction with endogenous proteins.
  • peptides or small molecules mimicking (P)RR interacting proteins can also be used to compete with endogenous proteins for the binding to (P)RR.
  • Inhibitors of (P)RR signaling e.g., inhibitors which block the phosphorylation of the receptor, are also examples of useful inhibitors in accordance with the present invention.
  • a "(P)RR inhibitor”, “(P)RR blocker”, “inhibitor of the renin/prorenin receptor activity” or “renin/prorenin receptor inhibitor” means an agent that inhibits the polypeptide or nucleic acid (e.g., mRNA) expression and/or one or more biological activities of the (P)RR.
  • Examples of particular (P)RR biological activities include binding of the receptor to renin and/or prorenin or other effector molecule, its capacity to increase the conversion of angiotensinogen to angiotensin I, the phosphorylation of the receptor, etc.
  • the inhibitors of the present invention may inhibit totally or partially the expression and/or activity of the (P)RR.
  • the (P)RR inhibitor of the present invention also has at least one of the following activities: a) increases the Ejection Fraction (EF) of the heart, b) reduces blood pressure, c) reduces free fatty acid level, d) reduces weight gain; e) reduces insulin level, f) reduces circulating triglyceride level, g) reduces glucose level or h) any combinations thereof.
  • EF Ejection Fraction
  • the (P)RR inhibitor is capable of normalizing the level of triglycerides, free fatty acids and/or glucose (Ae., returning to the normal levels found in a healthy subject).
  • the (P)RR inhibitor may also increase cardiac function and reduce vascular stiffness (improved stress/strain) and improve vascular reactivity (i.e., decrease response to endothelin and increase response to nitroprusside).
  • the methods of the present invention comprise a modulation of the expression of (P)RR in a cell or organism.
  • Such methods include, in particular embodiments, the use of an antisense nucleic acid of (P)RR, of (P)RR siRNAs or of a (P)RR specific ribozyme.
  • Other agents, which decrease the expression level and/or activity of (P)RR e.g., antibodies (vaccines), small molecules, peptides
  • agents useful for treating or preventing obesity and/or obesity-related hypertension are also encompassed as agents useful for treating or preventing obesity and/or obesity-related hypertension.
  • the present invention concerns antisense oligonucleotides hybridizing to a nucleic acid sequence encoding (P)RR protein, thereby enabling the control of the transcription or translation of the (P)RR gene in cells.
  • the antisense sequences of the present invention consist of all or part of the (P)RR nucleic acid sequence (SEQ ID NO:4, Genbank Accession number AF291814) in reverse orientation, and variants thereof.
  • the present invention further relates to small double stranded RNA molecules (siRNAs) derived from (P)RR nucleic acid sequence which also decrease (P)RR protein cell expression.
  • the present invention also relates to methods utilizing siRNA or antisense RNA to reduce (P)RR mRNA and/or protein expression and therefore, to prevent or treat obesity and/or obesity-related hypertension.
  • inhibition or reduction of (P)RR expression significantly decreases the production of Ang I, Ang Il and/or (P)RR signaling.
  • inhibition or reduction of (P)RR expression significantly decreases the level of circulating triglycerides.
  • the (P)RR complementary sequences of the present invention can either be directly transcribed in target cells or synthetically produced and incorporated into cells by well-known methods.
  • the present invention features a method of reducing (P)RR expression in a subject by administering thereto a RNA, or derivative thereof (e.g., siRNA, antisense RNA, etc), or vector producing same in an effective amount, to reduce (P)RR expression, thereby decreasing (P)RR function and treating or preventing obesity and/or obesity-related hypertension.
  • a RNA e.g., siRNA, antisense RNA, etc.
  • the RNA can be modified so as to be less susceptible to enzymatic degradation or to facilitate its delivery to a target cell (e.g., an adipocyte).
  • RNA interference toward a targeted DNA segment in a cell can be achieved by administering a double stranded RNA (e.g., siRNA) molecule to the cell, wherein the ribonucleotide sequence of the double stranded RNA molecule corresponds to the ribonucleotide sequence of the targeted DNA segment.
  • a double stranded RNA e.g., siRNA
  • the antisense region of the siRNAs or antisense RNA of the present invention is still capable of maintaining its ability to hybridize to the target sequence, of hybridizing to the ribonucleotide sequence of the targeted gene (e.g., (P)RR mRNA) and of inhibiting its expression (e.g., trigger RNAi).
  • the present invention concerns inhibition of of (P)RR expression.
  • the (P)RR inhibitor is selected from an antisense molecule, a siRNA or siRNA-like molecule.
  • the (P)RR inhibitor is a nucleic acid that is substantially complementary to a portion of an mRNA encoding a (P)RR (e.g., SEQ ID NO:5).
  • the (P)RR inhibitor is complementary to a portion of a nucleic acid sequence substantially identical to the nucleotide sequence of SEQ ID NO:4.
  • the portion of an mRNA comprises at least 5 contiguous bases.
  • the siRNA, siRNA-like molecule, or antisense molecule is substantially complementary to a portion of an mRNA encoding a (P)RR.
  • the siRNA, siRNA-like molecule, or antisense molecule is substantially complementary to a portion of an mRNA encoding the protein sequence of SEQ ID NO:5.
  • the siRNA or siRNA-like molecule comprises less than about 30 nucleotides. In embodiments, the siRNA or siRNA-like molecule comprises about 21 to about 23 nucleotides.
  • RNAi RNA interference
  • P polypeptide of interest
  • RNAi RNA interference
  • RNAi may be used to create a pseudo "knockout", i.e. a system in which the expression of the product encoded by a gene or coding region of interest is reduced, resulting in an overall reduction of the activity of the encoded product in a system.
  • RNAi may be performed to target a nucleic acid of interest or fragment or variant thereof, to in turn reduce its expression and the level of activity of the product which it encodes.
  • RNAi is described in for example Hammond et al. (2001), Sharp (2001), Caplen et al. (2001), Sedlak (2000) and published US patent applications 20020173478 (Gewirtz; published November 21 , 2002) and 20020132788 (Lewis et al.; published November 7, 2002).
  • Reagents and kits for performing RNAi are available commercially from for example Ambion Inc. (Austin, TX, USA) and New England Biolabs Inc. (Beverly, MA, USA).
  • the initial agent for RNAi in some systems is thought to be dsRNA molecule corresponding to a target nucleic acid.
  • RNAi short interfering RNAs
  • the enzyme thought to effect this first cleavage step has been referred to as "Dicer” and is categorized as a member of the RNase III family of dsRNA-specific ribonucleases.
  • RNAi may be effected via directly introducing into the cell, or generating within the cell by introducing into the cell a suitable precursor (e.g. vector encoding precursor(s), etc.) of such an siRNA or siRNA-like molecule.
  • RNA-induced silencing complex RISC
  • the RISC thus formed may subsequently target a transcript of interest via base- pairing interactions between its siRNA component and the target transcript by virtue of homology, resulting in the cleavage of the target transcript approximately 12 nucleotides from the 3' end of the siRNA.
  • RNAi may be effected by the introduction of suitable in vitro synthesized siRNA or siRNA-like molecules into cells. RNAi may for example be performed using chemically-synthesized RNA (Brown et al., 2002).
  • suitable expression vectors may be used to transcribe such RNA either in vitro or in vivo.
  • In vitro transcription of sense and antisense strands may be effected using for example T7 RNA polymerase, in which case the vector may comprise a suitable coding sequence operably-linked to a T7 promoter.
  • the in w ⁇ rotranscribed RNA may in embodiments be processed (e.g. using E. coli RNase III) in vitro to a size conducive to RNAi.
  • the sense and antisense transcripts are combined to form an RNA duplex which is introduced into a target cell of interest.
  • vectors may be used, which express small hairpin RNAs (sh RNAs) which can be processed into siRNA-like molecules.
  • sh RNAs small hairpin RNAs
  • Various vector-based methods are described in for example Brummelkamp et al. (2002), Lee et al. (2002), Miyagashi and Taira (2002), Paddison et al. (2002) Paul et al. (2002) Sui et al. (2002) and Yu et al. (2002).
  • Various methods for introducing such vectors into cells either in vitro or in vivo (e.g. gene therapy) are known in the art.
  • siRNA-like molecule refers to a nucleic acid molecule similar to an siRNA (e.g. in size and structure) and capable of eliciting siRNA activity, i.e. to effect the RNAi- mediated inhibition of expression.
  • such a method may entail the direct administration of the siRNA or siRNA-like molecule into a cell, or use of the vector-based methods described above.
  • the siRNA or siRNA-like molecule is less than about 30 nucleotides in length.
  • the siRNA or siRNA-like molecule is about 21-23 nucleotides in length.
  • siRNA or siRNA-like molecule comprises a 19-21 bp duplex portion, each strand having a 2 nucleotide 3' overhang.
  • the siRNA or siRNA-like molecule is substantially identical to a nucleic acid encoding a polypeptide of interest, or a fragment or variant (or a fragment of a variant) thereof. Such a variant is capable of encoding a protein having activity similar to the polypeptide of interest.
  • the sense strand of the siRNA or siRNA-like molecule is substantially identical to SEQ ID NO: 4, or a fragment thereof (RNA having U in place of T residues of the DNA sequence).
  • the therapeutic effect comprises one or more of a decrease/reduction in weight gain, decrease/reduction in circulating triglyceride, glucose, free fatty acid and/or insulin levels, an improvement of cardiac function (increase in the Ejection Fraction (EF) of the heart), an improvement of vascular reactivity (increased response to nitroprusside while decreasing response to endothelin), a decrease/reduction in vascular stiffness (increases stress and strain), a decrease/reduction in angiotensin I production, a decrease/reduction in angiotensin Il production and a decrease in the subject's blood pressure.
  • EF Ejection Fraction
  • compositions formulations and uses described herein are suitable for both humans and animals, preferably mammals.
  • the term "subject” in the context of the present invention relates to any mammal including a mouse, rat, pig, monkey and horse. In a specific embodiment, it refers to a human.
  • a "subject in need thereof " or a "patient” in the context of the present invention is intended to include any subject that will benefit or that is likely to benefit from the inhibition of the expression or activity of the (P)RR.
  • a subject in need thereof is a subject diagnosed with obesity and/or obesity-related hypertension or having a disease or condition that is likely to be associated with obesity and/or obesity-related hypertension. Subjects suffering from a metabolic syndrome and hypertension are examples of likely candidates.
  • a subject in need thereof is a subject suffering from obesity and/or obesity-related hypertension.
  • the subject in need thereof is a subject suffering from hypertension but who has not yet developed obesity.
  • the subject in need thereof is a subject who has gained at least 5%, 8%, 10%, 12%, 15% or more of his/her initial weight.
  • a subject in need thereof is a subject undergoing therapy for the underlying disease or condition which is associated with obesity and/or obesity-related hypertension or likely to be associated with obesity and/or obesity-related hypertension (hypertension; type 2 diabetes; dyslipidemia; coronary heart disease etc).
  • the pharmaceutical composition comprising a (P)RR inhibitor is administered prior to the onset of obesity and/or obesity-related hypertension as a preventive measure.
  • the pharmaceutical composition of the present invention is administered in combination with a drug or drugs used to treat an underlying disease or condition such as hypertension.
  • the composition of the present invention is administered once the subject has been diagnosed with obesity and/or obesity-related hypertension.
  • composition of the present invention is administered in combination with one or more other drugs used for the prevention and/or treatment of hypertension
  • drugs used for the prevention and/or treatment of hypertension such as inhibitors of angiotensin converting enzyme (ACE) (e.g., captopril (Capoten), benazepril (Lotensin), enalapril (Vasotec), lisinopril (Prinivil, Zestril) fosinopril (Monopril), ramipril (Altace), perindopril (Aceon), quinapril (Accupril), moexipril (Univasc), and trandolapril (Mavik) and inhibitors of angiotensin receitor (e.g., valsartan, losartan, telmisartan, candersartan).
  • ACE angiotensin converting enzyme
  • composition of the present invention is administered in combination with one or more other drugs used for the prevention and/or treatment of obesity such as leptin, orlistat (Xenical), sibutramine (Reductil, Meridia), rimonabant and metformin.
  • drugs used for the prevention and/or treatment of obesity such as leptin, orlistat (Xenical), sibutramine (Reductil, Meridia), rimonabant and metformin.
  • the invention relates to the use of (P)RR as a target in screening assays used to identify compounds that are useful for the prevention or treatment of obesity and/or obesity-related hypertension, said method comprising determining whether:
  • the above-mentioned method is an in vitro method.
  • the level of a molecule generated by a (P)RR activity is the level of angiotensin I.
  • the (P)RR activity is its binding to renin and/or prorenin.
  • the (P)RR activity is the increased in angiotensin I production.
  • a reporter assay-based method of selecting agents which modulate (P)RR expression includes providing a cell comprising a nucleic acid sequence comprising a (P)RR transcriptional regulatory sequence operably-linked to a suitable reporter gene. The cell is then exposed to the agent suspected of affecting (P)RR expression ⁇ e.g., a test/candidate compound) and the transcription efficiency is measured by the activity of the reporter gene. The activity can then be compared to the activity of the reporter gene in cells unexposed to the agent in question.
  • Suitable reporter genes include but are not limited to beta( ⁇ )-D-galactosidase, luciferase, chloramphenicol acetyltransferase and green fluorescent protein (GFP).
  • the present invention further provides a method of identifying or characterizing a compound for treating or preventing obesity and/or obesity-related hypertension, said method comprising: (a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a (P)RR gene (e.g., a promoter region naturally associated with a (P)RR gene), operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound, said decrease in reporter gene expression or reporter protein activity being an indication that said test compound may be used for treating or preventing obesity and/or obesity-related hypertension.
  • the above-mentioned method is an in vitro method.
  • the above-mentioned screening methods further comprise determining whether said test compound a) increases the Ejection Fraction (EF) of the heart, b) reduces blood pressure, c) reduces free fatty acid level, d) reduces weight gain; e) reduces insulin level, f) reduces circulating triglyceride level, g) reduces glucose level or h) any combinations thereof.
  • EF Ejection Fraction
  • the above-noted assays may be applied to a single test compound or to a plurality or "library" of such compounds (e.g., a combinatorial library). Any such compound may be utilized as lead compound and further modified to improve its therapeutic, prophylactic and/or pharmacological properties for the prevention and treatment of obesity and/or obesity-related hypertension.
  • Such assay systems may comprise a variety of means to enable and optimize useful assay conditions.
  • Such means may include but are not limited to: suitable buffer solutions, for example, for the control of pH and ionic strength and to provide any necessary components for optimal (P)RR activity and stability ⁇ e.g., protease inhibitors), temperature control means for optimal (P)RR activity and or stability, and detection means to enable the detection of the (P)RR/renin/prorenin reaction product, (e.g., Ang I).
  • a variety of such detection means may be used, including but not limited to one or a combination of the following: radiolabelling (e.g., 32 P, 14 C, 3 H), antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g., generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g., horseradish peroxidase, green fluorescent protein), specific binding reagents (e.g., biotin/streptavidin), and others.
  • radiolabelling e.g., 32 P, 14 C, 3 H
  • antibody-based detection e.g., fluorescence, chemiluminescence
  • spectroscopic methods e.g., generation of a product with altered spectroscopic properties
  • reporter enzymes or proteins e.g., horseradish peroxidase, green fluorescent protein
  • specific binding reagents e.g., biotin/streptavidin
  • the assay may be carried out in vitro utilizing a source of (P)RR which may comprise naturally isolated or recombinantly produced (P)RR, in preparations ranging from crude to pure.
  • Recombinant (P)RR may be produced in a number of prokaryotic or eukaryotic expression systems, which are well known in the art (see for example Martin F. et al., 2001. lmmunogenetics 53(4): 296-306) for the recombinant expression of (P)RR.
  • Such assays may be performed in an array format. In certain embodiments, one or a plurality of the assay steps are automated.
  • a homolog, variant and/or fragment of (P)RR which retains activity may also be used in the screening methods of the invention.
  • Homologues include protein sequences, which are substantially identical to the amino acid sequence of a (P)RR (e.g., SEQ ID NO:5, GeneBank Ace. No. NP_005756), sharing significant structural and functional homology with a (P)RR.
  • Variants include, but are not limited to, proteins or peptides, which differ from a (P)RR by any modifications, and/or amino acid substitutions, deletions or additions (e.g., fusion with another polypeptide).
  • Modifications can occur anywhere including the polypeptide backbone, ⁇ i.e., the amino acid sequence), the amino acid side chains and the amino or carboxy termini. Such substitutions, deletions or additions may involve one or more amino acids. Fragments include a fragment or a portion of a (P)RR or a fragment or a portion of a homologue or variant of a (P)RR which retains (P)RR activity, i.e., binds to renin and/or prorenin.
  • a “functional derivative” or “functional fragment” of a molecule ⁇ e.g., a
  • (P)RR blocker of the present invention refers to a molecule which retains the same activity as the original molecule but which differs by any modifications, and/or amino acid substitutions, deletions or additions ⁇ e.g., fusion with another polypeptide). Modifications can occur anywhere including the polypeptide backbone ⁇ i.e., the amino acid sequence, the amino acid side chains and the amino or carboxy termini). Such substitutions, deletions or additions may involve one or more amino acids. The substitutions are preferably conservative, i.e., an amino acid is replaced by another amino acid having similar physico-chemical properties (size, hydrophobicity, charge/polarity, etc.) as well known by those of ordinary skill in the art.
  • Functional fragments of the (P)RR blocker include a fragment or a portion of a (P)RR blocker or a fragment or a portion of a homologue or variant of a (P)RR blocker which retains inhibiting activity, i.e., binds to the (P)RR.
  • prodrug means a pharmacological substance that is administered in an inactive (or significantly less active) form. Once administered, the prodrug is metabolised in vivo into an active metabolite ⁇ e.g., a (P)RR blocker of the present invention).
  • active metabolite e.g., a (P)RR blocker of the present invention.
  • P PRR blocker of the present invention.
  • the rationale behind the use of a prodrug is generally for absorption, distribution, metabolism and excretion optimization.
  • Prodrugs are usually designed to improve oral bioavailability, with poor absorption from the gastrointestinal tract usually being the limiting factor. Additionally, the use of a prodrug strategy increases the selectivity of the drug for its intended target.
  • Prodrugs can be classified into two types based on their sites of conversion into the final active drug form: Type I, those that are converted intracellular ⁇ , and Type II, those that are converted extracellularly, especially in digestive fluids or the systemic circulation.
  • Type I those that are converted intracellular ⁇
  • Type II those that are converted extracellularly, especially in digestive fluids or the systemic circulation.
  • a "conjugate" of a drug refers to the union of a pharmacological substance (e.g., a (P)RR blocker of the present invention) to another molecule (peptide, antibody, chemical compound (e.g., polyethylene glycol PEG, etc.).
  • a pharmacological substance e.g., a (P)RR blocker of the present invention
  • another molecule peptide, antibody, chemical compound (e.g., polyethylene glycol PEG, etc.
  • Pharmaceutical substances are often conjugated to other molecules to increase their bioavailability by, for example, targeting specific cells or tissue (e.g., drug-antibody conjugates).
  • Conjugates may also be designed to increase drug stability or solubility, in particular solvents. PEG, for example, generally increases water solubility, which can be a great advantage when making pharmaceutical compositions.
  • the pharmacological substance may or may not be covalently linked to the other molecule to form a conjugate.
  • conjugates may be subject to hydrolysis under physiological conditions (e.g., pH 7.2-7.4) to release an active drug.
  • the conjugated drug may thus be a prodrug or an active drug.
  • an "analogue" of a molecule of the present invention is a molecule whose physical structure is related to that of another drug. Although they have similar physical properties and biological activities, analogues can have different chemical structures. It is a structural derivative of a parent compound that often differs from it by a single or very few elements.
  • Homology and “homologous” and “homologue” refer to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid sequence is "homologous” to or is a “homologue” of another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term 'homologous' does not infer evolutionary relatedness).
  • sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%, e.g., with any (P)RR (SEQ ID NOs:4 and 5).
  • a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences.
  • An "unrelated" or “non-homologous” sequence shares less than 40% identity, though preferably less than about 25% identity, with (P)RR sequences (SEQ ID NOs:4 and 5).
  • Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, e.g., with a (P)RR sequence (SEQ ID NO:4). Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J.
  • the BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold.
  • Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • W word length
  • B BLOSUM62 scoring matrix
  • E expectation
  • P(N) the smallest sum probability
  • nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1 , preferably less than about 0.1 , more preferably less than about 0.01 , and most preferably less than about 0.001.
  • An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, more preferably highly stringent conditions.
  • Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1% SDS at 42°C (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).
  • hybridization to filter- bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65 0 C, and washing in 0.1 x SSC/0.1% SDS at 68°C (see Ausubel, et al. (eds), 1989, supra).
  • Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York).
  • stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • the assay may in an embodiment be performed using an appropriate host cell comprising (P)RR activity.
  • a host cell may be prepared by the introduction of DNA encoding (P)RR (e.g., comprising the nucleotide sequence set forth in SEQ ID NO:4 or the coding sequence thereof (i.e., positions 90 to 1142 of SEQ ID NO:4 or a sequence encoding SEQ ID NO:5) or a fragment/variant thereof having (P)RR activity) into the host cell and providing conditions for the expression of (P)RR.
  • P DNA encoding
  • Such host cells may be prokaryotic or eukaryotic, bacterial, yeast, amphibian or mammalian.
  • Transcriptional regulatory sequence or “transcriptional regulatory element” as used herein refers to DNA sequences, such as initiation and termination signals, enhancers and promoters, splicing signals and polyadenylation signals, which induce or control the transcription of protein coding sequences with which they are operably linked.
  • a first nucleic acid sequence is "operably-linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
  • enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths
  • some polynucleotide elements may be operably-linked but not contiguous.
  • a transcriptionally regulatory element "normally" associated with, for example, a (P)RR gene refers to such an element or a functional portion thereof derived from sequences operably-linked to, for example, a (P)RR gene in its naturally-occurring state (i.e., as it occurs in a genome in nature).
  • the construct may comprise an in frame fusion of a suitable reporter gene within the open reading frame of a (P)RR gene.
  • the reporter gene may be chosen as such to facilitate the detection of its expression, e.g. by the detection of the activity of its gene product.
  • Such a reporter construct may be introduced into a suitable system capable of exhibiting a change in the level of expression of the reporter gene in response to exposure to a suitable biological sample.
  • Such an assay would also be adaptable to a possible large scale, high-throughput, automated format, and would allow more convenient detection due to the presence of its reporter component.
  • Expression levels may in general be detected by either detecting mRNA from the cells and/or detecting expression products, such as polypeptides and proteins. Expression of the transcripts and/or proteins encoded by the nucleic acids described herein may be measured by any of a variety of known methods in the art. In general, the nucleic acid sequence of a nucleic acid molecule (e.g., DNA or RNA) in a sample can be detected by any suitable method or technique of measuring or detecting gene sequence or expression.
  • a nucleic acid molecule e.g., DNA or RNA
  • Such methods include, but are not limited to, polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), in situ PCR, quantitative PCR (q-PCR), in situ hybridization, Southern blot, Northern blot, sequence analysis, microarray analysis, detection of a reporter gene, or other DNA/RNA hybridization platforms.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase-PCR
  • q-PCR quantitative PCR
  • in situ hybridization Southern blot
  • Northern blot sequence analysis
  • microarray analysis detection of a reporter gene, or other DNA/RNA hybridization platforms.
  • RNA expression preferred methods include, but are not limited to: extraction of cellular mRNA and Northern blotting using labelled probes that hybridize to transcripts encoding all or part of one or more of the genes of this invention; amplification of mRNA expressed from one or more of the genes of this invention, using gene-specific primers, polymerase chain reaction (PCR), quantitative PCR (q-PCR), and reverse transcriptase- polymerase chain reaction (RT-PCR), followed by quantitative detection of the product by any of a variety of means; extraction of total RNA from the cells, which is then labelled and used to probe cDNAs or oligonucleotides encoding all or part of the genes of this invention, arrayed on any of a variety of surfaces; in situ hybridization and detection of a reporter gene.
  • PCR polymerase chain reaction
  • q-PCR quantitative PCR
  • RT-PCR reverse transcriptase- polymerase chain reaction
  • Methods to measure protein expression levels of selected genes of this invention include, but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), flow cytometry, and assays based on a property of the protein, including but not limited to, DNA binding, ligand binding and interaction with other protein partners.
  • ELISA enzyme-linked immunosorbant assay
  • RIA radioimmunoassay
  • MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight
  • the expression level of a gene of the present invention can be normalized on the basis of the relative ratio of the mRNA level of this gene to the mRNA level of a housekeeping gene or the relative ratio of the protein level of the protein encoded by this gene to the protein level of the housekeeping protein, so that variations in the sample extraction efficiency among cells or tissues are reduced in the evaluation of the gene expression level.
  • a "housekeeping gene” is a gene the expression of which is substantially the same from sample to sample or from tissue to tissue, or one that is relatively refractory to change in response to external stimuli.
  • a housekeeping gene can be any RNA molecule other than that encoded by the gene of interest that will allow normalization of sample RNA or any other marker that can be used to normalize for the amount of total RNA added to each reaction.
  • the GAPDH gene, the G6PD gene, the actin gene, ribosomal RNA, 36B4 RNA, PGK1 , RPLPO, or the like may be used as a housekeeping gene.
  • Methods for calibrating the level of expression of a gene are well known in the art.
  • the expression of a gene can be calibrated using reference samples, which are commercially available.
  • reference samples include, but are not limited to: Stratagene® QPCR Human Reference Total RNA, ClontechTM Universal Reference Total RNA, and XpressRefTM Universal Reference Total RNA.
  • a “reference” or “control” level may be determined, for example, by measuring the level of expression of (P)RR nucleic acid or encoded polypeptide, or the level of (P)RR activity, in a corresponding biological sample obtained from one or more healthy subject(s) (i.e., not suffering from obesity and/or obesity-related hypertension or known not to be susceptible to obesity and/or obesity-related hypertension).
  • a control level i.e., not suffering from obesity and/or obesity-related hypertension or known not to be susceptible to obesity and/or obesity-related hypertension.
  • a substantially similar level refers to a difference in the level of expression or activity between the level determined in a first sample (i.e., test sample) and the reference level which is 15% or less; in a further embodiment, 10% or less; in a further embodiment, 5% or less.
  • a “lower” or “decreased” level refers to a level of expression or activity in a sample (i.e., test sample) which is at least 20% lower, in an embodiment at least 30% lower, in a further embodiment at least 40% lower; in a further embodiment at least 50% lower, in a further embodiment at least 100% lower (i.e., 2- fold), in a further embodiment at least 200% lower (i.e., 3-fold), in a further embodiment at least 300% lower (i.e., 4-fold), relative to the reference level (e.g., in the absence of an (P)RR inhibitor).
  • (P)RR antibody refers to an antibody that specifically binds to (interacts with) a (P)RR protein and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as the (P)RR protein.
  • (P)RR antibodies include polyclonal, monoclonal, humanized as well as chimeric antibodies.
  • the term antibody or immunoglobulin is used in the broadest sense, and covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies and antibody fragments so long as they exhibit the desired biological activity.
  • Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments.
  • Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodiesTM, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. Additionally, any secondary antibodies, either monoclonal or polyclonal, directed to the first antibodies would also be included within the scope of this invention.
  • antibody encompasses herein polyclonal, monoclonal antibodies and antibody variants such as single-chain antibodies, humanized antibodies, chimeric antibodies and immunologically active fragments of antibodies (e.g., Fab and Fab' fragments) which inhibit or neutralize their respective interaction domains in Hyphen and/or are specific thereto.
  • the present invention is illustrated in further details by the following non- limiting examples.
  • REN-eGFP transgenic mice provided by Dr. Kenneth Gross's laboratory at the Buffalo Cancer Institute.
  • the REN-eGFP mice were subsequently bred and maintained by backcross breeding to C57BL/6.
  • the animals were maintained on 12-h light/dark cycle with standard laboratory chow (2018; Teklab Premier Laboratory Diets, Madison, Wl) and water ad libitum. Mice used for these experiments were 12-15 weeks of age.
  • mice were separated into 4 groups: high-fat/high-carbohydrate diet (F3282; Bio-Serv, Frenchtown, NJ; HF/HC)- (P)RR blocker, HF/HC-saline, normal diet (N)-(P)RR blocker, and N-saline.
  • high-fat/high-carbohydrate diet F3282; Bio-Serv, Frenchtown, NJ
  • HF/HC)- (P)RR blocker HF/HC-saline
  • normal diet N-(P)RR blocker
  • N-saline normal diet
  • mice were implanted subcutaneously with an osmotic mini-pump (#1004; Alzet, Cupertino, CA) filled with either the (P)RR blocker (obtained from Dr. Peter Schiller's laboratory at the IRCM) at a dose of 0.1 mg/kg/day or saline under isoflurane anaesthesia.
  • mice received either a HF/HC or an N diet for 10 weeks. Mice were weighed weekly throughout the treatment. Food was weighed for 2 weeks on a daily basis to determine animal consumption. Care of the mice used in the experiments met the standard set forth by the Canadian Council in their guidelines for the care and use of experimental animals, and all procedures were approved by the University Animal Care and Use Committee at the CHUM Research Center.
  • IPPLKKMPS 11 (SEQ ID NO:2) was synthesized by the manual solid-phase technique using a published protocol 12 .
  • the product was purified by preparative reversed-phase HPLC (RP-HPLC) and its purity was established by thin layer chromatography and analytical RP-HPLC. Its structural identity was established by electrospray mass spectrometry. Tissue collection
  • REN-eGFP mice as well as their non-transgenic littermates were sacrificed. Multiple tissues (white adipose tissues from perirenal, reproductive, omental and subcutaneous depots, brown adipose tissue, kidney, heart, liver and submandibular gland) were collected, weighed and snap-frozen in liquid nitrogen until assayed. Blood was collected by intra-cardiac punction, placed in a tube containing EDTA and spun down to recuperate plasma which was also frozen until assayed. For the measurement of Free Fatty Acids (FFA), glucose and insulin, animals were fasted overnight before tissue collection.
  • FFA Free Fatty Acids
  • Protein samples were separated by electrophoresis and transferred to a nitrocellulose membrane. Proteins were detected using a GFP (Chemicon) or (P)RR (Abeam) antisera, depending on the study, as well as ⁇ -actin (Abeam) or GAPDH (Santa Cruz) to control for the quantity and quality of protein. This was accomplished using ECL West PicoTM kits (Pierce).
  • Plasma mouse renin activity was measured as described previously 13 .
  • RIA was performed on plasma with the Ang-1 125 l-labelled RIA kit (Diasorin). Metabolism
  • FFA Free Fatty Acids
  • mice from both groups did not differ in body weight initially as can be seen in Table 1.
  • the group placed on HF/HC diet was significantly larger at the end of the treatment compared to those maintained on normal diet (Table 1).
  • This increased weight gain was mainly due to an increased fat mass as all the adipose sites were significantly larger in the high-fat diet fed mice whereas the heart weight did not differ (Table 1).
  • Table 1 Animal and tissue weight
  • Renin and (P)RR mRNA expression could be detected in all adipose depots studied (perirenal, reproductive, omental and subcutaneous). However, diet had no effect on the renin expression, as can be seen in Table 2. Interestingly, (P)RR mRNA expression was significantly increased by the HF/HC diet in all white adipose tissue ( Figure 1). However, (P)RR protein was shown to be increased significantly only in subcutaneous white adipose tissue ( Figure 2) although there was a tendency for an increase in other depots. [0096] Table 2. Renin mRNA expression in different tissues
  • mice on a normal diet (circles) who received (white) or not (black) a (P)RR blocker.
  • mice administered the (P)RR blocker in concomitance with the HF/HC diet had a decreased Kcal daily consumption.
  • the increased Kcal consumption observed in the HF/HC group compared to the normal diet receiving saline was absent in the presence of the (P)RR blocker.
  • DSI TA11 PA-C10 probes
  • vascular structures lumen, medial and external diameter
  • stress and strain have been calculated according to previously- described formulae 18 .
  • Sodium nitroprusside serves as a source of nitric oxide, a potent peripheral vasodilator that affects both arterioles and venules (venules more than arterioles).
  • Sodium nitroprusside is often administered intravenously to patients who are experiencing a hypertensive emergency. Its mechanism of action appears to be the liberation of nitric oxide (NO), which causes relaxation of the vascular smooth muscle.
  • Endothelin-1 is a potent vasoconstrictor. Increased endothelin-1 -mediated vasoconstrictor tone has been linked to the etiology of a number of pathologies including hypertension, congestive heart failure, and coronary artery disease. Diet-induced obesity and the metabolic syndrome are major risk factors for the development of vascular diseases such as atherosclerosis and hypertension. The progression of these diseases is characterized by endothelial dysfunction leading to altered responses to vasoactive substances including nitric oxide (NO) and endothelin-1 (ET-1) as well as to ⁇ - adrenoceptor ( ⁇ -AR) activation.
  • NO nitric oxide
  • ET-1 endothelin-1
  • ⁇ -AR ⁇ - adrenoceptor
  • results presented in Figure 10 indicate that the (P)RR blocker has antihypertensive effects as mice receiving the HF/HC diet concomitantly with the blocker (black) tended to have lower mean arterial pressure than those receiving only saline (white).
  • results presented in Figures 11 and 12 indicate that administration of the blocker (black) to obese animals (receiving a HF/HC diet) improves their response to sodium nitroprusside (a vasodilatator, Figure 9) while decreasing their response to endothelin-1 (a vasoconstrictor, Figure 10) when compared to those receiving saline (white).
  • LVEDD LV end-diastolic diameter
  • LVESD LV end-systolic diameter
  • EF Ejection Fraction
  • mice diet-induced obesity mice
  • DIO mice diet-induced obesity mice
  • mice were purchased from JAX laboratories. These mice have received 18 weeks of HF diet, starting at 6 weeks of age, and thus, were 24 weeks of age when they were received. These mice were then implanted with mini-osmotic pumps for the delivery of the (P)RR blocker or saline. Two groups of mice were tested: 1. obese/blocker; and 2. obese/saline;.
  • mice and food were weighed and water consumption measured weekly.
  • Body composition was assessed using the EchoMRI (Echo Medical Systems), before and after 5 weeks of treatment. The use of the EchoMRI allowed to evaluate the percentage of fat tissue, lean tissue, body fluids and total body water without any invasive manipulation.
  • Blockade of the (P)RR Diet-induced obesity (DIO) mice were obtained from Jackson Laboratories. All mice were 24 weeks of age at the start of treatment and had received 18 weeks of high-fat diet starting at the age of 6 weeks. DIO male mice were randomly placed into 2 groups: 1) HF/HC-(P)RR blocker; 2) HF/HC-saline. The HF/HC diet used for these studies was the F3282 diet from Bio-Serv. For these experiments, under isofluorane anaesthesia, mice were implanted with a mini-osmotic pump (#1004; Alzet) filled with either the (P)RR blocker, at a dose of 0.1 mg/kg/day, or saline for 10 weeks.
  • DIO Diet-induced obesity mice were obtained from Jackson Laboratories. All mice were 24 weeks of age at the start of treatment and had received 18 weeks of high-fat diet starting at the age of 6 weeks. DIO male mice were randomly placed into 2 groups: 1) HF/HC-(P)RR block
  • mice received either a HF/HC or normal (N) diet for 10 weeks.
  • the chosen dose and route of administration are based on published literature demonstrating protective effects on cardiac and renal pathology in rodents 11 ' 15 .
  • mice whole body composition analyzer. To do so, mice were first weighed then placed in the supplied restrainers and in the instrument.
  • mice in both groups (1) HF/HC-(P)RR blocker; 2) HF/HC-saline) had a similar body weight (Figure 7), mice in the blocker group tended to have a higher fat body mass (Figure 8) and lower lean body mass ( Figure 9) then the saline group.
  • mice in the saline group increased their body weight while animals receiving the blocker tended to maintain their body weight ( Figure 7).
  • mice receiving the blocker tended to decrease their fat body mass and maintain their lean body mass while the animals receiving saline increased their fat body mass and decreased their lean body mass. This suggests that the blocker not only produces a decreased body fat but also preserves lean body mass which could contribute to maintaining the loss of body fat as lean body mass contributes to basal metabolism.
  • Nguyen G Delarue F, Burckle C, Bouzhir L, Giller T, Sraer JD. Pivotal role of the renin/prorenin receptor in angiotensin Il production and cellular responses to renin. J CHn Invest 2002 June; 109(11): 1417-27. 10. Achard V, Boullu-Ciocca S, Desbriere R, Nguyen G, Grino M. Renin receptor expression in human adipose tissue. Am J Physiol Regul lntegr Comp Physiol 2007 January;292(1):R274-R282.

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Abstract

L'invention porte sur un procédé de traitement ou de prévention de l'obésité et/ou de l'hypertension liée à l'obésité, comprenant l'administration à un sujet en ayant besoin d'une quantité thérapeutiquement efficace d'un inhibiteur de l'activité du récepteur de la rénine/prorénine, l'obésité et/ou l'hypertension liée à l'obésité étant ainsi traitée ou prévenue.
PCT/CA2009/000731 2008-05-27 2009-05-27 Procédés de traitement ou de prévention de l'obésité et de l'hypertension liée à l'obésité WO2009143619A1 (fr)

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WO2012045587A1 (fr) * 2010-10-06 2012-04-12 Celltrend Gmbh Nouvelle méthode de diagnostic de l'hypertension et des cardiomyopathies
CN108004310A (zh) * 2017-12-13 2018-05-08 深圳大学 肾素(原)受体(p)rr基因及其抑制剂的应用
CN108473988A (zh) * 2015-12-29 2018-08-31 国立大学法人北海道大学 抑制肾素原基因或肾素原受体基因表达的单链核酸分子及其用途
US10934542B2 (en) 2013-12-27 2021-03-02 Bonac Corporation Artificial match-type miRNA for controlling gene expression and use therefor
US11027023B2 (en) 2014-12-27 2021-06-08 Bonac Corporation Natural type miRNA for controlling gene expression, and use of same
US11142769B2 (en) 2015-03-27 2021-10-12 Bonac Corporation Single-stranded nucleic acid molecule having delivery function and gene expression regulating ability

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ICHIHARA, A. ET AL.: "Nonproteolytic Activation of Prorenin Contributes to Development of Cardiac Fibrosis in Genetic Hypertension.", HYPERTENSION, vol. 47, no. 5, May 2006 (2006-05-01), pages 894 - 900 *
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012045587A1 (fr) * 2010-10-06 2012-04-12 Celltrend Gmbh Nouvelle méthode de diagnostic de l'hypertension et des cardiomyopathies
US8846324B2 (en) 2010-10-06 2014-09-30 Celltrend Gmbh In vitro method for diagnosing or predicting hypertension and/or early-stage cardiovascular end-organ damage by determining the concentration of soluble prorenin receptor
AU2011311765B2 (en) * 2010-10-06 2015-07-16 Celltrend Gmbh A new method for diagnosing hypertension as well as cardiomyopathies
US10934542B2 (en) 2013-12-27 2021-03-02 Bonac Corporation Artificial match-type miRNA for controlling gene expression and use therefor
US11027023B2 (en) 2014-12-27 2021-06-08 Bonac Corporation Natural type miRNA for controlling gene expression, and use of same
US11142769B2 (en) 2015-03-27 2021-10-12 Bonac Corporation Single-stranded nucleic acid molecule having delivery function and gene expression regulating ability
CN108473988A (zh) * 2015-12-29 2018-08-31 国立大学法人北海道大学 抑制肾素原基因或肾素原受体基因表达的单链核酸分子及其用途
EP3399037A4 (fr) * 2015-12-29 2019-05-29 National University Corporation Hokkaido University Molécule d'acide nucléique monocaténaire inhibant l'expression d'un gène de la prorénine ou d'un gène récepteur de la prorénine, et son utilisation
CN108004310A (zh) * 2017-12-13 2018-05-08 深圳大学 肾素(原)受体(p)rr基因及其抑制剂的应用

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