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WO2007019283A2 - Identification des effets de 5-lipoxygenase sur l'obesite et la resistance a l'insuline - Google Patents

Identification des effets de 5-lipoxygenase sur l'obesite et la resistance a l'insuline Download PDF

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WO2007019283A2
WO2007019283A2 PCT/US2006/030386 US2006030386W WO2007019283A2 WO 2007019283 A2 WO2007019283 A2 WO 2007019283A2 US 2006030386 W US2006030386 W US 2006030386W WO 2007019283 A2 WO2007019283 A2 WO 2007019283A2
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mice
insulin
gene
aloxδ
lipoxygenase
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WO2007019283A3 (fr
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Margarete Mehrabian
Aldons Lusis
Hooman Alayee
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University of California Berkeley
University of California San Diego UCSD
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University of California San Diego UCSD
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Insulin resistance occurs in 25% of non-diabetic, non-obese, apparently healthy individuals, and predisposes them to both diabetes and coronary artery disease.
  • Diabetes mellitus is a major health problem in the United States affecting approximately 7% of the population.
  • the most common form of diabetes mellitus is non-insulin-dependent diabetes mellitus (NIDDM or type Il diabetes).
  • NIDDM non-insulin-dependent diabetes mellitus
  • Hyperglycemia in type Il diabetes is the result of both resistance to insulin in muscle and other key insulin target tissues, and decreased beta cell insulin secretion.
  • Longitudinal studies of individuals with a strong family history of diabetes indicate that the insulin resistance precedes the secretory abnormalities. Prior to developing diabetes these individuals compensate for their insulin resistance by secreting extra insulin. Diabetes results when the compensatory hyperinsulinemia fails.
  • the secretory deficiency of pancreatic beta cells then plays a major role in the severity of the diabetes.
  • Insulin resistance results in an increased risk for having elevated plasma triglycerides (TG), lower high density lipoproteins (HDL), and high blood pressure, a cluster of abnormalities that have been termed by different investigators as either Syndrome X, the insulin resistance syndrome, or the metabolic syndrome. It is believed that either the hyperinsulinemia, insulin resistance, or both play a direct role in causing these abnormalities. Data from ethnic, family, and longitudinal studies suggest that a major component of resistance is inherited.
  • IR-TK insulin receptor tyrosine kinase
  • the present invention provides methods and compositions for the treatment and diagnosis of diseases related to hyperglycemic conditions, including diabetes, insulin resistance, and the like. Alleles, including variations in the 5-lipoxygenase promoter region, are associated with disease susceptibility, and their detection is used in the diagnosis of a predisposition to these conditions.
  • Figure 1 is a graph depicting the correlation of 5-LO promoter genotypes with insulin levels in humans.
  • Figure 2 is a graph depicting the correlation of 5-LO genotypes with insulin resistance in humans.
  • FIG. 1 Differences in QTL traits as a function genotype in the BXD cross. F2 mice homozygous for DBA alleles at the chromosome 6 locus have significantly increased total fat mass, omental fat mass, leptin levels (expressed as a ratio of percent body fat), bone mineral density, and VLDL/LDL levels compared to mice homozygous for B6 alleles.
  • the Alox ⁇ ' ' " liver gene expression signature is even more significantly enriched for genes in the BXD liver expression data set whose expression values are linked to Alox ⁇ and correlated with omental fat mass (shown in parentheses).
  • omental fat mass shown in parentheses.
  • genes with eQTLs with lod scores greater than 2.0 1 ,177 (5.9%) are correlated with omental fat pad mass at the 0.05 significance level and map to the 18cM Alox ⁇ interval.
  • 87 genes (19.6%) have eQTL with lod scores greater than 2.0 that map to the interval and correlate with omental fat pad mass at the 0.05 significance level.
  • FIG. Histograms of correlation coefficients computed between omental fat mass and the gene expression levels in 5 different gene sets.
  • the Pearson correlation coefficient was computed between omental fat mass and every gene expression trait in the set of 23,574 genes on BXD microarray.
  • the histograms were plotted with the number of genes on the y-axis and the correlation coefficients on the x-axis. Highlighted in each plot is the percentage of genes in the given set that had statistically significant correlation coefficients at the 0.05 significance level.
  • the different sets of genes represented for each panel are A) a simulated set of 23,574 gene expression traits; B) the set of 23,574 gene expression traits represented in the BXD data set; C) the set of 1,991 genes whose eQTLs give lod scores greater than 2 and map to the 18cM Alox ⁇ interval; D) the set of 444 genes in the Alox ⁇ '1' liver expression signature; and E) the set of 104 genes from the Alox ⁇ '1' liver expression signature whose eQTLs give lod scores greater than 2 in the BXD cross and map within the 18cM interval flanking Alox ⁇ .
  • FIG. 8 Alox ⁇ 1' mice have delayed glucose clearance due to defective insulin secretion.
  • An IPGTT were performed as described in Methods.
  • FIG. 9 5-LO is expressed in ovaries and adrenals and Alox ⁇ ' ' mice exhibit adrenal lipid depletion.
  • A Cryosections of ovaries and adrenals from wildtype B6 stain positive for 5-LO whereas Alox ⁇ 1' mice exhibit minimal staining (100X magnification).
  • B Adrenal glands from mice maintained on either chow or HFC diets were cryosectioned and stained for cholesteryl esters using oil red O. The cortex normally stains intensely because of the presence of cholesteryl esters that serve as a pool of cholesterol for steroid hormone synthesis (4OX magnification).
  • the invention is based, in part, on the evaluation of the expression and role of 5-LO, which is both differentially expressed in disease models, and for which alleles predisposing to altered metabolic conditions are herein identified.
  • 5-LO is both differentially expressed in disease models, and for which alleles predisposing to altered metabolic conditions are herein identified.
  • This permits the definition of 5-LO and other leukotriene synthesis genes as part of a disease pathway and the identification of a target in the pathway that is useful both diagnostically, in drug screening, and therapeutically.
  • Alleles of 5-LO that predispose to coronary artery disease (CAD) are also associated with altered insulin metabolism and may be indicative of a predisposition to diabetes.
  • the leukotrienes constitute a group of arachidonic acid-derived compounds with biologic activities suggesting important roles in inflammation and immediate hypersensitivity.
  • the enzyme 5-lipoxygenase (EC 1.13.11.34) catalyzes 2 reactions in the formation of leukotrienes. Matsumoto et at. (1988) Proc. Nat. Acad. Sci. 85:26-30, herein incorporated by reference, isolated cDNA clones for human lung and placenta 5-lipoxygenase and deduced the complete amino acid sequence of the enzyme.
  • Alleles of the human 5-LO gene have a promoter polymorphism, in which there is a variable number of tandem binding sites for the transcription factors Sp1/Egr-1 (Drazen et a/. (1999) Nat Genet 22:168-70: and In et al. (1997) J. Clin. Invest. 99:1130-1137, herein incorporated by reference), where each repeat has the sequence motif GGGCGG.
  • the common allele in the human population consists of five repeated binding sites and has been termed the "5", or "N” allele. Alleles with less than 5 repeats, usually 3 repeats or 4 repeats, may be referred to numerically as "3" or "4", or collectively as deleted, or "D" alleles.
  • Alleles with expanded repeats greater than 5 in number, usually 6 or 7 repeats may be referred to collectively as "E" expanded or "A” addition alleles.
  • Four genotypic groups have been defined: homozygous 55 (indicating that both alleles consisted of five repeated binding sites); 33, 34, and 44 (one or two binding sites deleted); 35 and 45 (one allele deleted); and 56, 57, and 67 (one or both alleles expanded).
  • a comparison between the genotypic groups revealed that individuals carrying deleted repeat alleles (genotypes 33, 34, or 44) had greatly increased incidence of coronary artery disease compared to individuals with either wild type alleles or larger numbers of repeats.
  • the sequence is the human 5-LO gene is provided as SEQ ID NO:1, where the promoter region extends from nucleotides 1- 1844, and the coding sequence starts at nucleotide 1845.
  • Predisposing 5-LO allele can have one or more Sp1/Egr-1 binding sites deleted, usually at least one binding site deletion on each chromosome, relative to the common allele in the human population, which wild type allele consists of five repeated Sp1/Egr-1 binding sites. Typically such susceptible alleles will have not more than 4 Sp1/Egr-1 binding site repeats. Other predisposing alleles are those changes in the 5-LO DNA sequence that confer an increased susceptibility.
  • 5-LO cytosolic phospholipase A2
  • FLAP 5-lipoxygenase activating protein
  • LT A4 can then be converted to LTB4 via LTA4 hydrolase (LTA4H) or shunted into the cysteinyl leukotriene pathway and converted to LTC4 by LTC4 synthase (LTC4S), which is then converted to LTD4 and subsequently LTE4 by g-glutamyl transferase and LTD4 peptidase, respectively.
  • LTB4 binds to cell surface receptors known as LTB4 receptor 1 (LTB4R1) or LTB4 receptor 2 (LTB4R2) and the cysteinyl leukotrienes (LTC4, LTD4, and LTE4) bind to their respective receptors, CysLTRI and CysLTR2. As a result, these molecules stimulate proinflammatory signaling pathways in target cells.
  • the identification of the entire LT synthesis pathway and all the genes involved in this metabolic process are considered as having effects on atherosclerosis development, measures of adiposity (i.e. fat mass and leptin levels), lipid (ie. cholesterol and triglyceride) levels, insulin/glucose metabolism, and bone density.
  • adiposity i.e. fat mass and leptin levels
  • lipid ie. cholesterol and triglyceride
  • methods are provided for determining a predisposition to altered metabolic conditions involving insulin/glucose dynamics in an individual.
  • the methods comprise an analysis of genomic DNA in an individual for an allele of the 5- lipoxygenase promoter, which confers an increased susceptibility.
  • Individuals are screened by analyzing their genomic 5-LO gene sequence for the presence of a predisposing allele, as compared to a normal 5-LO sequence.
  • the normal 5-LO sequence shall be understood to include sequence variants in non-coding regions that do not affect the level of expression of the gene, and coding region variants that do not change the amino acid sequence, e.g. "third position" changes.
  • the methods also comprise the analysis of genomic DNA in an individual for other leukotriene synthesis genes, which can also confer increased risk.
  • the effect of a sequence variation on 5-LO expression or function can be determined by analysis for segregation of the sequence variation with the disease phenotype, e.g. presence of glucose tolerance, insulin levels, etc. A predisposing mutation will segregate with incidence of the disease.
  • biochemical studies are performed to determine whether a candidate sequence variation in the 5-LO coding region or control regions affects the quantity or function of the protein. Expression levels of a candidate variant allele are compared to expression levels of the normal allele by various methods known in the art.
  • Methods for determining promoter or enhancer strength include quantitation of the expressed natural protein; insertion of the variant control element into a vector with a reporter gene such as ⁇ -galactosidase, chloramphenical acetyltransferase, etc. that provides for convenient quantitation; and the like.
  • a reporter gene such as ⁇ -galactosidase, chloramphenical acetyltransferase, etc. that provides for convenient quantitation; and the like.
  • Genomic DNA is isolated from the individual or individuals that are to be tested.
  • DNA can be isolated from any nucleated cellular source such as blood, hair shafts, saliva, mucous, biopsy, feces, etc.
  • Methods using PCR amplification can be performed on the DNA from a single cell, although it is convenient to use at least about 10 5 cells. Where large amounts of DNA are available, the genomic DNA is used directly.
  • the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis, or amplified by conventional techniques. Of particular interest is the use of the polymerase chain reaction (PCR) to amplify the DNA that lies between two specific primers.
  • PCR polymerase chain reaction
  • a detectable label may be included in the amplification reaction. Suitable labels include fluorochromes, e.g.
  • fluorescein isothiocyanate FITC
  • rhodamine Texas Red
  • phycoerythrin allophycocyanin
  • 6-carboxyfluorescein 6-carboxyfluorescein
  • JE 6-carboxy-X-rhodamine
  • HEX ⁇ -carboxy-Z.'l'J ⁇ ?- hexachlorofluorescein
  • 5-carboxyfluorescein 5-FAM
  • N,N,N',N'-tetramethyl-6- carboxyrhodamine TAMRA
  • radioactive labels e.g.
  • the label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label.
  • the label may be conjugated to one or both of the primers.
  • the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.
  • Primer pairs are selected from the 5-LO genomic sequence using conventional criteria for selection.
  • the primers in a pair will hybridize to opposite strands, and will collectively flank the region of interest.
  • the primers will hybridize to the complementary sequence under stringent conditions, and will generally be at least about 16 nt in length, and may be 20, 25 or 30 nucleotides in length.
  • the primers will be selected to amplify the specific region of the 5-LO gene suspected of containing the predisposing mutation. Typically the length of the amplified fragment will be selected so as to allow discrimination between repeats of 2 to 8 units. Multiplex amplification may be performed in which several sets of primers are combined in the same reaction tube, in order to analyze multiple exons simultaneously. Each primer may be conjugated to a different label.
  • a diagnostic screening method of particular interest detects the number of SP-1 repeats in the promoter region of the human 5-LO gene.
  • the organization of the region comprises a repeat region of from about 2 to about 8 6 base pair repeats of the binding motif GGGCGG, flanked by unique sequences.
  • sequences are selected for amplification primers.
  • the exact composition of the primer sequences are not critical to the invention, but they must hybridize to the flanking sequences under stringent conditions. Criteria for selection of amplification primers are as previously discussed.
  • a primer sequence that is close to the repeat sequence such that the total amplification product is at least about 30, more usually at least about 50, preferably at least about 100 or 200 nucleotides in length, which will vary with the number of repeats that are present, to not more than about 500 nucleotides in length.
  • the number of repeats has been found to be polymorphic, as previously described, thereby generating individual differences in the length of DNA that lies between the amplification primers.
  • the primers are used to amplify the region of genomic DNA that contains the repeats. Conveniently, a detectable label will be included in the amplification reaction, as previously described. Multiplex amplification may be performed in which several sets of primers are T/US2006/030386
  • each of the sets of primers is labeled with a different fluorochrome.
  • the products are size fractionated. Fractionation may be performed by gel electrophoresis, particularly denaturing acrylamide or agarose gels.
  • gel electrophoresis particularly denaturing polyacrylamide gels in combination with an automated DNA sequencer, see Hunkapillar et al. (1991) Science 254:59-74. The automated sequencer is particularly useful with multiplex amplification or pooled products of separate PCR reactions.
  • Capillary electrophoresis may also be used for fractionation. A review of capillary electrophoresis may be found in Landers, et al. (1993) BioTechniques 14:98-111.
  • the size of the amplification product is proportional to the number of repeats (n) that are present at the locus specified by the primers. The size will be polymorphic in the population, and is therefore an allelic marker for that locus.
  • the amplified or cloned fragment may be sequenced by dideoxy or other methods, and the length of the amplified region, or the sequence of bases, is compared to the normal 5- LO sequence.
  • the predisposing mutation creates or destroys a recognition site for a restriction endonuclease
  • the fragment is digested with that endonuclease, and the products size fractionated to determine whether the fragment was digested. Fractionation is performed by gel electrophoresis, particularly acrylamide or agarose gels. Hybridization with the variant sequence may also be used to determine its presence, by Southern blots, dot blots, etc.
  • Single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), and heteroduplex analysis in gel matrices is used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility.
  • the hybridization pattern of a control and variant sequence to an array of oligonucleotide probes immobilised on a microarray may also be used as a means of detecting the presence of variant sequences.
  • the presence of a predisposing mutation is indicative that an individual is at increased risk of developing altered metabolic conditions relating to diabetes.
  • the diagnosis of a disease predisposition allows the affected individual to seek early treatment of potential lesions, and to avoid activities that increase risk.
  • Hyperglycemia in type Il diabetes is the result of both resistance to insulin in muscle and other key insulin target tissues, and decreased beta cell insulin secretion. Longitudinal studies of individuals with a strong family history of diabetes indicate that the insulin resistance precedes the secretory abnormalities. Prior to developing diabetes these individuals compensate for their insulin resistance by secreting extra insulin. Diabetes results when the compensatory hyperinsulinemia fails. The secretory deficiency of pancreatic beta cells then plays a major role in the severity of the diabetes.
  • Insulin resistance is an essential feature of a great variety of clinical disorders in addition to diabetes, including coronary artery disease, hyperlipidemia, obesity and hypertension. Individuals with non-insulin dependent diabetes have insulin resistance in peripheral tissues.
  • IGT Impaired glucose tolerance
  • Insulin resistance results in an increased risk for having elevated plasma triglycerides (TG), lower high density lipoproteins (HDL) 1 and high blood pressure, a cluster of abnormalities that have been termed by different investigators as either Syndrome X, the insulin resistance syndrome, or the metabolic syndrome. It is believed that either the hyperinsulinemia, insulin resistance, or both play a direct role in causing these abnormalities. Data from ethnic, family, and longitudinal studies suggest that a major component of resistance is inherited.
  • TG plasma triglycerides
  • HDL high density lipoproteins
  • the most widely accepted research method or 'gold standard 1 is the euglycemic insulin clamp technique.
  • exogenous insulin is infused, so as to maintain a constant plasma insulin level above fasting, while glucose is fixed at a basal level by infusing glucose at varying rates.
  • This glucose infusion is delivered via an indwelling catheter at a rate based on plasma glucose measurements every 5 min.
  • the glucose infusion rate is increased to return plasma glucose to basal levels and vice versa.
  • the amount of glucose infused over time (M value) is an index of insulin action on glucose metabolism. The more glucose that has to be infused per unit time, then the more sensitive the patient is to insulin.
  • the insulin-resistant patient requires much less glucose to maintain basal plasma glucose levels.
  • the effect of insulin on fuel metabolism can be assessed in the absence of the confounding effects of hypoglycemic counterregulation, endogenous insulin secretion, or variable levels of hyperglycemia, and multiple insulin actions can be assessed by using isotopes, including regulation of glucose uptake and production, inhibition of lipolysis, and changes in protein metabolism.
  • HOMA homeostasis model assessment
  • Comparison of a patient's fasting values with the model's predictions allows a quantitative assessment of the contributions of insulin resistance and deficient beta-cell function to the fasting hyperglycaemia.
  • the estimate of insulin resistance obtained by homeostasis model assessment correlates with estimates obtained by use of the euglycaemic clamp, the fasting insulin concentration, and the hyperglycaemic clamp.
  • the lower limit of the top quintile of HOMA distribution (i.e. 2.77) in nonobese subjects with no metabolic disorders has been chosen as the threshold for insulin resistance in some studies (Bonora et al. (1998) Diabetes 47:1643-9).
  • the measurement of insulin concentration can be done in the overnight fasted condition, since in the postprandial state, glucose levels are changing rapidly and the variable levels of glucose confound the simultaneous measure of insulin levels as an index of insulin action. There is a significant correlation between fasting insulin levels and insulin action as measured by the clamp technique. Very high plasma insulin values in the setting of normal glucose levels are very likely to reflect insulin resistance. As individuals develop diabetes, plasma glucose increases and plasma insulin decreases and so the plasma insulin level no longer reflects only insulin resistance because it becomes influenced by the appearance of a ⁇ -cell defect and hyperglycemia.
  • the genes in the 5-LO/leukotriene pathway have been found to be differentially expressed in an animal model for altered metabolic states relating to diabetes.
  • "Differential expression” as used herein refers to both quantitative as well as qualitative differences in the genes' temporal and/or tissue expression patterns.
  • a differentially expressed gene may have its expression diminished or inactivated in protective versus susceptible conditions.
  • the 5-LO gene therefore finds use in screening for agents that modulate expression or activity, and which find use in treatment of diabetes and insulin resistant states.
  • Drug candidates of interest include known 5-LO inhibitors, many of which are known in the art, for example zileuton, ABT-761 (see Drazen et al., supra.); 2,5-Diaryl tetrahydrofurans, 2,5-diaryl tetrahydrothiophenes, 2,4-diaryl tetrahydrofurans, 2,4-diaryl tetrahydrothiophenes, 1,3-diaryl cyclopentanes, 2,4-diaryl pyrrolidines, and 2,5-diaryl pyrrolidines as disclosed in U.S. Patent no. 6,294,574; compounds described in U.S. Patent no. 6,194,585, and the like.
  • a 5-LO inhibitor can, for example, act as the basis for amelioration of hyperglycemic disease.
  • Such compounds may include, but are not limited to peptides, antibodies, or small organic or inorganic compounds. Methods for the identification of such compounds are described below.
  • Cell- and animal-based systems can act as models for hyperglycemic disease and are useful in such drug screening.
  • the animal- and cell-based models may be used to identify drugs, pharmaceuticals, therapies and interventions that are effective in treating hyperglycemic disease.
  • animal models may be used to determine the LD 50 and the ED 50 in animal subjects, and such data can be used to determine the in vivo efficacy of potential treatments.
  • Animal-based model systems of disease may include, but are not limited to, non-recombinant and engineered transgenic animals.
  • animal models exhibiting hyperglycemic disease symptoms may be engineered by utilizing, for example, 5-LO gene sequences in conjunction with techniques for producing transgenic animals that are well known to those of skill in the art.
  • target gene sequences may be introduced into, and knocked out or overexpressed in the genome of the animal of interest.
  • Animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate cardiovascular disease animal models.
  • Any technique known in the art may be used to introduce a target gene transgene into animals to produce the founder lines of transgenic animals.
  • Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. ScL, USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al., 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, MoI Cell. Biol. 3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723); etc.
  • Specific cell types within the animals may be analyzed and assayed for cellular phenotypes characteristic of hyperglycemic disease. Further, such cellular phenotypes may include a particular cell type's fingerprint pattern of expression as compared to known fingerprint expression profiles of the particular cell type in animals exhibiting hyperglycemic disease symptoms. Cells that contain and express 5-LO and/or other leukotriene synthesis pathway genes can be utilized to identify compounds that exhibit anti-hyperglycemic disease activity.
  • Cells of a cell type known to be involved in hyperglycemic disease may be transfected with sequences capable of increasing or decreasing the amount of 5-LO gene expression within the cell.
  • 5-LO gene sequences may be introduced into, and overexpressed in, the genome of the cell of interest, or, if endogenous target gene sequences are present, they may be either overexpressed or, alternatively disrupted in order to underexpress or inactivate target gene expression.
  • Transfection of target gene sequence nucleic acid may be accomplished by utilizing standard techniques. Transfected cells can be evaluated for the presence of the recombinant 5-LO gene sequences, for expression and accumulation of 5-LO gene mRNA, and for the presence of recombinant 5-LO protein. Where a decrease in 5-LO gene expression is desired, standard techniques may be used to demonstrate whether a decrease in expression is achieved.
  • In vitro systems may be designed to identify compounds capable of inhibiting 5-LO.
  • Such compounds may include, but are not limited to, peptides made of D-and/or L- configuration amino acids, phosphopeptides, antibodies, and small organic or inorganic molecules.
  • the principle of the assays used to identify compounds that inhibit 5-LO involves preparing a reaction mixture of 5-LO and a test compound under conditions and for a time sufficient to allow the two components to interact, and detecting the resulting change in the catalytic activity in the formation of leukotrienes.
  • a simple binding assay can be used as an initial screening method.
  • one method to conduct such an assay would involve anchoring 5-LO protein or a test substance onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction.
  • the assay tests the presence of products catalyzed by 5-LO.
  • a routine assay of 5-LO activity can be performed in a mixture containing 50 mM potassium phosphate buffer at pH 7.4, 2 mM CaCI 2 , 2 mM ATP, 25 M arachidonic acid (0.1 Ci) and 5-LO enzyme (50-100 mg of protein) in a final volume of 200 ml.
  • the reaction is carried out at 24° C. for 3 minutes.
  • the mixture is extracted with 0.2 ml of an ice- cold mixture of ethyl ethe ⁇ methanol: 0.2 M citric acid (30:4:1).
  • the extract is subjected to thin-layer chromatography at -10° C. in a solvent system of petroleum ethe ⁇ ethyl ethe ⁇ acetic acid (15:85:0.1).
  • the silica gel zones corresponding to authentic arachidonic acid and its metabolites are scraped into scintillation vials for counting.
  • the enzyme activity is expressed in terms of the amount of arachidonic acid oxygenated for 3 minutes.
  • the reaction can be performed on a solid phase or in liquid phase.
  • the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed under conditions such that any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-lg antibody).
  • a binding reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for target gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.
  • Cell-based systems such as those described above may be used to identify compounds that act to ameliorate disease symptoms.
  • such cell systems may be exposed to a test compound at a sufficient concentration and for a time sufficient to elicit such an amelioration of disease symptoms in the exposed cells. After exposure, the cells are examined to determine whether one or more of the disease cellular phenotypes has been altered to resemble a more normal or more wild type, non-disease phenotype.
  • animal-based disease systems such as those described, above may be used to identify compounds capable of ameliorating disease symptoms.
  • Such animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies, and interventions, which may be effective in treating disease.
  • animal models may be exposed to a compound, suspected of exhibiting an ability to ameliorate disease symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of disease symptoms in the exposed animals. The response of the animals to the exposure may be monitored by assessing the reversal of disorders associated with disease.
  • any treatments that reverse any aspect of insulin resistance and other hyperglycemic conditions should be considered as candidates for human disease therapeutic intervention.
  • Dosages of test agents may be determined by deriving dose- response curves.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5O /ED 50 .
  • Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, ethy
  • compositions may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one specific 5-LO nucleic acid reagent described herein, which may be conveniently used, e.g., in clinical settings, for prognosis of patients susceptible to disease.
  • mice were purchased from the Jackson Laboratories, Bar Harbor, Maine, and housed 4 per cage at 25 0 C on a 12-hour light/dark cycle. They were maintained either on a chow diet or a high-fat, high-cholesterol diet containing 15% fat, 1.25% cholesterol, and 0.5% cholic acid (diet No. 90221 , Harlan-Teklad). The mice used in the experiments described below were of both sexes and between 4 to 6 months of age. All procedures were in accordance with current National Institutes of Health guidelines and were approved by the UCLA Animal Research Committee.
  • 5-LO "7" mice on a B6 background were generated as described previously.
  • 5-LO ⁇ ' ⁇ mice were first bred to LDLR ";” mice (also on a B6 background), and the F1 progeny were backcrossed to LDLR ⁇ ' ⁇ mice to produce 5-LO +/" / LDLR ⁇ ' ⁇ mice. These mice were then intercrossed to generate double knockout animals.
  • 5-LO ⁇ ' ⁇ I LDLR ⁇ ' ⁇ mice were obtained, they did not produce offspring. Therefore, the experiments described herein were performed with 5-LO +/' /LDLR ⁇ ' ⁇ mice.
  • PCR primers used for the LDLR +/+ allele were (SEQ ID NO:5) ⁇ '-ACCCCAAGACGTGCTCCCAGGATGA-S' and (SEQ ID NO:6) 5'- CGCAGTGCTCCTCATCTGACTTGT-3' and for the mutant allele were (SEQ ID NO:7) 5'- AGGATCTCGTCGTGACCCATGGCGA-3' and (SEQ ID NO:8) 5'- GAGCGGCGATACCGTAAAGCACGAGG-3'.
  • Plasma Lipid and Insulin Measurements Mice were fasted overnight and bled retro- orbitally under isoflurane anesthesia. Enzymatic assays for plasma cholesterol levels were performed as described previously. Insulin levels were measured in duplicate by ELISA (Crystal Chemical IUSKRO20).
  • LTB4 levels were determined in duplicate using a commercially available ELIZA kit (Cayman Chemical). Assays were performed on bone marrow cells (25 ⁇ g protein) homogenized in 10 mmol/L Tris, pH 8.0.
  • cDNA was prepared from peritoneal macrophage RNA of CAST and B6 mice using an Superscript rtPCR kit (Gibco BRL). The PCR primers used for sequencing were as follows: (SEQ ID NO:9) 5-
  • Bone Marrow Cells and Peritoneal Macrophages were flushed from mouse femurs with DMEM/5% fetal calf serum (FCS) and centrifuged at 1500 RPM for 15 minutes (3 repetitions of washing and centrifugation). Peritoneal macrophages were isolated after lavage with DMEM/5% FCS, as described for bone marrow cells.
  • FCS fetal calf serum
  • C0N6 Mice Have Reduced Expression of ⁇ -LO.
  • Quantitative trait locus mapping of a cross between resistant CAST and susceptible B6 mice for atherosclerotic lesion development revealed a locus with a powerful effect on atherosclerosis on mouse chromosome 6.
  • a congenic strain, CON6, containing the locus derived from CAST on the background of B6 was constructed.
  • the congenic strain was almost entirely resistant to atherosclerosis, even when an LDL receptor-null mutation was introduced.
  • These studies defined the critical region of the gene to between ⁇ 45 cM and 74 cM on mouse chromosome 6. To complement this approach, various candidate genes within the locus were tested.
  • the 5-LO gene is located near the middle of the congenic region, at -53 cM.
  • subject invention provides a convenient and effective way of determining whether a patient will be susceptible to hyperglycemic disease.
  • the subject methods will provide a number of benefits, including preventive treatment and diet. As such, the subject invention represents a significant contribution to the art.
  • mice congenic for the CAST allele of Alox ⁇ were markedly resistant to atherosclerosis and had significantly reduced expression of the enzyme, which could be attributed to amino acid substitutions in the c-terminus of 5-LO.
  • Alox ⁇ '1' mice on a hyperlipidemic background were resistant to aortic lesion formation and had reduced levels of plasma insulin, an observation supported by recent genetic studies in humans that have implicated the 5-LO pathway in atherosclerosis.
  • peroxisome proliferator activated receptor gamma ⁇ Ppar ⁇ This transcription factor is involved in adipocyte differentiation, insulin sensitivity, and is the target of pharmaceutical agonists, such as rosiglitazone, that are used to treat type 2 diabetes.
  • pharmaceutical agonists such as rosiglitazone
  • FIG. 4 shows that, of the 23,574 genes on the BXD microarray, 20,107 genes had expression QTLs (eQTLs) with lod scores > 2 (point-wise significant at the 0.01 significance level). Nearly 10% (1 ,991) of these eQTLs mapped to an 18cM window encompassing Alox ⁇ (Table 1). Since roughly 1% (236 genes) would have been expected by chance, this region of the mouse genome is considered a hotspot for eQTL activity in this cross.
  • AloxS 1' mice exhibit altered glucose-stimulated insulin secretion.
  • B6 and CAST we observed a QTL for insulin levels over Alox ⁇ .
  • IPGTTs intraperitoneal glucose tolerance tests
  • the wild type mice had doubled the amount of plasma insulin as compared with Alox ⁇ '1' mice, which only became similar to control mice after 30 min.
  • Alox ⁇ 1' mice we isolated ⁇ -cells from Alox ⁇ " ' ' and wildtype mice and measured insulin secretion in vitro after incubating in 4, 12, and 16mM glucose. However, there were no differences in intracellular insulin levels or its secretion by the ⁇ -cells in these experiments.
  • Alox ⁇ 1 mice have elevated leptin, they do not consume less food than their wild-type littermates. Given that LEPRs are present in other tissues, it is possible that 5-LO deficiency decreases their peripheral expression, which could, in turn, lead to elevated plasma leptin. This notion is supported by the decreased expression of LEPR we observed in the livers of Alox ⁇ 1" mice. Moreover, since leptin is also known to inhibit insulin secretion, the hyperleptinemia in Alox ⁇ '1' mice could further contribute to the altered glucose-stimulated insulin response. Interestingly, Mancuso and colleagues have also demonstrated that leptin can induce LT synthesis 2-4-fold in murine macrophages, which, by extension, could increase inflammation and atherogenesis in the artery wall.
  • mice on a B6 background were bred in house from known homozygous parental breeders, which were backcrossed to B6 for more that 10 generations.
  • Control B6 mice were either bred in house or purchased from the Jackson Laboratories (Bar Harbor, Maine). All animals were housed 4 per cage at 25 0 C on a 10-hr dark/14-hr light cycle and maintained on either on a chow diet (Purina diet # 5015) or a HFC containing 15%fat, 1.25% cholesterol, and 0.5% cholic acid (Harlan-Teklad diet # 90221 ).
  • the mice used in the experiments were of both sexes and age matched between 4-7 months of age. All procedures were in accordance with current the National Research Council, Guide for the Care and Use of Laboratory Animals and were approved by the UCLA Animal Research Committee.
  • Competitive hybridizations were performed by mixing fluorescently labeled cRNA (5mg) from Alox ⁇ 1' mice and wildtype controls or from rosiglitazone-treated mice and B6 controls.
  • the sections were blocked using 10% Normal Serum (Vector Laboratories, Inc., CA), and antigens were detected with affinity-purified polyclonal antibodies against 5-LO (Cayman Chemical, Ann Arbor, Ml), which were used at 1 :100 dilution and applied for 1.5 hrs.
  • the tissue sections were washed and the primary antibodies were detected using biotin conjugated secondary antibodies (Vector Laboratories, Burlingame, CA), incubated in HRP- Aviden (Vector Laboratories, Burlingame, CA), detected by DAB (Vector Laboratories, Burlingame, CA) and lightly counterstained with hematoxylin. Each tissue was stained in duplicate.
  • the cortex normally stains intensely because of the presence of cholesteryl esters that serve as a pool of cholesterol for steroid hormone synthesis.

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Abstract

Selon cette invention, il est prouvé que la déficience de 5-LO exacerbe des marqueurs du syndrome métabolique. Cette enzyme présente des effets significatifs sur l'adiposité et le métabolisme ainsi que sur la densité osseuse et la fertilité.
PCT/US2006/030386 2005-08-04 2006-08-02 Identification des effets de 5-lipoxygenase sur l'obesite et la resistance a l'insuline Ceased WO2007019283A2 (fr)

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