EP1686998A1 - Inhibition de la phosphodiesterase 9 comme traitement d'etats associes a l'obesite - Google Patents

Inhibition de la phosphodiesterase 9 comme traitement d'etats associes a l'obesite

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Publication number
EP1686998A1
EP1686998A1 EP04769658A EP04769658A EP1686998A1 EP 1686998 A1 EP1686998 A1 EP 1686998A1 EP 04769658 A EP04769658 A EP 04769658A EP 04769658 A EP04769658 A EP 04769658A EP 1686998 A1 EP1686998 A1 EP 1686998A1
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Prior art keywords
pde9
cell
gene
mouse
cells
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EP04769658A
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German (de)
English (en)
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Shawn Clive Black
Earl Michael Gibbs
John Douglas Mcneish
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Pfizer Products Inc
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Pfizer Products Inc
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Publication of EP1686998A1 publication Critical patent/EP1686998A1/fr
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    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • A—HUMAN NECESSITIES
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2227/105—Murine
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Definitions

  • the present invention provides methods to decrease body weight and/or body fat in the treatment, for example, of overweight or obese patients, and methods to treat eating disorders (e.g., binge eating disorder and bulimia), by administering a phosphodiesterase 9 (PDE9) inhibitor.
  • PDE9 phosphodiesterase 9
  • the invention also features genetically- modified mammalian cells, and genetically-modified mice, with a disruption of the PDE9 gene.
  • Cyclic nucleotide phosphodiesterases catalyze the hydrolysis of cyclic nucleotides, such as the second messengers cAMP (cyclic adenosine 3'5'- monophosphate) and cGMP (cyclic guanine 3'5'-monophosphate).
  • PDEs play a pivotal regulatory role in a wide variety of signal transduction pathways (Beavo, Physiol. Rev. 75: 725-748, 1995).
  • PDEs mediate processes involved in vision (McLaughlin et al., Nat. Genet. 4: 130-134, 1993), olfaction (Yan et al., Proc. Natl. Acad. Sci. USA 92: 9677-81 , 1995), platelet aggregation (Dickinson et al. Biochem. J. 323: 371-377, 1997), aldosterone synthesis (MacFarland et al., J. Biol. Chem.
  • PDEs form a superfamily of enzymes that are subdivided into 11 major gene families (Beavo, Physiol. Rev. 75: 725-748, 1995; Beavo et al., Mol. Pharmacol.
  • Each PDE gene family encodes a phosphodiesterase distinguished functionally by unique enzymatic characteristics and pharmacological profiles. In addition, each family exhibits distinct tissue, cell, and subcellular expression patterns (Beavo et al., Mol. Pharmacol. 46: 399-405, 1994; Soderling et al., Proc. Natl. Acad. Sci. USA 95: 8991-8996, 1998; Fisher et al., Biochem. Biophys. Res. Commun. 246: 570-577, 1998; Hayashi et al., Biochem.
  • PDE9 is present in a variety of human tissues, including testes, brain, small intestine, skeletal muscle, heart, lung, thymus, and spleen. PDE9 inhibitors have bee reported as useful to treat cardiovascular disorders (WO 03/037899), and insulin resistance syndrome, hypertension, and/or type 2 diabetes (WO 03/037432).
  • the invention features a method of treating an animal to reduce body fat comprising administering to an animal in need thereof a therapeutically effective amount of a PDE9 inhibitor.
  • the animal is a human or companion animal (e.g., dog, cat, horse) and is overweight, more preferably, the animal is obese.
  • the animal is a food stock animal (e.g., chicken, cattle, pig) and such treatment is rendered to produce leaner meat.
  • the PDE9 inhibitor is a PDE9 selective inhibitor or the PDE9 inhibitor is administered orally.
  • the invention features a method of treating an animal for an eating disorder comprising administering to an animal in need thereof a therapeutically effective amount of a PDE9 inhibitor.
  • the eating disorder is binge eating disorder or bulimia
  • the PDE9 inhibitor is a PDE9 selective inhibitor
  • the PDE9 inhibitor is administered orally.
  • the invention features a method of treating an animal for metabolic syndrome comprising administering to an animal in, need thereof a therapeutically effective amount of a PDE9 inhibitor.
  • the PDE9 inhibitor is a PDE9 selective inhibitor, or the PDE9 inhibitor is administered orally.
  • the invention also features a genetically-modified mouse, wherein the mouse is homozygous for disruption of the PDE9 gene and wherein the mouse, following a six week high fat diet, exhibits reduced body weight or reduced fat mass in an adipose depot, as compared to a wild type mouse following a six week high fat diet.
  • the mouse expresses an exogenous reporter gene under the control of the regulatory sequences of the PDE9 gene or the mouse exhibits nondetectable PDE9 activity.
  • the invention provides a cultured genetically-modified murine cell derived from the above-described mouse.
  • the invention provides a method for producing the above-described mouse comprising: (a) introducing a DNA sequence into a mouse ES cell, wherein the DNA sequence comprises a PDE9 gene targeting construct, which, upon recombination with the PDE9 gene, disrupts the PDE9 gene; (b) selecting a mouse ES cell whose genome comprises a disruption of the PDE9 gene; (c) introducing an ES cell selected in step (b) into a mouse blastocyst or morulae; (d) transplanting the blastocyst or morulae of step (c) into a pseudopregnant mouse; (e) developing the transferred blastocyst or morulae to term to produce a chimeric mouse; and (f) mating sexually mature chimeric mice and mice heterozygous for the PDE9 disruption to obtain a mouse homozygous for the PDE9 gene disruption; wherein the mouse, following a six week high fat diet, exhibits reduced body weight or reduced
  • the invention also features a genetically-modified cultured mammalian cell, wherein the cell is homozygous for disruption of the PDE9 gene and wherein the cell, or a progeny cell derived from the cell, exhibits nondetectable PDE9 polypeptide activity wherein the cell or progeny cell would exhibit PDE9 polypeptide activity absent the homozygous disruption.
  • the cell is an embryonic stem (ES) cell, more preferably, the cell is a murine ES cell or a human ES cell.
  • the invention provides an isolated nucleic acid molecule comprising a PDE11 gene targeting construct, wherein, upon recombination with the PDE9 gene, the construct disrupts the PDE9 gene.
  • PDE9 inhibitor an agent that reduces or attenuates the biological activity of the PDE9 polypeptide.
  • Such agents may include proteins, such as anti-PDE9 antibodies, nucleic acids, e.g., PDE9 antisense or RNA interference (RNAi) nucleic acids, amino acids, peptides, carbohydrates, small molecules (organic or inorganic), or any other compound or composition which decreases the activity of a PDE9 polypeptide either by effectively reducing the amount of PDE9 present in a cell, or by decreasing the enzymatic activity of the PDE9 polypeptide.
  • RNAi RNA interference
  • Compounds that are PDE9 inhibitors include all solvates, hydrates, pharmaceutically acceptable salts, tautomers, stereoisomers, and prodrugs of the compounds.
  • a small molecule PDE9 inhibitor used in the present invention has an IC 50 of less that 10 ⁇ M, more preferably, less than 1 ⁇ M, and, even more preferably, less than 0.1 ⁇ M.
  • Any PDE9 inhibitor used in the present invention is preferably also selective against some or all other PDEs, preferably, against PDE1A, PDE1 B, PDE1C, PDE2, PDE3A, PDE3B, PDE4A, PDE4B, PDE4C, PDE4D, PDE5, PDE6, PDE7A, PDE7B, PDE8A, PDE8B, PDE10, and/or PDE11.
  • a “selective" PDE9 inhibitor is meant an agent that inhibits PDE9 activity with an IC 50 at least 10-fold less, preferably, at least 100-fold less, than the IC 50 for inhibition of one or more other PDEs.
  • agents are combined with a pharmaceutically acceptable delivery vehicle or carrier.
  • An antisense oligonucleotide directed to the PDE9 gene or mRNA to inhibit its expression is made according to standard techniques (see, e.g., Agrawal et al. Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs, Vol. 20, 1993).
  • RNA molecule that functions to reduce the production of PDE9 enzyme in a cell can be produced according to standard techniques known to those skilled in the art (see, e.g., Hannon, Nature 418: 244-251 , 2002; Shi, Trends in Genetics 19: 9-12, 2003; Shuey et al., Drug Discovery Today 7: 1040-1046, 2002).
  • Examples of PDE9 inhibitors are provided herein and in WO 03/037899, in WO 03/037432, and in U.S. Provisional Patent Appl. No. 60/466,639, filed April 30, 2003, incorporated herein by reference.
  • Decreased PDE9 activity means a manipulated decrease in the polypeptide activity of the PDE9 enzyme as a result of genetic disruption or manipulation of the PDE9 gene function that causes a reduction in the level of functional PDE9 polypeptide in a cell, or as the result of administration of a pharmacological agent that inhibits PDE9 activity.
  • pharmaceutically acceptable indicates that the designated carrier, vehicle, diluent, excipient(s), and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
  • prodrug refers to a compound that is a drug precursor which, following administration, releases the drug in vivo via a chemical or physiological process (e.g., upon being brought to physiological pH or through enzyme activity).
  • a discussion of the synthesis and use of prodrugs is provided by Higuchi and Stella, Prodrugs as Novel Delivery Systems, vol. 14 of the ACS Symposium Series, and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.
  • salts and “pharmaceutically acceptable salts” refer to organic and inorganic salts of a compound, a stereoisomer of the compound, or a prodrug of the compound.
  • BMI body mass index
  • Obesity is typically defined as a BMI of 30 or greater (see, e.g., National Heart, Lung, and Blood Institute, Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults, The Evidence Report, Washington, DC: U.S. Department of Health and Human Services, NIH publication no. 98-4083,1998).
  • BMI National Heart, Lung, and Blood Institute
  • Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults The Evidence Report, Washington, DC: U.S. Department of Health and Human Services, NIH publication no. 98-4083, 1998.
  • BMI body fat
  • NIH publication no. 98-4083 International Health and Human Services
  • a “high fat diet” as administered to a genetically-modified or wild type mouse, is meant a diet composed of at least 45% kcal fat, and, preferably, at least 58% fat.
  • Exemplary diets include the Surwit diet (Surwit et al., Metabolism 47: 1354-1359; Surwit et al., Metabolism 47: 1089-1096, 1998; Surwit et al., J. Biol. Chem.
  • High blood pressure ⁇ 60/90 mmHg
  • Hyperlipidemia triglyceride concentration ⁇ 50 mg/dl (1.695 mmol/l) and/or HDL cholesterol ⁇ 35 mg/dl (0.9 mmol/l) in men and ⁇ 39 mg/dl (1.0 mmol/l) in women;
  • Microalbuminuria urinary albumin excretion rate ⁇ >0 g/min or an albumin-to- creatinine ratio ⁇ 0 mg/kg.
  • therapeutically effective is meant resulting in a decrease in body fat.
  • a “disrupted PDE9 gene” refers to a PDE9 gene that is genetically-modified such that the cellular activity of the PDE9 polypeptide encoded by the disrupted gene is decreased or, preferably, eliminated in cells that normally express a wild type version of the PDE9 gene.
  • the genetic modification When the genetic modification effectively eliminates all wild type copies of the PDE9 gene in a cell (e.g., the genetically-modified, non-human mammal or animal cell is homozygous for the PDE9 gene disruption or the only wild type copy of the PDE9 gene originally present is now disrupted), the genetic modification results in a reduction in PDE9 polypeptide activity as compared to a control cell that expresses the wild type PDE9 gene.
  • PDE9 polypeptide activity results from either reduced PDE9 gene expression (i.e., PDE9 mRNA levels are effectively reduced resulting in reduced levels of PDE9 polypeptide) and/or because the disrupted PDE9 gene encodes a mutated polypeptide with altered, e.g., reduced, function as compared to a wild type PDE9 polypeptide.
  • the activity of PDE9 polypeptide in the genetically-modified, non-human mammal or animal cell is reduced to 50% or less of wild type levels, more preferably, to 25% or less, and, even more preferably, to 10% or less of wild type levels.
  • the homozygous PDE9 gene disruption results in non-detectable PDE9 activity in cells of a type that demonstrate wild type PDE9 activity.
  • a "genetically-modified, non-human mammal" containing a disrupted PDE9 gene refers to a non-human mammal created by genetic engineering to contain a disrupted PDE9 gene, as well as a progeny of such non-human mammal that inherits the disrupted PDE9 gene.
  • a genetically-modified non-human mammal may be produced, for example, by creating a blastocyst or embryo carrying the desired genetic modification and then implanting the blastocyst or embryo in a foster mother for in utero development.
  • the genetically-modified blastocyst or embryo can be made, in the case of mice, by implanting a genetically-modified embryonic stem (ES) cell into a mouse blastocyst or by aggregating ES cells with tetraploid embryos.
  • ES embryonic stem
  • various species of genetically-modified embryos can be obtained by nuclear transfer.
  • the donor cell is a somatic cell or a pluripotent stem cell, and it is engineered to contain the desired genetic modification that disrupts the PDE9 gene.
  • the nucleus of this cell is then transferred into a fertilized or parthenogenetic oocyte that is enucleated; the resultant embryo is reconstituted and developed into a blastocyst.
  • a genetically-modified blastocyst produced by either of the above methods is then implanted into a foster mother according to standard methods well known to those skilled in the art.
  • a "genetically- modified, non-human mammal” includes all progeny of the non-human mammals created by the methods described above, provided that the progeny inherit at least one copy of the genetic modification that disrupts the PDE9 gene. It is preferred that all somatic cells and germline cells of the genetically-modified non-human mammal contain the modification.
  • Preferred non-human mammals that are genetically- modified to contain a disrupted PDE9 gene include rodents, such as mice and rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, and ferrets.
  • a "genetically-modified animal cell" containing a disrupted PDE9 gene refers to an animal cell (preferably a mammalian cell), including a human cell, created by genetic engineering to contain a disrupted PDE9 gene, as well as daughter cells and cells differentiated from a genetically-modified parent ES or stem cell, that inherit the disrupted PDE9 gene. These cells may be genetically-modified in culture according to any standard method known in the art.
  • non-human mammalian cells may also be isolated from a genetically-modified, non-human mammal that contains a PDE9 gene disruption.
  • the animal cells of the invention may be obtained from primary cell or tissue preparations as well as culture-adapted, tumorigenic, or transformed cell lines.
  • These cells and cell lines are derived, for example, from endothelial cells, epithelial cells, islets, neurons and other neural tissue-derived cells, mesothelial cells, osteocytes, lymphocytes, chondrocytes, hematopoietic cells, immune cells, cells of the major glands or organs (e.g., testicle, liver, lung, heart, stomach, pancreas, kidney, and skin), muscle cells (including cells from skeletal muscle, smooth muscle, and cardiac muscle), exocrine or endocrine cells, fibroblasts, and embryonic and other totipotent or pluripotent stem cells (e.g., ES cells, ES-like cells, embryonic germline cells, and other stem cells, such as progenitor cells and tissue-derived stem cells).
  • endothelial cells e.g., endothelial cells, epithelial cells, islets, neurons and other neural tissue-derived cells, mesothelial cells, osteocytes,
  • the preferred genetically-modified cells are ES cells, more preferably, mouse or rat ES cells, and, most preferably, human ES cells, as well as cells differentiated from the genetically-modified ES cells.
  • a non-human mammal or a animal cell that is "genetically-modified” is heterozygous or homozygous for a modification that is introduced into the non-human mammal or animal cell, or into a progenitor non-human mammal or animal cell, by genetic engineering.
  • the standard methods of genetic engineering that are available for introducing the modification include homologous recombination, viral vector gene trapping, irradiation, chemical mutagenesis, and the transgenic expression of a nucleotide sequence encoding antisense RNA alone or in combination with catalytic ribozymes.
  • Preferred methods for genetic modification to disrupt a gene are those which modify an endogenous gene by inserting a "foreign nucleic acid sequence" into the gene locus, e.g., by homologous recombination or viral vector gene trapping.
  • a "foreign nucleic acid sequence” is an exogenous sequence that is non-naturally occurring in the gene.
  • This insertion of foreign DNA can occur within any region of the PDE9 gene, e.g., in an enhancer, promoter, regulator region, noncoding region, coding region, intron, or exon.
  • the most preferred method of genetic engineering for gene disruption is homologous recombination, in which the foreign nucleic acid sequence is inserted in a targeted manner either alone or in combination with a deletion of a portion of the endogenous gene sequence.
  • "Homozygosity" when referring to PDE9 gene disruption in a non-human mammal or an animal cell means a non-human mammal or animal cell having disruption of all alleles of the PDE9 gene.
  • the PDE9 gene sequences of each of these disrupted alleles need not be identical.
  • a non-human mammal may be homozygous for PDE9 disruption wherein one allele of PDE9 is disrupted as a result of deletion of one region of the gene sequence and the other allele is disrupted as a result of deletion of another region of the gene sequence.
  • ES cell or an "ES-like cell” means a pluripotent stem cell derived from an embryo, from a primordial germ cell, or from a teratocarcinoma, that is capable of indefinite self-renewal as well as differentiation into cell types that are representative of all three embryonic germ layers.
  • “Microarray” means an arrangement of distinct polynucleotides or polypeptides on a substrate, as more fully described herein.
  • Wild type when referring to a non-human mammal or an animal cell, means a non-human mammal or an animal cell, as the case may be, that does not comprise a disrupted PDE9 gene.
  • wild type in a comparison of a particular characteristic of a non-human mammal of this invention to that characteristic in a wild type mammal, the term wild type refers to non-human mammal that does not comprise a disrupted PDE9 gene (i.e., a mammal whose PDE9 gene is wild type).
  • a wild type non-human mammal is substantially similar, and, more preferably, substantially identical, to a non-human mammal of the invention, except for the non- disruption or disruption of the PDE9 gene, respectively.
  • wild type refers to an animal cell that does not comprise a disrupted PDE9 gene (i.e., a cell whose PDE9 gene is wild type).
  • a wild type animal cell is substantially similar, and, more preferably, substantially identical, to an animal cell of the invention, except for the non-disruption or disruption of the PDE9 gene, respectively.
  • nucleic acids and polypeptides are found in standard textbooks of molecular biology, protein science, and immunology (see, e.g., Davis et al., Basic Methods in Molecular Biology, Elsevir Sciences Publishing, Inc., New York, NY, 1986; Hames et al., Nucleic Acid Hybridization, IL Press, 1985; Molecular
  • FIG. 1 is a schematic of the targeting construct used to disrupt the PDE9 gene.
  • a portion of the genomic sequence of each homology arm is shown as SEQ ID NO: 1 and SEQ ID NO: 2.
  • Fig. 2 shows the cDNA sequence for a murine PDE9 (SEQ ID NO: 3).
  • the underlined sequence, base pairs 142-175 was deleted and replaced with LacZ-Neo.
  • FIG. 3 is a line graph detailing the body weight change in wild type (WT) and genetically-modified mice homozygous for disruption of the PDE9 gene (PDE9 knockout (KO) mice) during the course of a six week high fat diet.
  • Fig. 4A male
  • Fig. 4B female
  • SC subcutaneous
  • TBW total body weight
  • Fig. 5 is a bar graph comparing the body weight of female WT and PDE9 KO mice following a six week control chow diet.
  • Fig. 6A baseline
  • Fig. 6A baseline
  • 6B post-six week chow diet are bar graphs showing the mass of adipose depots in female WT and PDE9 KO mice. (Ing - inguinal subcutaneous; Gon - gonadal; RP - retroperitoneal; Mes - mesenteric) -13-
  • PDE9 inhibitors are known to those skilled in the art and may be determined by standard assays known to those in the art, such as in WO 03/037899 and WO 03/037432.
  • the PDE9 inhibitors used in the methods of the invention include those disclosed in WO 03/037899 and WO 03/037432, as well as in U.S. Provisional Appl. No. 60/466,639, filed April 30, 2003, incorporated hereinbefore by reference.
  • Compound A 3-isopropyl-5-[2-(2-morpholin-4-yl-ethoxy)-benzyl]-1 ,6-dihydro-pyrazolo[4,3- d] pyrimidin-7-one (hereinafter referred to as "Compound A"); 1- ⁇ [2-(3-isopropyl-7-oxo-6,7-dihydro-1 H-pyrazolo[4,3-d]pyrimidin-5-ylmethyl)- phenoxy]-acetyl ⁇ -pyrrolidine-2-carboxylic acid; 3-isopropyl-5-[2-(2-oxo-2-piperazin-1-yl-ethoxy)-benzyl]-1 ,6-dihydro- pyrazolo[4,3-d]pyrimidin-7-one trifluoro acetate; 3-isopropyl-5-[2-(2-morpholin-4-yl-2-oxo-ethoxy)-benzyl]-1 ,6-dihydro
  • Fig. 7 is a line graph detailing the time course for body weight gain in female ob/ob mice in Control, Compound A-treated (100 mg/kg/day), and Darglitazone- treated groups.
  • Fig. 8A is a bar graph showing the Compound A dose effect on body weight at Days 2 and 4 in female ob/ob mice.
  • Fig. 8B is a bar graph showing the Compound A dose effect on food consumption at Days 2 and 4 in female ob/ob mice.
  • Fig. 9 is a bar graph comparing the time course for food consumption between Control, Compound A-treated (100 mg/kg/day), and Darglitazone-treated female ob/ob mice.
  • Fig. 8A is a bar graph showing the Compound A dose effect on body weight at Days 2 and 4 in female ob/ob mice.
  • Fig. 8B is a bar graph showing the Compound A dose effect on food consumption at Days 2 and 4 in female ob/ob mice.
  • Fig. 10 is a line graph comparing plasma glucose in Control, Compound A- treated (100 mg/kg/day), and Darglitazone-treated female ob/ob mice.
  • Fig. 11 is a line graph showing plasma triglycerides in Control and Compound A-treated (50 and 100 mg/kg/day) female ob/ob mice at Days 1 , 2, and 4.
  • Fig. 12 is a bar graph comparing plasma fructosamine in Control, Compound A (100 mg/kg/day), and Darglitazone-treated female ob/ob mice at Day 16.
  • the present invention is directed to methods to decrease body weight and/or body fat in an animal, e.g., in the treatment of overweight or obese patients (e.g., humans or companion animals), or as a means to produce leaner meat in food stock animals (e.g., cattle, chickens, pigs), and methods to treat eating disorders (e.g., binge eating disorder and bulimia) in patients in need thereof by administering a PDE9 inhibitor.
  • the invention also features biological tools to further study PDE9 function, i.e., genetically-modified mice and animal cells having a PDE9 gene disruption.
  • a PDE9 inhibitor reduces weight gain in the ob/ob mouse model of obesity
  • PDE9 knockout mice are relatively resistant to developing increased body weight and increased adiposity subsequent to exposure to a high fat diet.
  • Both Examples demonstrate that causing a decrease in PDE9 activity is an effective method to reduce body weight and/or body fat, and can be used, e.g., to treat animal patients that are overweight, obese, or suffer from an eating disorder, and can be used in animal food stock species to produce leaner meat.
  • An agent identified as a PDE9 inhibitor is administered in a dose sufficient to reduce body weight or body fat, e.g., by reducing the mass of one or more adipose depots.
  • Such therapeutically effective amounts will be determined using routine optimization techniques that are dependent on, for example, the condition of the patient or animal, the route of administration, the formulation, the judgment of the practitioner, and other factors evident to those skilled in the art in light of this disclosure.
  • the PDE9 inhibitors suitable for use in accordance with the present invention can be administered alone but, in human therapy, will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the PDE9 suitable for use in accordance with the present invention or salts or solvates thereof can be administered orally, buccally or sublingually in the form of tablets, capsules (including soft gel capsules), multi- particulate, gels, films, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, dual-, controlled-release or pulsatile delivery applications.
  • Such compounds may also be administered via fast dispersing or fast dissolving dosages forms or in the form of a high energy dispersion or as coated particles.
  • Suitable pharmaceutical formulations may be in coated or un-coated form as desired.
  • Such solid pharmaceutical compositions for example, tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine and starch (preferably corn, potato or tapioca starch), disintegrants such as sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC), hydroxypropylcellulose (HPC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), sucrose, gelatin and acacia.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, gly
  • lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules or HPMC capsules.
  • Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the PDE9 inhibitor compounds may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • Modified release and pulsatile release dosage forms may contain excipients such as those detailed for immediate release dosage forms together with additional excipients that act as release rate modifiers, these being coated on and/or included in the body of the device.
  • Release rate modifiers include, but are not exclusively limited to, HPMC, HPMCAS, methyl cellulose, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer, ammonio methacrylate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer and mixtures thereof.
  • Modified release and pulsatile release dosage forms may contain one or a combination of release rate modifying excipients.
  • Release rate modifying excipients maybe present both within the dosage form, i.e., within the matrix, and/or on the dosage form, i.e., upon the surface or coating.
  • Fast dispersing or dissolving dosage formulations may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, gelatin, hydroxypropylmethyl cellulose, magnesium stearate, mannitol, methyl methacrylate, mint flavouring, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol.
  • FDDFs dispersing or dissolving as used herein to describe FDDFs are dependent upon the solubility of the drug substance used i.e., in cases where the drug substance is insoluble, a fast dispersing dosage form can be prepared, and, in cases where the drug substance is soluble, a fast dissolving dosage form can be prepared.
  • the PDE9 inhibitors suitable for use in accordance with the present invention can also be administered parenterally, for example, intracavernosally, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion or needle-free techniques.
  • a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from about 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
  • the daily dosage level of the PDE9 inhibitors for use in the present invention will usually be from 1 to 500 mg (in single or divided doses). A preferred dosage range is about 1 mg to about 100 mg. The dosage may by via single dose, divided daily dose, or multiple daily dose.
  • tablets or capsules of the PDE9 inhibitors suitable for use in accordance with the present invention may contain from 1 mg to 250 mg of active compound for administration singly or two or more at a time, as appropriate.
  • Preferred tablets or capsules will contain about 1 mg to about 50 mg of active compound for administration singly or two or more at a time, as appropriate.
  • the physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient.
  • PDE9 inhibitors suitable for use in accordance with the present invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray or nebuliser with the use of a suitable propellant, e.g.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g.
  • Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch. Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff' contains from 1 to 50 mg of a PDE9 inhibitor for delivery to the animal to be treated.
  • the overall daily dose with an aerosol will be in the range of from 1 to 50 mg which may be administered in a single dose or, more usually, in divided doses throughout the day.
  • the PDE9 inhibitors suitable for use in accordance with the present invention may also be formulated for delivery via an atomiser.
  • Formulations for atomiser devices may contain the following ingredients as solubilisers, emulsifiers or suspending agents: water, ethanol, glycerol, propylene glycol, low molecular weight polyethylene glycols, sodium chloride, fluorocarbons, polyethylene glycol ethers, sorbitan trioleate, oleic acid.
  • the PDE9 inhibitors suitable for use in accordance with the present invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder.
  • the PDE9 inhibitors suitable for use in accordance with the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. The PDE9 inhibitors may also be administered by the ocular route.
  • the compounds can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
  • the PDE9 inhibitors suitable for use in accordance with the present invention can be formulated as a suitable ointment containing the active ingredient or agent suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • Suitable lotion or cream suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the PDE9 inhibitors suitable for use in accordance with the present invention may also be used in combination with a cyclodextrin. Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules.
  • Formation of a drug-cyclodextrin complex may modify the solubility, dissolution rate, bioavailability and/or stability property of a drug molecule.
  • Drug-cyclodextrin complexes are generally useful for most dosage forms and administration routes.
  • the cyclodextrin may be used as an auxiliary additive, e.g. as a carrier, diluent or solubiliser.
  • Alpha-, beta- and gamma- cyclodextrins are some of the most commonly used and suitable examples are described in WO 91/11172, WO 94/02518 and WO 98/55148.
  • oral administration is the preferred route, being the most convenient.
  • the drug may be administered parenterally, sublingually, or buccally.
  • a PDE9 inhibitor is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
  • Such animals include companion animals who are overweight, obese, or at risk of being overweight or obese.
  • Other animals that may be treated according to the present invention are foodstock animals in order to obtain leaner meat than would be obtained absent treatment according to the present invention.
  • Therapeutic efficacy of such PDE9 inhibitors can be determined in light of this disclosure by standard therapeutic procedures in cell cultures or experimental animals, e.g., for determining the ED 50 (the dose therapeutically effective in 50% of the population).
  • 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 may vary, for example, depending upon the formulation and the route of administration.
  • 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 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the PDE9 inhibitors used in accordance with the present invention may also be used in conjunction with other pharmaceutical agents for the treatment of the diseases, conditions and/or disorders described herein. Therefore, methods of treatment that include administering PDE9 inhibitors in combination with other pharmaceutical agents are also provided.
  • Suitable pharmaceutical agents that may be used in combination with the compounds of the present invention include anti- obesity agents such as ⁇ 3 adrenergic receptor agonists, apolipoprotein-B secretion/microsomal triglyceride transfer protein (apo-B/MTP) inhibitors, peptide YY 3- 36 and analogs thereof, MCR-4 agonists, cholecystokinin-A (CCK-A) agonists, monoamine reuptake inhibitors (e.g., sibutramine), sympathomimetic agents, cannabinoid receptor antagonists (e.g., rimonabant (SR-141,716A)), dopamine agonists (e.g., bromocriptine), melanocyte-stimulating hormone receptor analogs, 5HT2c agonists, melanin concentrating hormone antagonists, leptin (the OB protein), leptin analogs, leptin receptor agonists, galanin antagonists, lipase inhibitors
  • anti-obesity agents including the preferred agents set forth hereinbelow, are well known, or will be readily apparent in light of the instant disclosure, to one of ordinary skill in the art.
  • anti-obesity agents selected from the group consisting of orlistat, sibutramine, bromocriptine, ephedrine, leptin, pseudoephedrine, and peptide YY 3-36 (including analogs thereof).
  • compounds of the present invention and combination therapies are administered in conjunction with exercise and a sensible diet.
  • anti-obesity agents for use in the combinations, pharmaceutical compositions, and methods of the invention can be prepared using methods known to one of ordinary skill in the art, for example, sibutramine can be prepared, e.g., as described in U.S. Pat. No. 4,929,629; bromocriptine can be prepared, e.g., as described in U.S. Pat. Nos. 3,752,814 and 3,752,888; orlistat can be prepared, e.g., as described in U.S. Pat. Nos. 5,274,143; 5,420,305; 5,540,917; and 5,643,874; and PYY 3-36 (including analogs) can be prepared, e.g., as described in U.S. Patent Appl.
  • treatment of a mammal with a therapeutically effective amount of a PDE9 inhibitor can include a single treatment or, preferably, can include a series of treatments.
  • the genetically-modified, non-human mammals and genetically-modified animal cells, including human cells, of the invention are heterozygous or homozygous for a modification that disrupts the PDE9 gene.
  • the animal cells may be derived by genetically engineering cells in culture, or, in the case of non-human mammalian cells, the cells may be isolated from genetically-modified, non-human mammals.
  • the PDE9 gene locus may be disrupted using techniques for genetic modification known in the art, including chemical mutagenesis (Rinchik, Trends in Genetics 7: 15-21 , 1991 , Russell, Environmental & Molecular Mutagenesis 23 (Suppl. 24): 23-29, 1994), irradiation (Russell, supra), transgenic expression of PDE9 gene antisense RNA, either alone or in combination with a catalytic RNA ribozyme sequence (Luyckx et al., Proc. Natl. Acad. Sci.
  • the disruption of the PDE9 gene by the insertion of a foreign nucleic acid sequence into the PDE9 gene locus.
  • the foreign sequence is inserted by homologous recombination or by the insertion of a viral vector.
  • the method of PDE9 gene disruption to create the genetically modified non-human mammals and animal cells of the invention is homologous recombination and includes a deletion of a portion of the endogenous PDE9 gene sequence.
  • the integration of the foreign sequence disrupts the PDE9 gene through one or more of the following mechanisms: by interfering with the PDE9 gene transcription or translation process (e.g., by interfering with promoter recognition, or by introducing a transcription termination site or a translational stop codon into the PDE9 gene); or by distorting the PDE9 gene coding sequence such that it no longer encodes a PDE9 polypeptide with normal function (e.g., by inserting a foreign coding sequence into the PDE9 gene coding sequence, by introducing a frameshift mutation or amino acid(s) substitution, or, in the case of a double crossover event, by deleting a portion of the PDE9 gene coding sequence that is required for expression of a functional PDE9 protein).
  • the foreign DNA sequence is introduced into the cell according to a standard method known in the art such as electroporation, calcium-phosphate precipitation, retroviral infection, microinjection, biolistics, liposome transfection, DEAE-dextran transfection, or transferrinfection , (see, e.g., Neumann et al., EMBO J. 1 : 841-845, 1982; Potter et al., Proc. Natl. Acad. Sci USA 81 : 7161-65, 1984; Chu et al., Nucleic Acids Res.
  • a standard method known in the art such as electroporation, calcium-phosphate precipitation, retroviral infection, microinjection, biolistics, liposome transfection, DEAE-dextran transfection, or transferrinfection , (see, e.g., Neumann et al., EMBO J. 1 : 841-845, 1982; Potter et al., Proc. Natl. Acad
  • the preferred method for introducing foreign DNA into a cell is electroporation.
  • Homologous Recombination targets the PDE9 gene for disruption by introducing a PDE9 gene targeting vector into a cell containing a PDE9 gene.
  • the ability of the vector to target the PDE9 gene for disruption stems from using a nucleotide sequence in the vector that is homologous, i.e., related, to the PDE9 gene.
  • This homology region facilitates hybridization between the vector and the endogenous sequence of the PDE9 gene.
  • the probability of a crossover event between the targeting vector and genomic sequences greatly increases. This crossover event results in the integration of the vector sequence into the PDE9 gene locus and the functional disruption of the PDE9 gene.
  • General principles regarding the construction of vectors used for targeting are reviewed in Bradley et al. (Biotechnol. 10: 534, 1992).
  • Two different types of vector can be used to insert DNA by homologous recombination: an insertion vector or a replacement vector.
  • An insertion vector is circular DNA which contains a region of PDE9 gene homology with a double stranded break.
  • the more preferred vector to create the genetically modified non-human mammals and animals cells of the invention by homologous recombination is a replacement vector, which is colinear rather than circular.
  • Replacement vector integration into the PDE9 gene requires a double crossover event, i.e., crossing over at two sites of hybridization between the targeting vector and the PDE9 gene.
  • This double crossover event results in the integration of a vector sequence that is sandwiched between the two sites of crossover into the PDE9 gene and the deletion of the corresponding endogenous PDE9 gene sequence that originally spanned between the two sites of crossover (see, e.g., Thomas and Capecchi et al., Cell 51 : 503-12, 1987; Mansour et al., Nature 336: 348-52, 1988; Mansour et al., Proc. Natl. Acad. Sci. USA 87: 7688-7692, 1990; and Mansour, GATA 7: 219- 227, 1990).
  • a region of homology in a targeting vector used to create the genetically modified non-human mammals and animal cells of the invention is generally at least 100 nucleotides in length. Most preferably, the homology region is at least 1- 5 kilobases (kb) in length. Although there is no demonstrated minimum length or minimum degree of relatedness required for a homology region, targeting efficiency for homologous recombination generally corresponds with the length and the degree of relatedness between the targeting vector and the PDE9 gene locus. In the case where a replacement vector is used, and a portion of the endogenous PDE9 gene is deleted upon homologous recombination, an additional consideration is the size of the deleted portion of the endogenous PDE9 gene.
  • cloning vectors may be used as vector backbones in the construction of the PDE9 gene targeting vectors of the present invention, including pBluescript-related plasmids (e.g., Bluescript KS+11 ), pQE70, pQE60, pQE-9, pBS, pD10, phagescript, phiX174, pBK Phagemid, pNH8A, pNH16a, pNH18Z, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 PWLNEO, pSV2CAT, pXT1 , pSG (Stratagene), pSVK3, PBPV, PMSG, and pSVL, pBR322 and pBR322-based vectors, pMB9, pBR325, pKH47, p
  • pBluescript-related plasmids e.g.,
  • vectors are available from a variety of commercial sources (e.g., Boehringer Mannheim Biochemicals, Indianapolis, IN; Qiagen, Valencia, CA; Stratagene, La Jolla, CA; Promega, Madison, Wl; and New England Biolabs, Beverly, MA).
  • any other vectors e.g. plasmids, viruses, or parts thereof, may be used as long as they are replicable and viable in the desired host.
  • the vector may also comprise sequences which enable it to replicate in the host whose genome is to be modified. The use of such a vector can expand the interaction period during which recombination can occur, increasing the efficiency of targeting (see Molecular Biology, ed. Ausubel et al, Unit 9.16, Fig. 9.16.1).
  • the specific host employed for propagating the targeting vectors of the present invention is not critical. Examples include E. coli K12 RR1 (Bolivar et al., Gene 2: 95, 1977), E co// K12 HB101 (ATCC No. 33694), E. coli MM21 (ATCC No. 336780), E. coli DH1 (ATCC No. 33849), E. coli strain DH5 ⁇ , and E. coli STBL2.
  • hosts such as C. cerevisiae or ⁇ . subtilis can be used. The above-mentioned hosts are available commercially (e.g., Stratagene, La Jolla, CA; and Life Technologies, Rockville, MD).
  • a PDE9 gene targeting construct is added to an above-described vector backbone.
  • the PDE9 gene targeting constructs of the invention have at least one PDE9 gene homology region.
  • a PDE9 genomic or cDNA sequence is used as a basis for producing PCR primers. These primers are used to amplify the desired region of the PDE9 sequence by high fidelity PCR amplification (Mattila et al., Nucleic Acids Res. 19: 4967, 1991; Eckert and Kunkel 1 : 17, 1991 ; and U.S. Pat. No. 4,683, 202).
  • the genomic sequence is obtained from a genomic clone library or from a preparation of genomic DNA, preferably from the animal species that is to be targeted for PDE9 gene disruption, a PDE9 cDNA sequence can be used in making a PDE9 targeting vector (e.g., GenBank® NM008804 (murine) or GenBank® NM002606 (human)).
  • a PDE9 targeting vector e.g., GenBank® NM008804 (murine) or GenBank® NM002606 (human)
  • the targeting constructs of the invention also include an exogenous nucleotide sequence encoding a positive marker protein.
  • the stable expression of a positive marker after vector integration confers an identifiable characteristic on the cell, ideally, without compromising cell viability.
  • the marker gene is positioned between two flanking homology regions so that it integrates into the PDE9 gene following the ' double crossover event in a manner such that the marker gene is positioned for expression after integration.
  • the positive marker protein is a selectable protein; the stable expression of such a protein in a cell confers a selectable phenotypic characteristic, i.e., the characteristic enhances the survival of the cell under otherwise lethal conditions.
  • the selectable condition one can isolate cells that stably express the positive selectable marker-encoding vector sequence from other cells that have not successfully integrated the vector sequence on the basis of viability.
  • positive selectable marker proteins examples include neo (G418 or kanomycin), hyg (hygromycin), hisD (histidinol), gpt (xanthine), ble (bleomycin), and hprt (hypoxanthine) (see, e.g., Capecchi and Thomas, U.S. Pat. No. 5,464,764, and Capecchi, Science 244: 1288-92, 1989).
  • positive markers that may also be used as an alternative to a selectable marker include reporter proteins such as ⁇ - galactosidase, firefly luciferase, or green fluorescent protein (see, e.g., Current Protocols in Cytometry, Unit 9.5, and Current Protocols in Molecular Biology, Unit 9.6, John Wiley & Sons, New York, NY, 2000).
  • reporter proteins such as ⁇ - galactosidase, firefly luciferase, or green fluorescent protein
  • PDE9 gene locus versus random, non-homologous integration of vector sequence into any chromosomal position. Therefore, when using a replacement vector for homologous recombination to make the genetically modified non-human mammals and animal cells of the invention, it is also preferred to include a nucleotide sequence encoding a negative selectable marker protein. Expression of a negative selectable marker causes a cell expressing the marker to lose viability when exposed to a certain agent (i.e., the marker protein becomes lethal to the cell under certain selectable conditions).
  • negative selectable markers examples include herpes simplex virus thymidine kinase (gancyclovir or 1 ,2-deoxy-2-fluoro-c/-d-arabinofuransyl-5-iodouracil), Hprt (6- thioguanine or 6-thioxanthine), and diphtheria toxin, ricin toxin, and cytosine deaminase (5-fluorocytosine).
  • the nucleotide sequence encoding the negative selectable marker is positioned outside of the two homology regions of the replacement vector.
  • cells will only integrate and stably express the negative selectable marker if integration occurs by random, non-homologous recombination; homologous recombination between the PDE9 gene and the two regions of homology in the targeting construct excludes the sequence encoding the negative selectable marker from integration.
  • cells that have integrated the targeting vector by random, non- homologous recombination lose viability.
  • a targeting construct used to make the genetically modified non-human mammals and animal cells of the invention because a series of positive and negative selection steps can be designed to more efficiently select only those cells that have undergone vector integration by homologous recombination, and, therefore, have a potentially disrupted PDE9 gene.
  • positive-negative selection schemes, selectable markers, and targeting constructs are described, for example, in U.S. Pat. No. 5,464,764, WO 94/06908, U.S. Pat. No. 5,859,312, and Valancius and Smithies, Mol. Cell. Biol. 11: 1402, 1991.
  • the targeting vector may be designed so that the marker coding sequence is operably linked to the endogenous PDE9 gene promoter upon vector integration. Expression of the marker is then driven by the PDE9 gene promoter in cells that normally express the PDE9 gene.
  • each marker in the targeting construct of the vector may contain its own promoter that drives expression independent of the PDE9 gene promoter. This latter scheme has the advantage of allowing for expression of markers in cells that do not typically express the PDE9 gene (Smith and Berg, Cold Spring Harbor Symp. Quant. Biol. 49: 171 , 1984; Sedivy and Sharp, Proc. Natl. Acad. Sci.
  • Exogenous promoters that can be used to drive marker gene expression include cell-specific or stage-specific promoters, constitutive promoters, and inducible or regulatable promoters.
  • Non-limiting examples of these promoters include the herpes simplex thymidine kinase promoter, cytomegalovirus (CMV) promoter/enhancer, SV40 promoters, PGK promoter, PMC1-neo, metallothionein promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, avian beta globin promoter, histone promoters (e.g., mouse histone H3-614), beta actin promoter, neuron-specific enolase, muscle actin promoter, and the cauliflower mosaic virus 35S promoter (see generally, Sambrook et al., Molecular Cloning, Vols.
  • primers or genomic probes that are specific for the desired vector integration event can be used in combination with polymerase chain reaction (PCR) or Southern blot analysis to identify the presence of the desired vector integration into the PDE9 gene locus (Erlich et al., Science 252: 1643-51 , 1991; Zimmer and Gruss, Nature 338: 150, 1989; Mouellic et al., Proc. Natl.
  • PCR polymerase chain reaction
  • Gene Trapping Another method available for inserting a foreign nucleic acid sequence into the PDE9 gene locus to disrupt the PDE9 gene, based on the present description, is gene trapping. This method takes advantage of the cellular machinery present in all mammalian cells that splices exons into mRNA to insert a gene trap vector coding sequence into a gene in a random fashion. Once inserted, the gene trap vector creates a mutation that may disrupt the trapped PDE9 gene.
  • Individual mutant cell lines containing a disrupted PDE9 gene are identified in a population of mutated cells using, for example, reverse transcription (RT) and PCR to identify a mutation in a PDE9 gene sequence.
  • RT reverse transcription
  • This process can be streamlined by pooling clones. For example, to find an individual clone containing a disrupted PDE9 gene, RT-PCR is performed using one primer anchored in the gene trap vector and the other primer located in the PDE9 gene sequence.
  • a positive RT-PCR result indicates that the vector sequence is encoded in the PDE9 gene transcript, indicating that the PDE9 gene has been disrupted by a gene trap integration event (see, e.g., Sands et al., WO 98/14614, U.S. Pat. No. 6,080,576).
  • Temporal, Spatial, and Inducible PDE9 Gene Disruptions In certain embodiments of the present invention, a functional disruption of the endogenous PDE9 gene occurs at specific developmental or cell cycle stages (temporal disruption) or in specific cell types (spatial disruption). In other embodiments, the PDE9 gene disruption is inducible when certain conditions are present.
  • a recombinase excision system such as a Cre-Lox system, may be used to activate or inactivate the PDE9 gene at a specific developmental stage, in a particular tissue or cell type, or under particular environmental conditions.
  • Cre-Lox technology methods utilizing Cre-Lox technology are carried out as described by Torres and Kuhn, Laboratory Protocols for Conditional Gene Targeting, Oxford University Press, 1997. Methodology similar to that described for the Cre-Lox system can also be employed utilizing the FLP-FRT system. Further guidance regarding the use of recombinase excision systems for conditionally disrupting genes by homologous recombination or viral insertion is provided, for example, in U.S. Pat. No. 5,626,159; U.S. Pat. No. 5,527,695; U.S. Pat. No. 5,434,066; WO 98/29533; U.S. Pat. No. 6,228,639; Orban et al., Proc. Nat.
  • More than one recombinase system can be used to genetically modify a non-human mammal or animal cell of the present invention.
  • a portion of the PDE9 gene coding region is replaced by a targeting construct comprising the PDE9 gene coding region flanked by loxP sites.
  • Non-human mammals and animal cells carrying this genetic modification contain a functional, loxP-flanked PDE9 gene.
  • the temporal, spatial, or inducible aspect of the PDE9 gene disruption is caused by the expression pattern of an additional transgene, a Cre recombinase transgene, that is expressed in the non-human mammal or animal cell under the control of the desired spatially-regulated, temporally-regulated, or inducible promoter, respectively.
  • a Cre recombinase targets the loxP sites for recombination. Therefore, when Cre expression is activated, the LoxP sites undergo recombination to excise the sandwiched PDE9 gene coding sequence, resulting in a functional disruption of the PDE9 gene (Rajewski et al., J. Clin. Invest.
  • a cell containing both a Cre recombinase transgene and loxP-flanked PDE9 gene can be generated through standard transgenic techniques or, in the case of genetically-modified, non-human mammals, by crossing genetically- modified, non-human mammals wherein one parent contains a loxP flanked PDE9 gene and the other contains a Cre recombinase transgene under the control of the desired promoter. Further guidance regarding the use of recombinase systems and specific promoters to temporally, spatially, or conditionally disrupt the PDE9 gene is found, for example, in Sauer, Meth. Enz.
  • This system involves genetically modifying a cell to introduce a Tet promoter into the endogenous PDE9 gene regulatory element and a transgene expressing a tetracycline-controllable repressor (TetR).
  • TetR tetracycline-controllable repressor
  • the administration of tetracycline activates the TetR which, in turn, inhibits PDE9 gene expression and, therefore, disrupts the PDE9 gene (St-Onge et al., Nucleic Acids Res. 24: 3875-77, 1996; U.S. Patent No. 5,922,927).
  • Genetically- modified animal cells of the invention include, but are not limited to, mammalian cells, including human cells, and avian cells. These cells may be derived from genetically engineering any animal cell line, such as culture-adapted, tumorigenic, or transformed cell lines, differentiated genetically-engineered ES cells, or they may be isolated from a genetically-modified, non-human mammal carrying the desired PDE9 genetic modification. The cells may be heterozygous or homozygous for the disrupted PDE9 gene.
  • the method uses a scheme in which PDE9+/- targeted clones that express a selectable drug resistance marker are selected against a very high drug concentration; this selection favors cells that express two copies of the sequence encoding the drug resistance marker and are, therefore, homozygous for the PDE9 gene disruption (Mortensen et al., Mol. Cell. Biol. 12: 2391-95, 1992).
  • genetically-modified animal cells can be obtained from genetically-modified PDE9-/- non-human mammals that are created by mating non-human mammals that are PDE9+/- in germline ' cells, as further discussed below.
  • the PDE9 gene locus can be confirmed as the site of modification by PCR analysis according to standard PCR or Southern blotting methods known in the art (see, e.g., U.S. Pat. No. 4,683,202; and Erlich et al., Science 252: 1643, 1991). Further verification of the functional disruption of the PDE9 gene may also be made if PDE9 gene messenger RNA (mRNA) levels and/or PDE9 polypeptide levels are reduced in cells that normally express the PDE9 gene. Measures of PDE9 gene mRNA levels may be obtained by using RT-PCR, Northern blot analysis, or in situ hybridization.
  • mRNA messenger RNA
  • the quantification of PDE9 polypeptide levels produced by the cells can be made, for example, by standard immunoassay methods known in the art.
  • immunoassays include, but are not limited to, competitive and non- competitive assay systems using techniques such as RIAs (radioimmunoassays), ELISAs (enzyme-linked immunosorbent assays), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using, for example, colloidal gold, enzymatic, or radioisotope labels), Western blots, 2-dimensional gel analysis, precipitation reactions, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays.
  • Preferred genetically-modified animal cells of the invention are embryonic stem (ES) cells and ES-like cells. These cells are derived from the preimplantation embryos and blastocysts of various species, such as mice (Evans et al., Nature 129:154-156, 1981 ; Martin, Proc. Natl. Acad. Sci., USA, 78: 7634-7638, 1981), pigs and sheep (Notanianni et al., J. Reprod. Fert. Suppl., 43: 255-260, 1991 ; Campbell et al., Nature 380: 64-68,1996) and primates, including humans (Thomson et al., U.S. Patent No.
  • a sample of ES cells can be cultured indefinitely as stem cells, allowed to differentiate into a wide variety of different cell types within a single sample, or directed to differentiate into a specific cell type, such as macrophage-like cells, neuronal cells, cardiomyocytes, chondrocytes, adipocytes, smooth muscle cells, endothelial cells, skeletal muscle cells, keratinocytes, and hematopoietic cells, such as eosinophils, mast cells, erythroid progenitor cells, or megakaryocytes.
  • Directed differentiation is accomplished by including specific growth factors or matrix components in the culture conditions, as further described, for example, in Keller et al., Curr. Opin.
  • exemplary murine ES cell lines include AB-1 (McMahon and Bradley, Cell 62:1073-85, 1990), E14 (Hooper et al., Nature 326: 292-95, 1987), D3 (Doetschman et al., J. Embryol. Exp. Morph. 87: 27-45, 1985), CCE (Robertson et al, Nature 323: 445-48, 1986), RW4 (Genome Systems, St. Louis, MO), and DBA/1 lacJ (Roach et al., Exp. Cell Res. 221 : 520-25, 1995); an exemplary human ES cell line is H1.1 cells (Zwaka and Thomson, Nature Biotech. 21 : 319-321 ,
  • Genetically-modified murine ES cells may be used to generate genetically- modified mice, according to published procedures (Robertson, 1987, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Ed. E. J. Robertson, Oxford: IRL Press, pp. 71-112, 1987; Zjilstra et al., Nature 342: 435- 438, 1989; and Schwartzberg et al., Science 246: 799-803, 1989). Following confirmation that the ES cells contain the desired functional disruption of the PDE9 gene, these ES cells are then injected into suitable blastocyst hosts for generation of chimeric mice according to methods known in the art (Capecchi, Trends Genet. 5: 70, 1989).
  • mice employed in the present invention are not critical.
  • blastocysts include those derived from C57BL6 mice, C57BL6 Albino mice, Swiss outbred mice, CFLP mice, and MFI mice.
  • ES cells may be sandwiched between tetraploid embryos in aggregation wells (Nagy et al., Proc. Natl. Acad. Sci. USA 90: 8424-8428, 1993).
  • the blastocysts or embryos containing the genetically-modified ES cells are then implanted in pseudopregnant female mice and allowed to develop in utero (Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory press, Cold Spring Harbor, NY 1988; and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed., IRL Press, Washington, D.C., 1987).
  • the offspring born to the foster mothers may be screened to identify those that are chimeric for the PDE9 gene disruption.
  • offspring contain some cells that are derived from the genetically-modified donor ES cell as well as other cells derived from the original blastocyst.
  • offspring may be screened initially for mosaic coat color, where a coat color selection strategy has been employed, to distinguish cells derived from the donor ES cell from the other cells of the blastocyst.
  • DNA from tail tissue of the offspring can be used to identify mice containing the genetically-modified cells. The mating of chimeric mice that contain the PDE9 gene disruption in germ line cells produces progeny that possess the PDE9 gene disruption in all germ line cells and somatic cells.
  • mice that are heterozygous for the PDE9 gene disruption can then be crossed to produce homozygotes (see, e.g., U.S. Pat. No. 5,557,032; and U.S. Pat. No. 5,532,158).
  • An alternative to the above-described ES cell technology for transferring a genetic modification from a cell to a whole animal is to use nuclear transfer.
  • This method can be employed to make other genetically-modified, non-human mammals besides mice, for example, sheep (McCreath et al., Nature 29: 1066-69, 2000; Campbell et al., Nature 389: 64-66, 1996; and Schnieke et al., Science 278: 2130-33, 1997) and calves (Cibelli et al., Science 280: 1256-58, 1998).
  • somatic cells e.g., fibroblasts
  • pluripotent stem cells e.g., ES-like cells
  • a promoterless marker be used in the vector such that vector integration into the PDE9 gene results in expression of the marker under the control of the PDE9 gene promoter (Sedivy and Dutriaux, T.I.G. 15: 88-90, 1999; McCreath et al., Nature 29: 1066-69, 2000). Nuclei from donor cells which have the appropriate PDE9 gene disruption are then transferred to fertilized or parthenogenetic oocytes that are enucleated (Campbell et al., Nature 380: 64, 1996; Wilmut et al., Nature 385: 810, 1997).
  • the present invention also encompasses the progeny of the genetically- modified, non-human mammals and genetically-modified animal cells. While the progeny are heterozygous or homozygous for the genetic modification that disrupts the PDE9 gene, they may not be genetically identical to the parent non- human mammals and animal cells due to mutations or environmental influences, besides that of the original genetic disruption of the PDE9 gene, that may occur in succeeding generations.
  • the cells from a non-human genetically modified animal can be isolated from tissue or organs using techniques known to those of skill in the art.
  • the genetically modified cells of the invention are immortalized.
  • cells can be immortalized by genetically engineering the telomerase gene, an oncogene, e.g., mos or v-src, or an apoptosis-inhibiting gene, e.g., bcl-2, into the cells.
  • cells can be immortalized by fusion with a hybridization partner utilizing techniques known to one of skill in the art.
  • “Humanized” Non-human Mammals and Animal Cells The genetically-modified non-human mammals and animal cells (non- human) of the invention containing a disrupted endogenous PDE9 gene can be further modified to express the human PDE9 sequence (referred to herein as "humanized”).
  • a preferred method for humanizing cells involves replacing the endogenous PDE9 sequence with nucleic acid sequence encoding the human PDE9 sequence (Jakobsson et al., Proc. Natl. Acad. Sci. USA 96: 7220-25, 1999) by homologous recombination.
  • the vectors are similar to those traditionally used as targeting vectors with respect to the 5' and 3' homology arms and positive/negative selection schemes.
  • the vectors also include sequence that, after recombination, either substitutes the human PDE9 coding sequence for the endogenous sequence, or effects base pair changes, exon substitutions, or codon substitutions that modify the endogenous sequence to encode the human PDE9.
  • the human sequence can be the full length human cDNA sequence with a polyA tail attached at the 3' end for proper processing or the whole genomic sequence (Shiao et al., Transgenic Res. 8: 295-302, 1999). Further guidance regarding these methods of genetically modifying cells and non-human mammals to replace expression of an endogenous gene with its human counterpart is found, for example, in Sullivan et al., J. Biol. Chem. 272: 17972-17980, 1997, Reaume et al., J. Biol. Chem. 271 : 23380-23388, 1996, and Scott et al., U.S. Pat. No. 5,777,194).
  • Another method for creating such "humanized” organisms is a two step process involving the disruption of the endogenous gene followed by the introduction of a transgene encoding the human sequence by pronuclear microinjection into the knock-out embryos.
  • Uses for the Genetically-Modified Non-human Mammals and Animal Cells PDE9 function and therapeutic relevance can be further elucidated by additional investigation into the phenotype of PDE9-/- non-human mammals and animals cells of the invention, as illustrated, for example, in the Examples hereinbelow.
  • the genetically-modified PDE9-/- non-human mammals and animal cells can be used to determine whether the PDE9 plays a role in causing or preventing symptoms or phenotypes to develop in certain models of disease, e.g., obesity, eating disorders, cardiovascular disorders, insulin resistance syndrome, hypertension, and/or type 2 diabetes. If a symptom or phenotype is different in a PDE9-/- non-human mammal or animal cell as compared to a wild type (PDE9+/+) or PDE9+/- non-human mammal or animal cell, then the PDE9 polypeptide plays a role in regulating functions associated with the symptom or phenotype.
  • diseases or phenotypes e.g., obesity, eating disorders, cardiovascular disorders, insulin resistance syndrome, hypertension, and/or type 2 diabetes.
  • Examples of testing that can be used to assess PDE9 function include comparing PDE9-/- mice to wild type mice in terms of body weight, body fat, blood pressure, glucose/insulin metabolism (e.g., glucose uptake in isolated tissues, alterations in the activity of glycogen metabolism enzymes, alterations in glycogen levels in liver or muscle, and/or alterations in body composition), and changes in the activity or phosphorylation state of components in the insulin signaling pathway.
  • glucose/insulin metabolism e.g., glucose uptake in isolated tissues, alterations in the activity of glycogen metabolism enzymes, alterations in glycogen levels in liver or muscle, and/or alterations in body composition
  • the genetically-modified PDE9-/- non-human mammals and animal cells of the invention are useful to characterize any other effects caused by the agent besides those known to result from the (ant)agonism of PDE9 (i.e., the non-human mammals and animal cells can be used as negative controls).
  • the administration of the agent causes an effect in a PDE9+/+ non-human mammal or animal cell that is not known to be associated with PDE9 polypeptide activity
  • the agent exerts this effect solely or primarily through modulation of PDE9 by administering the agent to a corresponding PDE9-/- non-human mammal or animal cell. If this effect is absent, or is significantly reduced, in the PDE9-/- non- human mammal or animal cell, then the effect is mediated, at least in part, by PDE9.
  • the PDE9-/- non-human mammal or animal cell exhibits the effect to a degree comparable to the PDE9+/+ or PDE9+/- non-human mammal or animal cell, then the effect is mediated by a pathway that does not involve PDE9 signaling.
  • an agent is suspected of possibly exerting an effect predominantly via a PDE9 pathway, then the PDE9-/- non-human mammals and animal cells are useful as negative controls to test this hypothesis. If the agent is indeed acting through PDE9, then the PDE9-/- non-human mammals and animal cells, upon administration of the agent, should not demonstrate the same effect observed in the PDE9+/+ non-human mammals or animal cells.
  • the genetically modified non-human mammals and animal cells of the invention can also be used to identify genes whose expression is differentially regulated in PDE9+/- or PDE9-/- non-human mammals or animal cells relative to their respective wild type control. Techniques known to those of skill in the art can be used to identify such genes based upon the present description. For example, DNA arrays can be used to identify genes whose expression is differentially regulated in PDE9+/- or PDE9-/- mice to compensate for a deficiency in PDE9 expression. DNA arrays are known to those of skill in the art (see, e.g., Aigner et al., Arthritis and Rheumatism 44: 2777-89, 2001 ; U.S. Pat. No.
  • a chemical coupling procedure and an ink jet device may be used to synthesize array elements on the surface of a substrate.
  • An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements. After hybridization, nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence.
  • Full-length cDNAs, expressed sequence tags (ESTs), or fragments thereof may comprise the elements of a microarray. Fragments suitable for hybridization may be selected using software well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide.
  • the cDNA is fixed to the slide using, e.g., ultra-violet cross-linking followed by thermal and chemical treatments and subsequent drying. Fluorescent probes are prepared and used for hybridization to the elements on the substrate.
  • the substrate is analyzed by procedures well known in the art, for example, by scanning and analyzing images of a microarray.
  • the genetically modified non-human mammals and animal cells of the invention can also be used to identify proteins whose expression profile or postranslational modification is altered in PDE9+/- or PDE9-/- non-human mammals or animal cells relative to their respective wild type control. Techniques known to those of skill in the art can be used to identify such proteins based upon the present description.
  • proteomic assays can be used to identify proteins whose expression profile or postranslational modification is altered in PDE9+/- or PDE9-/- mice to compensate for a deficiency in PDE9 expression.
  • Proteomic assays are known to those of skill in the art (see, e.g., Conrads et al., Biochem. Biophys. Res. Commun.
  • PDE9 Targeting Vector A targeting vector construct was designed according to the scheme shown in Fig. 1. The construct contained two arms homologous to the murine PDE9 genomic sequence: a 0.9 kb 5' homology arm and a 4.3 kb 3' homology arm. These arms sandwiched a LacZ-Neo construct. DNA containing the targeting construct was inserted into ES R1 cells by electroporation (Deng et al., Dev. Biol. 185: 42-54, 1997; Udy et al., Exp. Cell Res. 231 : 296-301 , 1997). Upon homologous recombination, base pairs 142-175 of the PDE9 cDNA coding sequence shown in Fig.
  • ES cells that were neomycin resistant were analyzed by Southern blot to confirm disruption of a PDE9 gene. These targeted ES cells were then used for generation of chimeric mice by injecting the cells into blastocysts and implanting the blastocysts into pseudopregnant female mice (Capecchi et al., Trends Genet. 5: 70, 1989, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J.
  • mice of each gender remained on D11 mouse chow and the remaining groups of each gender were switched to a diet composed of 58 kcal% fat (D12331 Rodent Diet, Research Diets, Inc., New Brunswick, NJ) for the duration of the 6 week study.
  • Body weight was determined on Day 0 and monitored weekly.
  • Adipose depot mass was analyzed on Day 0 and at the end of the study, as further described below.
  • plasma glucose was determined via retro-orbital blood samples. 25 ⁇ L of blood was added to 100 ⁇ L of 0.025 percent heparinized-saline in microtubes (Denville Scientific, Inc., Metuchen, NJ).
  • the tubes were spun at the highest setting in a Beckman Microfuge 12 for 2 minutes. Plasma was collected for plasma glucose and triglyceride determination, as further described below. During the course of the study, body weight and food consumption were assessed, and blood samples were taken at approximately 8 am for plasma glucose and triglyceride measures, as further described below. On the morning of the last day of the study, blood samples were taken via retro-orbital sinus for plasma glucose and triglyceride determination. The mice were then sacrificed and about one milliliter of blood was collected in Microtainer® plasma separator tubes with lithium heparin (Becton-Dickinson, Inc., Franklin Lakes, NJ). The tubes were spun in a Beckman Microfuge 12 at the maximum setting for five minutes.
  • Plasma cGMP was measured using the BioTrakTM enzyme- immunoassay system (Amersham, Piscataway, NJ). Plasma insulin was assessed via a similar technique using the Mercodia ELISA Insulin kit supplied by ALPCO (Uppsala, Sweden). All assays were conducted according to each manufacturer's instructions. Quantification of adipose depot mass was done five days prior to the end of the study. To assess the adipose depot mass, 360° radioscopic images of the mice were obtained using a commercially available micro computed tomography (CT) system (MicroCAT ® , ImTek Inc., Oak Ridge, TN) with a high-resolution CCD/phosphor screen detector.
  • CT micro computed tomography
  • the scanner consisted of a cylindrical diameter/long field view of 36mm/36mm with a spatial resolution of less than 50 ⁇ M.
  • the X-ray source was biased at 40 KeV with the anode current set to 0.4 mA.
  • Anesthetized mice were placed on a radiotransparent mouse bed in an anatomically correct supine position, caudal end closest to the micro CT with the rostral end held in place against an anesthesia delivery tube.
  • An initial radiographic image was acquired at 90° to the plane of the mouse bed to allow correct positioning of the mouse by centering the scan acquisition area at the level of the iliac crest of each mouse. Once correct alignment was assured, each animal was scanned.
  • Each scan consisted of 196 individual projections with an exposure time of 250 ⁇ s/projection; total image acquisition time was approximately 12 minutes at 145 ⁇ M resolution.
  • Image reconstruction whereby the 196 projections acquired in the micro CT scan of the mouse were manipulated to produce two-dimensional cross sectional images of the mouse, was performed using the MicroCAT ® Reconstruction, Visualization, and Analysis Software (ImTek Inc., Oak Ridge, TN) (Paulus et al., Neoplasia 2: 62-70, 2000). Two sets of reconstructed images per scan were generated for each mouse for the determination of individual fat depot mass. The first set of six reconstructed images provided a montage for the analysis of inguinal and epididymal adipose tissue depot mass.
  • the second reconstruction set consisted of nine slices, determined by both intervertebral and midvertebral landmarks, and was used to determine retroperitoneal and mesenteric adipose tissue depot mass.
  • reconstructed bitmap images were converted to TIFF images.
  • the TIFF images were subsequently analyzed and fat depot mass determined using Scion Image for Windows® (Scion Corporation, Frederick MD). Demarcation lines separating individual fat depots were placed using the paintbrush tool (pixel size #3) and total pixel counts of each adipose region determined by the Scion Image software.
  • An upper and lower pixel intensity threshold was chosen, in this study, a look-up-table (LUT) of between 115-187 was determined to be optimal for capturing the adipose depot. Average pixel number between each slice was calculated (slice n +slice n+1 )/2).
  • the first factor corrects for specific gravity of glyceryl trioleate, representative of the density of the primary storage form of lipid in adipose tissue, i.e., triglyceride.
  • the second factor is the volume per pixel and the third factor converts the resulting mass into mg units.
  • mice also demonstrated a 6% decrease in body length.
  • the male and female KO mice also demonstrated decreased fat mass in various adipose depots (Fig. 4).
  • male KO mice significant decreases were seen in the retroperitoneal and mesenteric adipose depots; in female KO mice, significant decreases were seen in the inguinal, gonadal, and retroperitoneal depots.
  • female mice fed a standard chow diet no differences in body weight were observed between KO and wild type mice (Fig.
  • mice Female ob/ob mice obtained from Jackson Laboratories (Bar Harbor, ME) were used at 6 to 10 weeks of age. Mice were housed five per cage and allowed free access to water and, initially, to D11 mouse chow. Following a one week acclimation period, mice were switched to a powdered diet (Mouse Breeder/Auto-JL K20 mouse chow, PMI Feeds, Inc., St. Louis, MO) for three days and allowed to adapt to the diet prior to the start of the PDE9 inhibitor dosing period.
  • a powdered diet (Mouse Breeder/Auto-JL K20 mouse chow, PMI Feeds, Inc., St. Louis, MO) for three days and allowed to adapt to the diet prior to the start of the PDE9 inhibitor dosing period.
  • the PDE9 inhibitor compound (Compound A) was administered in powdered mouse chow that was custom ground (Research Diets, Inc., New Brunswick, NJ) as a compound/chow admixture; compounds were mixed with the chow to achieve consumption of the specified doses ranging from 1-200 mg/kg/day.
  • a group consuming darglitazone (1 mg/kg/day) was also included as a positive control.
  • Mice were randomly assigned to groups of ten with five mice per cage. Body weight was determined on Day 0 and weekly thereafter. On Day 1 , retro- orbital blood samples were obtained and plasma glucose was determined as previously described.
  • Fig. 7 shows a reduced body weight gain in ob/ob mice fed 100 mg/kg/day of the PDE9 inhibitor Compound A as compared to the mice fed either a compound-free control diet or a darglitazone-treated diet.
  • Compound A elicited a dose-dependent effect following 2 and 4 days of treatment, both in terms of reducing the normal body weight gain (Fig. 8A) and also in terms of reducing food intake (Fig. 8B).
  • the PDE9 effect on food intake could be transient, given that no effect on food intake was observed in the later stages of the study (Fig. 9) with the intermediate dose of 100 mg/kg/day.
  • the intermediate dose of 100 mg/kg/day of Compound A also resulted in decreased glucose, triglycerides and fructosamine.
  • Representative results are shown in Fig. 10, Fig. 11, and Fig. 12, respectively.
  • Both Examples demonstrate that causing a decrease in PDE9 activity is an effective method to reduce body weight and/or body fat, and can be used, e.g., to treat animal patients that are overweight, obese, or suffer from an eating disorder, and can be used in animal food stock species to produce leaner meat.

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Abstract

L'invention concerne des méthodes de diminution du poids du corps ou de la graisse contenue dans le corps d'un animal, par exemple lors du traitement de patients souffrant de surcharge pondérale ou d'obésité (que ce soient des êtres humains ou des animaux), ou afin de produire de la viande plus maigre chez des animaux d'élevage (par exemple du bétail, des poulets ou des cochons), ainsi que des méthodes de traitement de troubles de l'alimentation (tels que la frénésie alimentaire ou la boulimie) chez des patients qui en ont besoin, par administration d'un inhibiteur de la PDE 9. L'invention concerne également des outils biologiques qui permettent d'approfondir l'étude de la fonction de la PDE 9, c'est-à-dire des souris et des cellules animales génétiquement modifiées ayant une disruption génétique de la PDE 9.
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US9434733B2 (en) 2012-01-26 2016-09-06 H. Lundbeck A/S PDE9 inhibitors with imidazo triazinone backbone
SI3865484T1 (sl) 2015-07-07 2024-05-31 H. Lundbeck A/S Zaviralec pde9 z imidazo pirazinonsko hrbtenico za zdravljenje perifernih bolezni
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