MXPA06004887A - Phosphodiesterase 9 inhibition as treatment for obesity-related conditions - Google Patents

Abstract

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.

Description

Published: - wilh intemational search repon For two-leler codes and olher abbrevialions. Refer to the "Guid-ance Notes on Codes and Abbreviations" appearing to the begin-ning ofeach regular issue of the PCT Gazette.

INHIBITION OF PHOSPHODIESTERASE 9 AS A TREATMENT FOR STATES RELATED TO OBESITY FIELD OF THE INVENTION The present invention provides methods for decreasing body weight and / or body fat in the treatment, for example, of overweight or obese patients, and to methods for treating eating disorders (eg, binge eating and bulimia), by administering an inhibitor of phosphodiesterase 9 (PDE9). The present invention also deals with genetically modified mammalian cells and genetically modified mice, with an alteration of the PDE9 gene.

BACKGROUND OF THE INVENTION Individuals diagnosed as obese or overweight are at increased risk of developing other health conditions such as coronary heart disease, stroke, hypertension, type 2 diabetes mellitus, dyslipidemia, sleep apnea, osteoarthritis, gallbladder disease, depression, and some forms of cancer (for example, endometrium, breast, prostate, and colon). The negative consequences of obesity for health make it the second leading cause of death that can be avoided in the United States, and it has a significant economic and psychosocial effect on society (see, for example, McGinnis and Foege, JAMA 270: pages 2207 to 2212, 1993). Obesity has become a major problem of concern for public health due to its increasing frequency, and now it is recognized as a chronic disease that requires treatment to reduce the associated health risks. Although weight loss itself is an important result of treatment, one of the main objectives of controlling obesity is to improve cardiovascular and metabolic values to reduce the morbidity and mortality related to obesity. It has been shown that 5 to 10% loss of body weight can substantially improve metabolic values, such as blood glucose, blood pressure and lipid concentrations. Therefore, it is considered that an intentional reduction of 5 to 10% of body weight can reduce morbidity and mortality. Phosphodiesterases (PDE) of cyclic nucleotides catalyze the hydrolysis of cyclic nucleotides, such as second messengers cAMP (3 ', 5'-cyclic adenosine monophosphate) and cGMP (3', 5'-cyclic guanine monophosphate). Therefore, PDEs play a central regulatory role in a wide variety of signal transduction trajectories (Beavo, Physioi, Rev. 75: pages 725 to 748, 1995). For example, PDEs mediate the procedures involved in vision (McLaughlin et al., Nat. Genet 4: pages 130 to 134, 1993), olfaction (Yan et al., Proc. Nati. Acad. Sci. USA 92: pages 9677 to 9681, 1995), platelet aggregation (Dickinson et al., Biochem. J. 323: pages 371 to 377, 1997), aldosterone synthesis (MacFariand et al., J. Biol. Chem. 266: pages 136 to 142, 1991), insulin secretion (Zhao et al., J. Clin. Invest. 102: 869 to 873, 1998), T cell activation (Li et al., Science 283: pages 848 to 851, 1999). and smooth muscle relaxation (Boolell et al., Int.J. Impot. Res. 8: pages 47 to 52.1996; Ballard et al., J. Urol. 159: pages 2164-271, 1998). The PDEs form a superfamily of enzymes that are subdivided into 11 major gene families (Beavo, Physioi, Rev. 75: pages 725 to 748, 1995, Beavo et al., Mol.Pharmacol, 46: pages 399 to 405, 1994; Soderiing et al., Proc. Nati, Acad. Sci. USA 95: pages 8991 to 8996, 1998, Fisher et al., Biochem. Biophys. Res. Commun. 246: pages 570-57, 1998; Hayashi et al., Biochem. Biophys. Res. Commun. 250: pages 751 to 756, 1998; Soderiing et al., J. Biol. Chem. 273: pages 15553 to 15558, 1998; Fisher et al., J. Biol. Chem. 273: pages 15559 to 15564, 1998; Soderiing et al., Proc. Nati Acad. Sci. E.U.A. 96: pages 7071 to 7076, 1999; and Fawcett et al., Proc. Nati Acad. Sci. E.U.A. 97: pages 3702 to 3707, 2000). Each family of PDE genes encodes a phosphodiesterase that is functionally differentiated by enzymatic characteristics and unique pharmacological profiles. In addition, each family has different patterns of tissue, cellular and subcellular expression (Beavo et al., Mol.Pharmacol. 46: pages 399 to 405, 1994, Soderiing et al., Proc. Nati. Acad. Sci. USA 95: pages 8991 to 8996, 1998, Fisher et al., Biochem. Biophys. Res. Commun. 246: pages 570 to 577, 1998; Hayashi et al., Biochem. Biophys. Res.

Commun. 250: pages 751 to 756, 1998; Soderiing et al., J. Biol. Chem. 273: pages 15553 to 15558, 1998; Fisher et al., J. Biol. Chem. 273: pages 15559 to 15564, 1998; Soderiing et al., Proc. Nati Acad. Sci. E.U.A. 96: pages 7071 to 7076, 1999; Fawcett et al., Proc. Nati Acad. Sci. E.U.A. 97: pages 3702 to 3707, 2000; Boolell et al., Int. J. Impot. Res. 8: pages 47 to 52, 1996; Ballard et al., J. Urol. 159: pages 2164 to 2171, 1998; Houslay, Semin. Cell Dev. Biol. 9: pages 161 to 167, 1998; and Torphy et al., Pulm. Pharmacol. Ther. 12: pages 131 to 135, 1999). Therefore, by administering a compound that selectively regulates the activity of a family or subfamily of PDE enzymes, the transduction pathways of cAMP and / or cGMP signals can be regulated in a cell-or tissue-specific manner. Fisher et al. (J. Biol. Chem. 273: pages 15559 to 15564, 1998) identified the PDE9 enzyme as a new member of the PDE enzyme family that selectively hydrolyzes cGMP against cAMP. PDE9 is present in a variety of human tissues, including testes, brain, small intestine, skeletal muscle, heart, lung, thymus and spleen. It has been described that PDE9 inhibitors are useful for treating cardiovascular disorders (WO 03/037899), and insulin resistance syndrome, hypertension, and / or type 2 diabetes (WO 03/037432).

BRIEF DESCRIPTION OF THE INVENTION In a first aspect, the present invention is directed to a method of treating an animal to reduce body fat, which comprises administering to an animal in need thereof a therapeutically effective amount of a PDE9 inhibitor. Preferably, the animal is a human or companion animal (eg, dog, cat, horse) and is overweight, more preferably, the animal is obese. In another preferred embodiment, the animal is an animal for feeding (eg, chicken, cattle, pig) and said treatment is provided to produce lean meat. In another preferred embodiment, the PDE9 inhibitor is a selective inhibitor of PDE9 or the PDE9 inhibitor is administered orally. In a second aspect, the present invention is directed to a method for treating an animal of a feeding disorder, comprising administering to an animal in need thereof a therapeutically effective amount of a PDE9 inhibitor. Preferably, the eating disorder is a binge eating or bulimia disorder, the PDE9 inhibitor is a selective inhibitor of PDE9, or the PDE9 inhibitor is administered orally. In a third aspect, the present invention is directed to a method for treating an animal of metabolic syndrome, comprising administering to an animal in need, a therapeutically effective amount of a PDE9 inhibitor. Preferably, the PDE9 inhibitor is a selective inhibitor of PDE9, or the PDE9 inhibitor is administered orally. The present invention also deals with a genetically modified mouse, wherein the mouse is homozygous for the alteration of the PDE9 gene, and where the mouse, after a high-fat diet of six weeks, has lower body weight or lower fat mass in an adipose deposit, compared to a wild mouse after a six-week high-fat diet. In a preferred embodiment, the mouse expresses an exogenous reporter gene controlled by the regulatory sequences of the PDE9 gene, or the mouse exhibits no PDE9 activity that can be detected. In a related aspect, the present invention provides a cultured genetically modified murine cell obtained from the mouse described above. In another related aspect, the present 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 locallzation construct of the PDE9, which, after recombination with the PDE9 gene, alters the PDE9 gene; (b) selecting a mouse ES cell whose genome comprises an alteration of the PDE9 gene; (c) introducing an ES cell selected in step (b) into a blastocyst or mouse morulae; (d) transplanting the blastocyte or morulae of step (c) to a pseudopregnant mouse; (e) developing the blastocyte or morulae transferred to the end to produce a chimeric mouse; and (f) pairing sexually mature chimeric mice with heterozygous mice for the alteration of PDE9, to obtain a mouse homozygous for the alteration of the PDE9 gene; where the mouse, after a high-fat diet of six weeks, has lower body weight or lower fat mass in an adipose deposit, compared with a wild mouse after a six-week high-fat diet. The present invention also concerns a genetically modified mammalian cell, wherein the cell is homozygous for the alteration of the PDE9 gene and wherein the cell or a cell of the progeny obtained from the cell does not have activity that is it can detect PDE9 polypeptide, wherein the cell or cell of the progeny would present PDE9 polypeptide activity if the homozygous alteration is absent. In a preferred embodiment, the cell is an embryonic stem cell (ES), more preferably, the cell is a murine ES cell or a human ES cell. In another aspect, the present invention provides an isolated nucleic acid molecule comprising a PDE11 gene localization construct, in which, upon recombination with the PDE9 gene, the construct alters the PDE9 gene. Those skilled in the art will fully understand the terms used in the present disclosure and the appended claims to describe the present invention. However, unless otherwise provided herein, the following terms are as described immediately below.

By "PDE9 inhibitor" is meant 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, for example, PDE9 antisense nucleic acids or interfering RNA (RNAi), amino acids, peptides, carbohydrates, small molecules (organic or inorganic), or any other a compound or composition that decreases the activity of a PDE9 polypeptide by efficiently reducing the amount of PDE9 present in a cell, or by decreasing the enzymatic activity of the PDE9 polypeptide. Compounds which are PDE9 inhibitors include all solvates, hydrates, pharmaceutically acceptable salts, tautomers, stereoisomers and prodrugs of the compounds. Preferably, a small PDE9 inhibitor molecule used in the present invention has an Cl50 less than 10 μM, more preferably, less than 1 μM, even more preferably, less than 0.1 μM. Any PDE9 inhibitor used in the present invention is preferably also selective against some or all other PDEs, preferably, against PDE1A, PDE1B, PDE1C, PDE2, PDE3A, PDE3B, PDE4A, PDE4B, PDE4C, PDE4D, PDE5, PDE6, PDE7A, PDE7B, PDE8A, PDE8B, PDE10, and / or PDE11. By a "selective" PDE9 inhibitor is meant an agent that inhibits PDE activity with an Cl50 at least 10 times smaller, preferably at least 100 times lower, than Cl50 to inhibit one or more of the other PDEs. Preferably, said agents are combined with a pharmaceutically acceptable carrier or excipient for administration. An antisense oligonucleotide directed to the PDE9 gene or mRNA is prepared to inhibit its expression, according to standard techniques (see, eg, Agrawal et al., Methods in Molecular Biology; Protocols for Oligonucleotides and Analogs, Vol. 20, 1993). ). Likewise, an RNA molecule that functions to reduce the production of the PDE9 enzyme in a cell can be prepared according to standard techniques known to those skilled in the art (see, for example, Hannon, Nature 418: pages 244 a 251, 2002; Shi, Trends in Genetics 19: pages 9 to 12, 2003; Shuey et al., Drug Discovery Today 7: pages 1040 to 1046, 2002). Examples of PDE9 inhibitors are provided herein and in WO 03/037899, WO 03/037432, and in the provisional patent application of E.U.A. No. 60 / 466,639, filed on April 30, 2003, incorporated herein by reference. "Lower PDE9 activity" means a manipulated decrease in the polypeptide activity of the PDE9 enzyme as a result of the alteration or genetic manipulation of the function of the PDE9 gene, which results in a reduction of the level of functional PDE9 polypeptide in a cell, or as a result of administering a pharmacological agent that inhibits the activity of PDE9. The phrase "pharmaceutically acceptable" indicates that the carrier, carrier, diluent, excipient (s), and / or designated salt, is generally chemically and / or physically compatible with the other ingredients comprising the formulation, and is physiologically compatible with its receiver.

The term "prodrug" refers to a compound that is a precursor of the drug which, after administration, releases the drug in vivo by a chemical or physiological method (eg, after being brought to a physiological pH or by enzymatic activity) . A discussion of the synthesis and use of prodrugs Higuchi and Stella, Prodrugs as Novel Delivery Systems, vol. 14 of the ACS Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987. The terms "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. The states of "overweight" and the most serious of "obese", in an adult person 18 years of age or older, constitute having a body weight greater than the ideal (more specifically, greater than the ideal body fat) and in general are defined by the body mass index (BMI), which correlates with total body fat and the relative risk of premature death or disability due to illness as a result of being overweight or obese. The health risks increase with the increase of excess body fat. The BMI is calculated by weight in kilograms divided by height in meters squared (kg / m2), or alternatively, by weight in pounds, multiplied by 703, divided by height in square inches (Ibs x 703 / in2). "Overweight" typically constitutes a BMI between 25.0 and 29.9. "Obesity" is typically defined as a BMI of 30 O greater (see, for example, 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: USA Department of Health and Human Services, NIH publication No. 98-4083, 1998). In very muscular individuals, the correlation between BMI, body fat, and disease risk is weaker than in other individuals. Therefore, the assessment of whether such more muscular individuals are, in fact, overweight or obese, can be made more accurately by another measurement such as a direct measurement of total body fat or evaluation of waist-to-hip ratio . By a "high-fat diet", when administered to a genetically modified or wild-type mouse, is meant a diet composed of at least 45% in kcal of fat, and preferably, at least 58% of fat. Among the sample diets, the Surwit diet is included (Surwit et al., Metabolism 47: pages 1345 to 1359; Surwit et al., Metabolism 47: pages 1089 to 1096, 1998; Surwit et al., J. Biol. Chem. 271: pages 9437 to 9440, 1996; and Surwit et al., Metabolism 44: 645 to 651, 1995), Rodent Diet D12451 (45% of fat kcal, Research Diets, Inc., New Brunswick, NJ), and Rodent Diet D12331 (58% of fat kcal, Research Diets, Inc.). "Metabolic syndrome", as defined herein, and according to the Adult Treatment Panel III (ATP III, National Institutes of Health: Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III), Executive Summary, Bethesda, MD, National Institutes of Health, National Heart, Lung and Blood Institute, 2001 (NIH Pub. No. 01-3670)), occurs when a person has three or more of the following criteria: 1. Abdominal obesity: waist circumference > 102 cm in men and > 88 cm in women; 2. Hypertriglyceridemia: > 150 mg / dl (1695 mmol / l); 3. Low HDL cholesterol: < 40 mg / dl (1.036 mmol / l) in men and < 50 mg / dl (1,295 mmol / l) in women; 4. High blood pressure: > 130/85 mm Hg; 5. Fasting high glucose: > 110 mg / dL (> 6.1 mmol / L); or according to the criteria of the World Health Organization (Alberti and Zimmet, Diabet.Med.15: pages 539 to 553, 1998), when a person has diabetes, impaired glucose tolerance, impaired fasting glucose, or Insulin resistance plus two or more of the following abnormalities: 1. High blood pressure: > 160/90 mm Hg; 2. Hyperlipidemia: concentration of triglycerides > 150 mg / dL (1695 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; 3. Central obesity: waist-to-hip ratio > 0.90 for men and > 0.85 in women and / or BMI > 30 kg / m2; 4. Microalbuminuria: index of urinary excretion of albumin > 0 μg / min or a ratio of albumin to creatinine > 20 mg / kg.

By "therapeutically effective" it is understood that it results in a decrease in body fat. An "altered PDE9 gene" refers to a gene of PDE9 that is genetically modified in such a way that the cellular activity of the PDE9 polypeptide encoded by the altered gene is less, or preferably, or has been eliminated in cells that normally express a wild of the PDE9 gene. When the genetic modification efficiently eliminates all wild copies of the PDE9 gene in a cell (for example, the non-human mammalian or genetically modified animal cell is homozygous for the alteration of the PDE9 gene or the only wild copy of the gene for PDE9 originally present which is now altered), the genetic modification results in a reduction in PDE9 polypeptide activity compared to a control cell expressing the wild-type PDE9 gene. This reduction in PDE9 polypeptide activity is the result of expression of the PDE9 minor gene (ie, the levels of PDE9 mRNA are actually reduced, resulting in lower PDE9 polypeptide levels) and / or because the PDE9 gene altered encodes a mutated polypeptide with altered, eg reduced, function compared to a wild-type PDE9 polypeptide. Preferably, the activity of the PDE9 polypeptide in the non-human mammal or genetically modified animal cell is reduced to 50% or less of the wild-type levels, more preferably, to 25% or less, and even more preferably, to 10% or less of wild type levels. More preferably, alteration of the homozygous PDE9 gene results in PDE9 activity that can not be detected in cells of a type demonstrating wild-type PDE9 activity. A "non-human, genetically modified mammal" containing an altered PDE9 gene, refers to a non-human mammal genetically engineered to contain an altered PDE9 gene, as well as a progeny of said non-human mammal that inherits the gene of PDE9 altered. A genetically modified non-human mammal can be produced, for example, by creating a blastocyst or embryo carrying the desired genetic modification and then implanting the blastocyst or embryo in a host mother for development in the uterus. The blastocyst or genetically modified embryo can be elaborated, in the case of mice, by implanting a genetically modified embryonic stem cell (ES) in a mouse blastocyst or adding ES cells to tetraploid embryos. Alternatively, different species of genetically modified embryos can be obtained by nuclear transfer. In the case of nuclear transfer, the donor cell is a somatic cell or a pluripotent stem cell, and is genetically engineered to contain the desired genetic modification that alters the PDE9 gene. Then, the nucleus of this cell is transferred to a fertilized or parthenogenetic oocyte that is enucleated; the resulting embryo was reconstituted and developed into a blastocyst. Then, a genetically modified blastocyst produced by any of the above methods is implanted in a host mother according to standard methods well known to those skilled in the art. A "non-human, genetically modified mammal" includes all the progeny of non-human mammals created by the methods described above, with the proviso that the progeny inherit at least one copy of the genetic modification that alters 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 an altered 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 an altered PDE9 gene refers to an animal cell (preferably a mammalian cell), including a human cell, genetically engineered to contain an altered PDE9 gene, as well as daughter cells and differentiated cells from a genetically modified parental ES cell or cell, which inherit the altered PDE9 gene. These cells can be genetically modified in culture according to any standard method known in the art. As an alternative for genetically modifying cells in culture, non-human mammalian cells can also be isolated from a genetically modified non-human mammal that contains an alteration of the PDE9 gene. The animal cells of the present invention can be obtained from primary cells or tissue preparations, as well as from cell lines adapted to culture, tumorogenic or transformed. These cells and cell lines are obtained, for example, from endothelial cells, epithelial cells, islets, neurons and other cells obtained from neural tissue, mesothelial cells, osteocytes, lymphocytes, chondrocytes, hematopoietic cells, immune cells, cells of the glands or organs. major (eg, testicles, liver, lung, heart, stomach, pancreas, kidney and skin), muscle cells (including skeletal muscle cells, smooth muscle, and cardiac muscle), exocrine or endocrine cells, fibroblasts, and embryonic stem cells and other totipotent or pluripotent (e.g., ES cells, ES-type cells, embryonic germline cells, and other stem cells, such as progenitor cells and stem cells obtained from tissues). Preferred genetically modified cells are ES cells, more preferably, mouse or rat ES cells, more preferably, human ES cells, as well as cells differentiated from genetically modified ES cells. A non-human mammal or an 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 non-human mammal or progenitor animal cell, by genetic engineering. Standard methods of genetic engineering that are available to introduce the modification include homologous recombination, viral vector capture, irradiation, chemical mutagenesis, and transgene expression of a nucleotide sequence encoding the antisense RNA alone or in combination with catalytic ribozymes. Preferred methods for genetic modification to alter a gene are those that modify an endogenous gene by inserting a "foreign nucleic acid sequence" into the locus of the gene, for example, by homologous recombination or capture of a gene with a viral vector. A "foreign nucleic acid sequence" is an exogenous sequence that is not found naturally in the gene. This insertion of foreign DNA can occur within any region of the PDE9 gene, for example, in an enhancer, promoter, regulatory, non-coding 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, alone or in combination with a deletion of a part of the endogenous gene sequence. "Homocigosidad", when it refers to the alteration of the gene of PDE9 in a non-human mammal or animal cell means a non-human mammal or animal cell that has alteration of all alleles of the PDE9 gene. However, it is not necessary that the sequences of the PDE9 gene of each of these altered alleles be identical. For example, a non-human mammal can be homozygous for the PDE9 alteration in which one allele of PDE9 is altered as a result of the deletion of one region of the gene sequence and the other allele is altered as a result of the deletion of another region. of the gene sequence.

"ES cell" or an "ES cell" means a pluripotent stem cell obtained from an embryo, from a primordial germ cell, or from a teratocarcinoma, which has the capacity of indefinite self-renewal as well as of differentiation into cell types that are representative of the three embryonic germ layers. "Micromatrix" means an arrangement of different polynucleotides or polypeptides on a substrate, as described more fully herein. "Wild", when referring to a non-human mammal or an animal cell, means a non-human animal or an animal cell, as the case may be, that does not comprise an altered PDE9 gene. For example, in a comparison of a particular ccteristic of a non-human mammal of the present invention with this ccteristic in a wild mammal, the wild-type term refers to a non-human mammal that does not comprise an altered PDE9 gene (i.e. mammal whose PDE9 gene is wild).

Preferably, a wild nonhuman mammal is substantially similar, and more preferably, substantially identical, to a non-human mammal of the present invention, except for the non-alteration or alteration of the PDE9, respectively. Likewise, for example, in a comparison of a particular ccteristic of an animal cell of the present invention with this ccteristic in a wild animal cell, the wild-type term refers to an animal cell that does not comprise an altered PDE9 gene (i.e. a cell whose PDE9 gene is wild). Preferably, a wild animal cell is substantially similar, and more preferably, substantially identical, to an animal cell of the present invention, except for the non-alteration or alteration of the PDE9 gene, respectively. Other features and advantages of the present invention will become even more apparent from the following detailed description and from the Claims. Although the present invention is described in connection with specific embodiments, it will be understood that other changes and modifications that may be practiced are also part of the present invention and are also within the scope of the appended claims. It is intended that this application cover any equivalents, variants, uses or adaptations of the present invention that follows, in general, the principles of the present invention, including deviations from the present disclosure that are within known or customary practice. in the matter, and that can be determined without undue experimentation. Additional guidance is found with respect to the preparation and use of nucleic acids and polypeptides in standard textbooks of molecular biology, protein science and immunology (see, eg, 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 Cloning, Sambrook et al., Current Protocols in Molecular Biology, Eds. Ausubel et al., John Wiley and Sons, 2001, Current Protocols in Human Genetics, Eds. Dracopoli et al., John Wiley and Sons, 1994, Current Protocols in Protein Science, Eds. John E. Coligan et al., John Wiley and Sons, 2002, and Current Protocols. in Immunology, Eds. John E. Coligan et al., John Wiley and Sons, 1994). All publications, including published patent applications and issued Patents, mentioned in this report, are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic of the location construct used to alter the PDE9 gene. The 5 'and 3' homology arms complementary to the PDE9 genomic sequence, 0.9 kb and 4.3 kb in length respectively, flank a LacZ-Neo cassette. A part of the genomic sequence of each homology arm is shown as SEQ ID NO: 1 and SEQ ID NO: 2. Figure 2 shows the cDNA sequence for a murine PDE9 (SEQ ID NO: 3). After homologous recombination with the localization construct, the underlined sequence, base pairs 142-175, were deleted and replaced by LacZ-Neo. Figure 3 is a line graph detailing the change in body weight in wild (WT) and genetically modified mice homozygous for the PDE9 gene alteration (mouse that does not express (knockout, KO) PDE9) during the course of a diet high in fat for six weeks.

Figure 4A- (male) and Figure 4B (female) are bar graphs showing the mass of various adipose deposits in WT and KO PDE9 mice, after a six-week high-fat diet. SC - subcutaneous; PCT-total body weight. Figure 5 is a bar graph comparing the body weight of WT and KO PDE9 female mice after a six week control diet. Figure 6A, (baseline) and Figure 6B (after a six-week diet) are bar graphs showing the mass of adipose deposits in WT and KO PDE9 female mice (subcutaneous inguinal gonadotropic; - retroperitoneal, mes - mesenteric). Figure 7 is a line graph detailing the time course for body weight gain in female ob / ob mice in Control groups, treated with Compound A (100 mg / kg / day), and treated with Darglitazone. Figure 8A is a bar graph showing the effect of the dose of Compound A on body weight on Days 2 and 4 in female ob / ob mice. Figure 8B is a graph showing the effect of compound A dose on feed consumption on Days 2 and 4 in female ob / ob mice. Figure 9 is a bar graph that compares the time course for food consumption between control Ob / ob female mice, treated with compound A (100 mg / kg / day), and treated with Darglitazone.

Figure 10 is a line graph comparing plasma glucose in Control female ob / ob mice, treated with Compound A (100 mg / kg / day), and treated with Darglitazone. Figure 11 is a line graph showing the triglycerides in the plasma in Control female ob / ob mice and treated with compound A (50 and 100 mg / kg / day), on days 1, 2 and 4. Figure 12, is a bar graph comparing fructosamine in plasma in control Ob / ob female mice, treated with compound A (100 mg / kg / day), and treated with Darglitazone, on day 16.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to methods for decreasing body weight and / or body fat in an animal, for example, in the treatment of overweight or obese patients (e.g., humans or companion animals), or as a means to producing lean meat in animals for food (eg, livestock, chickens, pigs) and methods of treating eating disorders (eg, binge eating and bulimia) in patients in need thereof, by administration of a PDE9 inhibitor. The present invention also deals with biological tools to study in more detail the function of PDE9, ie, mice and genetically modified animal cells that have an alteration of the PDE9 gene. As described in the Examples herein, administration of a PDE9 inhibitor reduces weight gain in mouse ob / ob obesity models, and mice that do not express PDE9 are relatively resistant to developing higher body weight and greater adiposity after exposure to a high-fat diet. Both Examples demonstrate that producing a decrease in the activity of PDE9 is an effective method for reducing body weight and / or body fat, and can be used, for example, to treat animal patients who are overweight, obese or suffer from a disorder of food, and can be used in animal species for food to produce lean meat.

Exemplary PDE9 inhibitors Any PDE9 inhibitor can be used in the present invention. PDE9 inhibitors are known to those skilled in the art and can be determined by standard assays known to those skilled in the art, such as in WO 03/037899 and WO 03/037432. The PDE9 inhibitors used in the methods of the present invention include those described in WO 03/037899 and WO 03/037432, as well as in the provisional application of E.U.A. No. 60 / 466,639, filed on April 30, 2003, hereinafter incorporated by reference. The compounds described as inhibitors of PDE9 in the provisional patent application of E.U.A. cited above, include: 3-isopropyl-5- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one (in thereafter referred to as "Compound A"); acid 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 trifluoroacetate; 3-isopropyl-5- [2- (2-morpholin-4-yl-2-oxo-ethoxy) -benzyl] -1,6-dihydropyridolo [4,3-d] pyrimidin-7-one; 3-isopropyl-5- [2- (2-oxo-2-pyrrolidin-1-yl-ethoxy) -benzyl] -1,6-dihydro-prazolo [4,3-d] pyrimidin-7-one; N, N-diethyl-2- [2- (3-isopropyl-7-oxo-6,7-dihydro-1 H -pyrazolo [4,3-d] pyrimidin-5-ylmethyl) -phenoxy] -acetamide; 1- methyl acid ester. { [2- (3-isopropyl-7-oxo-6,7-dihydro-1 H -pyrazolo [4,3-d] pyrimidin-5-ylmethyl) -phenoxy] -acetyl} -pyrrolidine-2-carboxylic acid; 4- tertiary butyl ester. { [2- (3-isopropyl-7-oxo-6,7-dihydro-1 H -pyrazolo [4,3-d] pyrimidin-5-ylmethyl) -phenoxy] -acetyl} -piperazine-1-carboxylic acid; N- (2-dimethylamino-ethyl) -2- [2- (3-isopropyl-7-oxo-6,7-dihydro-1 H -pyrazolo [4,3-d] pyrimidin-5-ylmethyl) - phenoxy] -acetamide; [2- (3-isopropyl-7-oxo-6,7-dihydro-1 H -pyrazolo [4,3-djpyrimidin-5-ylmethyl] -phenoxy] -acetic acid; 3-isopropyl-5- [2- (5-chloro-2-morpholin-4-yl-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one; 3-isopropyl-5- [2- (2-pyrrolidin-1-yl-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one; 3-isopropyI-5- [2- (2-morpholin-4-yl-ethoxy) -cyclohexylmethyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidn-7-one; 5- [5-fiuoro-2- (2-morpholin-4-yl-ethoxy) -benzyl] -3-isopropyl-1,6-dihydropyridolo [4,3-d] pyrimidin-7-one; 3-cyclopentyl-5- [5-fluoro-2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one; 9- (1, 2-dimethyl-propyl) -2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one; 2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -9- (tetrahydro-furan-3-yl) -1,9-dihydro-purin-6-one; 5- [2- (2-diethylamino-ethoxy) -benzyl] -3-isopropyl-1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one; 3-cyclopentyl-5- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one; 9- (1 (R), 2-dimethyl-propyl) - [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one; 9- (2-methyl-butyl) -2- [2- (2-morfoin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one; 9-Cyclopentyl-2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one; - [2- (2-morfoHn-4-yl-ethoxy) -benzyl] -3-pyridin-3-yl-1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one; 9- (1, 2-dimethyl-propyl) -2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one; 9 -sopropyl-2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one; 2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -9- (tetrahydro-furan-2-ylmethyl) -1,9-dihydro-purin-6-one; 9- (1-Isopropyl-2-methyl-propyl) -2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one; 9- (1-ethyl-propyl) -2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one; and N- [2- (3-isopropyl-7-oxo-6,7-dihydro-1 H -pyrazolo [4,3-d] pyrimidin-5-ylmethyl) -cyclohexyl] - 2-pyrroidin-1-yl-acetamide. Those skilled in the art will understand that all stereoisomers, tautomers, solvates, hydrates, prodrugs, and pharmaceutically acceptable salts of the compounds listed above are also included.

Therapeutic methods An agent identified as a PDE9 inhibitor is administered in a dose sufficient to reduce body weight or body fat., for example, reducing the mass of one or more adipose deposits. Said therapeutically effective amounts will be determined using routine optimization techniques that depend, for example, on the condition of the patient or animal, the route of administration, the formulation, the physician's judgment, and other factors evident to those skilled in the art to the patient. light of this description. PDE9 inhibitors suitable for use in accordance with the present invention can be administered alone, although in human therapy, they will generally be administered in admixture with a suitable excipient, diluent or carrier selected with respect to the intended route of administration and practice. standard pharmaceutical For example, PDE9 suitable for use in accordance with the present invention or its salts or solvates can be administered orally, buccally, or sublingually in the form of tablets, capsules (including soft gel capsules), multiparticles, gels, films , ovules, elixirs, solutions or suspensions, which may contain flavor agents or dyes, for applications of immediate, delayed, modified, sustained, double or pulsed administration. Such compounds can also be administered by rapid dispersion or rapid dissolution dosage forms either in the form of a high energy dispersion or as coated particles. Suitable pharmaceutical formulations may be in coated or uncoated form as desired. Said solid pharmaceutical compositions, for example, the tablets, may contain excipients such as microcrystalline cellulose, lactose, sodium eitrate, calcium carbonate, calcium phosphate dibasic, glycine and starch (preferably corn starch, potato or tapioca), disintegrants such as starch. sodium glycolate, croscarmellose sodium and certain complex silicates, and granulation binders, such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxypropylmethyl cellulose acetate succinate (HPMCAS), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type can also be used 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. For aqueous suspensions and / or elixirs, the PDE9 inhibitor compounds can be combined with different sweetening or flavoring agents, coloring matter or colorants, with emulsifying and / or suspending agents, and with diluents such as water, ethanol, propylene glycol and glycerin , and their combinations. The modified release and pulsed release dosage forms may contain excipients, such as those detailed for immediate release dosage forms with additional excipients that act as release index modifiers, these being coated and / or included in the body of the delivery. device. The release index modifiers include, but are not limited to, HPMC, HPMCAS, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, polyethylene oxide, xanthan gum, Carbomer, ammonium methacrylate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, methacrylic acid copolymer and mixtures thereof. The modified release and pulsed release dosage forms may contain one or a combination of excipients that modify the rate of release. The excipients that modify the rate of release may be present both within the dosage form, ie, within the matrix, and / or on the dosage form, that is, on the surface or coating. The dispersion or rapid dissolution dosage formulations (FDDFs) may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethylcellulose, gelatin, hydroxypropylmethylcellulose, magnesium stearate, mannitol , methyl methacrylate, mint flavor, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol. The terms "dispersion" or "solution" are used herein to describe FDDFs that depend on the solubility of the drug substance used, that is, in cases where the drug substance is insoluble, a form of rapid dispersion dosage, and, in cases where the drug substance is soluble, a rapid dissolution dosage form can be prepared. PDE9 inhibitors suitable for use in accordance with the present invention can also be administered parenterally, for example, intracavernously, intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasthemally, intracranially, intramuscularly, or subcutaneously, or they can be administered by infusion or needleless techniques. For such parenteral administration they are best used in the form of a sterile aqueous solution, which may contain other substances, for example, enough salts or glucose to produce the solution isotonic with the blood. The aqueous solutions should be suitably regulated (preferably at a pH of about 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is easily accomplished by standard pharmaceutical techniques known to those skilled in the art. For oral and parenteral administration to human patients, the daily dosage level of the PDE9 inhibitors for use in the present invention will normally be from 1 to 500 mg (in a divided dose or doses). A preferred dosage range is from about 1 mg to about 100 mg. The dosage may be by a single dose, or divided daily dose, or multiple daily doses. Alternatively, continuous dosing, such as for example, by a controlled release dosage form wherein said continuous dosage form can be administered on a daily basis or in which said continuous dosing can be performed by a slow release formulation , whose doses are for more than one day at a time. Thus, for example, tablets or capsules of PDE9 inhibitors suitable for use in accordance with the present invention, may contain from 1 mg to 250 mg of the active compound to be administered one by one, or two or more at a time, according to be suitable Preferred tablets or capsules will contain from about 1 mg to about 50 mg of the active compound to be administered one by one, or two or more at a time, as appropriate. In any case, the doctor will determine the actual dosage that will be most suitable for any individual patient, and will vary with the age, weight and response of the particular patient. Of course, there may be individual cases in which greater or lesser dosage intervals are deserved, and these are within the scope of the present invention. PDE9 inhibitors suitable for use in accordance with the present invention may 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 container pressurized, pump, spray or nebulizer, using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane, such as 1, 1, 1, 2-tetrafluoroethane (HFA 134A® or 1, 1, 1, 2,3,3,3-heptafluoropropane (HFA 227EA®), carbon dioxide or other suitable gas In the case of a pressurized aerosol, the dosing unit can be determined by providing a valve for supplying a measured quantity The pressurized container, pump, sprayer or nebulizer, can contain a solution or suspension of the active compound, for example, using a mixture of ethanol and the propellant as solvent, which additionally contains a lubricant, for example, sorbitan trioleate Capsules and cartridges (made, for example, of gelatin) for use in an inhaler or insufflator can be formulated to contain a powder mixture of a compound of the present invention and a suitable powder base such as lactose or starch The dry powder or aerosol formulations are preferably arranged in such a way that each measured or "puff" dose contains from 1 to 50 mg of a PDE9 inhibitor for supply the animal to be treated. The overall daily dose with an aerosol will be within the range of 1 to 50 mg, which can be administered in a single dose or, more usually, in divided doses throughout the day. PDE9 inhibitors suitable for use in accordance with the present invention can also be formulated for delivery with an atomizer. The formulations for atomizing devices may contain the following ingredients as solubilizing agents, emulsifiers or suspending agents: water, ethanol, glycerol, propylene glycol, low molecular weight polyethylene glycols, sodium chloride, fluorocarbons, polyethylene glycol ethers, sorbitan trioleate, oleic acid.

Alternatively, PDE9 inhibitors suitable for use in accordance with the present invention can be administered in the form of a suppository or pessary, or they can be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or powdery dust. PDE9 inhibitors suitable for use in accordance with the present invention can also be administered dermally or transdermally, for example, by using a skin patch. They can also be administered through the pulmonary or rectal routes. PDE9 inhibitors can also be administered via the eye. For ophthalmic use, the compounds can be formulated as micronized suspensions in isotonic, pH adjusted and sterile saline, or preferably, as solutions in saline, isotonic, pH adjusted and sterile, optionally combined with a preservative, such as sodium chloride. benzalum Alternatively, they can be formulated into an ointment such as petrolatum. For topical application to the skin, PDE9 inhibitors suitable for use in accordance with the present invention can be formulated in the form of a suitable ointment containing the suspended or dissolved active ingredient or agent, for example, in a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene-polyoxypropylene compound, emulsifying wax and water. Alternatively, they may be formulated in the form of a suitable lotion or cream, suspended or dissolved, for example, in a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, wax esters of cetyl, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. PDE9 inhibitors suitable for use in accordance with the present invention can also be used in combination with a cyclodextrin. It is known that cyclodextrins form inclusion and non-inclusion complexes with the drug molecules. The formation of a drug-cyclodextrin complex can modify the property of solubility, dissolution index, bioavailability and / or stability of a drug molecule. Drug-cyclodextrin complexes are generally useful for most dosage forms and routes of administration. As an alternative to the direct complex formation with the drug, the cyclodextrin can be used as an auxiliary additive, for example, as a carrier, diluent or solubilizer. 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. In general, in humans, oral administration is the preferred route, being the most convenient. In cases where the recipient suffers from a swallowing disorder or drug absorption deficiency after oral administration, the drug can be administered parenterally, sublingually or buccally. For veterinary use, a PDE9 inhibitor is administered in the form of a suitably acceptable formulation according to normal veterinary practice, and the veterinarian will determine the dosage regimen and route of administration that will be most suitable for a particular animal. These animals include companion animals that are overweight, obese, or at risk of being overweight or obese. Other animals that can be treated according to the present invention are animals for feeding, in order to obtain meat more lean than would be obtained in the absence of treatment according to the present invention. The therapeutic efficiency of said PDE9 inhibitors can be determined in light of this description, by standard therapeutic procedures in cell cultures or with experimental animals, for example, to determine the ED50 (the therapeutically effective dose in 50% of the population ). The data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. Dosage may vary, for example, depending on the formulation and the route of administration. For any PDE9 inhibitor used in the method of the present invention, the therapeutically effective dose can be calculated initially from the cell culture assays. A dose can be formulated in animal models to achieve a concentration range circulating in the plasma, which includes the Cl50 determined in cell culture. This information can be used to determine more precisely the useful doses in humans. The levels in the plasma can 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 to treat the diseases, conditions and / or disorders described herein. Therefore, treatment methods are also provided which include administering PDE9 inhibitors in combination with other pharmaceutical agents. Suitable pharmaceutical agents that can be used in combination with the compounds of the present invention include antiobesity agents, such as β3 adrenergic receptor agonists, inhibitors of apolipoprotein-B secretion / microsomal triglyceride transfer protein (apo-B / MTP), peptide YY3-36 and its analogs, MCR-4 agonists, cholecystokinin-A (CCK-A) agonists, monoamine reuptake inhibitors (eg, sibutramine), sympathomimetic agents, cannabinoid receptor antagonists ( for example, rimonabant (SR-141.716A)), dopamine agonists (eg, bromocriptine), melanocyte-stimulating hormone receptor analogues, 5HT2c agonists, melamine hormone-binding antagonists, leptin (OB protein) ), leptin analogs, leptin receptor agonists, galanin antagonists, lipase inhibitors (eg, tetrahydrolipstatin, ie, orlistat), abnormal agents exicos (eg, a bombesin agonist), neuropeptide Y antagonists, thyromimetic agents, dehydroepiandrosterone or its analogs, glucocorticoid receptor agonists or antagonists, orexin receptor antagonists, glucagon-like peptide-1 receptor agonists, ciliary neurotrophic factors (eg, Axokine® available from Regeneran Pharmaceuticals, Inc., Tarrytown, NY and Procter & amp;; Gamble Company, Cincinnati, OH), human agouti-related proteins (AGRP), ghrelin receptor antagonists, histamine 3 receptor antagonists or inverse agonists, neuromedine U receptor agonists, llß-hydroxy-steroid-dehydrogenase inhibitors- 1 and the like. Other anti-obesity agents, including the preferred agents set forth below, are well known, or will be readily apparent in the light of the present disclosure, to one skilled in the art. Especially preferred are anti-obesity agents selected from the group consisting of orlistat, sibutramine, bromocriptine, ephedrine, leptin, pseudoephedrine, and peptide YY3-36 (including their analogues). Preferably, the compounds of the present invention and combination therapies are administered together with exercise and a sensible diet. Representative anti-obesity agents for use in the combinations, pharmaceutical compositions, and methods of the present invention, can be prepared using methods known to those skilled in the art, for example, sibutramine can be prepared, for example, as described in the US Patent No. 4,929,629; Bromocriptine can be prepared, for example, as described in US Patents. No. 3,752,814 and 3,752,888; Orlistat can be prepared, for example, as described in the patents of E.U.A. No. 5,274,143; 5,420,305; 5,540,917; and 5,643,874; and PYY3-36 (including analogs) can be prepared, for example, as described in the publication of the patent application of E.U.A. No. 2002/0141985 and WO 03/027637. The person skilled in the art will understand that some factors may influence the dosage and time required to efficiently treat a mammal including, but not limited to, the severity of the disease or disorder, previous treatments, general health and / or age of the mammal and other diseases present. In addition, treatment of a mammal with a therapeutically effective amount of a PDE9 inhibitor can include a single treatment, or preferentially, can include a series of treatments.

Non-human Mammals and Genetically Modified Cells Non-human mammals and genetically modified animal cells, including human, genetically modified cells, of the present invention are heterozygous or homozygous for a modification that alters the PDE9 gene. The animal cells can be obtained from genetically engineered cells in culture, or in the case of non-human mammalian cells, the cells can be isolated from non-human mammals genetically modified.

Alteration of the PDE9 gene In order to create the non-human mammals and genetically modified mammalian cells of the present invention, the PDE9 gene locus can be altered using the techniques for genetic modification known in the art, including chemical mutagenesis ( Rinchik, Trends in Genetics 7: pages 15 to 21, 1991, Russell, Environmental &Molecular Mutagenesis 23 (Suppl 24): pages 23 to 29, 1994), irradiation (Russell, see above), transgenic expression of the antisense RNA of the PDE9 gene, alone or combined with a catalytic ribozyme RNA sequence (Luyckx et al., Proc. Nati, Acad. Sci. 96: pages 12174 to 12179, 1999; Sokol et al., Transgenic Research 5: pages 363 a 371, 1996, Efrat et al., Proc. Nati, Acad. Sci. USA 91: pages 2051 to 2055, 1994, Larsson et al., Nucleic Acids Research 22: pages 2242 to 2248,1994), and as discussed with more detail below, the alteration of the PDE9 gene by ins of a foreign nucleic acid sequence at the locus of the PDE9 gene. Preferably, the foreign sequence is inserted by homologous recombination or by insertion of a viral vector. More preferably, the method of altering the PDE9 gene to create the non-human mammals and genetically modified animal cells of the present invention, is the homologous recombination and includes a deletion of a part of the endogenous PDE9 gene sequence. The integration of the foreign sequence alters the PDE9 gene through one or more of the following mechanisms: interfering with the process of transcription or translation of the PDE9 gene (for example, interfering with the recognition of the promoter, or introducing a site of transcription termination or stop codon translation in the PDE9 gene); or by distorting the coding sequence of the PDE9 gene such that it no longer encodes a PDE9 polypeptide with normal function (e.g., by inserting a foreign coding sequence into the coding sequence of the PDE9 gene, introducing a frame shift mutation). of reading or substitution of amino acid (s), or in the case of a double cross-linking event, by removing a part of the coding sequence of the PDE9 gene that is necessary for the expression of a functional PDE9 protein). To insert a foreign sequence into a locus of the PDE9 gene in the genome of a cell to create the non-human mammals and genetically modified animal cells of the present invention based on the present disclosure, the foreign DNA sequence is introduced into the cell of according to a standard method known in the art, such as electroporation, calcium phosphate precipitation, retroviral infection, microinjection, biolistics, liposome transfection, transfection of DEAE-dextran, or transfer of infection (see, for example, Neumann et al. , EMBO J. 1: pages 841 to 845, 1982, Potter et al., Proc. Nati, Acad. Sci USA 81: pages 7161 to 7165, 1984; Chu et al., Nucleic Acids Res. 15: pages 1311 to 1326 , 1987, Thomas and Capecchi, Cell 51: pages 503 to 512, 1987, Baum et al., Biotechniques 17: pages 1058 to 1062, 1994; Biewenga et al., J. Neuroscience Methods 71: pages 67 to 75, 1997; Zhang et al., Biotechniques 15: pages 868 to 872, 1993; Ray and Gage, Biotechniques 13: pages 598 to 603, 1992; Lo, Mol. Cell. Biol. 3: pages 1803 1814, 1983; Nickoloff et al., Mol. Biotech 10: pages 93 to 101, 1998; Linney et al., Dev. Biol. (Orlando) 213: pages 207 to 216, 1999; Zimmer and Gruss, Nature 338: pages 150 to 153, 1989; and Robertson et al., Nature 323: pages 445 to 448, 1986). The preferred method for introducing foreign DNA into a cell is electroporation.

Homologous Recombination The homologous recombination method localizes the PDE9 gene for alteration by introducing a PDE9 gene localization vector into a cell containing a PDE9 gene. The ability of the vector to locate the PDE9 gene for alteration is the result of the use of a nucleotide sequence in the vector that is homologous, ie, related to the PDE9 gene. This region of homology facilitates hybridization between the vector and the endogenous sequence of the PDE9 gene. After hybridization, the probability of a cross-linking event between the localization vector and genomic sequences increases greatly. This cross-linking event results in the integration of the vector sequence into the locus of the PDE9 gene and the functional alteration of the PDE9 gene. The general principles regarding the construction of the vectors used to locate are reviewed by Bradley et al. (Biotechnol. : page 534, 1992). Two different types of vectors can be used to insert DNA by homologous recombination: an insertion vector or a substitution vector. An insertion vector is circular DNA that contains a region of homology with the PDE9 gene with a break in the double strand. After hybridization between the region of homology and the endogenous PDE9 gene, a single event of crosslinking at the break of the double strand results in the insertion of the entire vector sequence into the endogenous gene at the crosslinking site. The most preferred vector for creating non-human mammals and genetically modified animal cells of the present invention by homologous recombination is a substitution vector, which is colinear instead of circular. The integration of the substitution vector in the PDE9 gene requires a double cross-linking event, i.e., cross-linking at two hybridization sites between the localization vector and the PDE9 gene. This double cross-linking event results in the integration of a vector sequence that is sandwiched between the two cross-linking sites in the PDE9 gene and the deletion of the corresponding endogenous PDE9 gene sequence that originally extended between the two sites of cross-linking (see, for example, Thomas and Capecchi et al., Cell 51: pages 503 to 512, 1987; Mansour et al., Nature 336: pages 348 to 352, 1988; Mansour et al., Proc. Nati. Acad. Sci. USA 87: pages 7688 to 7692, 1990, and Mansour, GATA 7: pages 219 to 227, 1990).

A region of homology in a localization vector used to create the non-human mammals and genetically modified animal cells of the present invention is generally at least 100 nucleotides in length. More preferably, the region of homology is at least 1 to 5 kilobases (kb) in length. Although a minimum length or minimum degree of ratio necessary for a region of homology has not been demonstrated, the localization efficiency for homologous recombination generally corresponds to the length and degree of relationship between the localization vector and the gene locus. of PDE9. In the case where a substitution vector is used, and a part of the endogenous PDE9 gene is deleted after homologous recombination, an additional consideration is the size of the deleted part of the endogenous PDE9 gene. If this part of the endogenous PDE9 gene is greater than 1 kb in length, then a localization cassette with regions of homology that are longer than 1 kb is recommended to enhance the efficiency of the recombination. In the bibliography, additional guidance is described in relation to the. selection and use of sequences effective for homologous recombination, based on the present disclosure (see, for example, Deng and Capecchi, Mol Cell. Biol. 12: pages 3365 to 3371, 1992; Bollag et al., Annu. Genet, 23: pages 199 to 225, 1989, and Waldman and Liskay, Mol. Cell, Biol. 8: pages 5350 to 5357, 1988). As will be recognized by those skilled in the art based on the present invention, a wide variety of cloning vectors may be used as chains -mainly in the construction of PDE9 gene localization vectors of the present invention, including plasmids related to pBluescript (for example, Bluescript KS + 11), pQE70, pQE60, pQE-9, pBS, pD10, Phagescript, phiX174, phagemid pBK, pNH8A, pNH16a, pNH18Z, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 PWLNEO, pSV2CAT, pXT1, pSG (Stratagene), pSVK3, PBPV, PMSG, and pSVL, pBR322 and vectors based on pBR322, pMB9, pBR325, pKH47, pBR328, pHC79, Charon phage 28, pKB11, pKSV-10, related plasmids with pK19, pUC plasmids, and the pGEM series of plasmids. These vectors are available in 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). However, any other vectors may be used, for example, plasmids, viruses, or their parts, provided they are replicable and viable in the desired host. The vector can also comprise sequences that allow it to replicate in the host whose genome is to be modified. The use of said vector can lengthen the period of interaction during which recombination occurs, increasing the efficiency of localization (see, Molecular Biologv, ed. Ausubel et al, Unit 9.16, Fig. 9.16.1). The specific host used to propagate the localization vectors of the present invention is not critical. Examples include E. coli K12 RR1 (Bolivar et al., Gene 2: page 95, 1977), E. coli K12 HB101 (ATCC No. 33694), E. coli MM21 (ATCC No. 336780), E. coli DH1 (ATCC No. 33849), strain of E coli DH5a, and E. coli STBL2. Alternatively, hosts such as C. cerevisiae or B. subtilis may be used. The aforementioned guests are commercially available (e.g., Stratagene, La Jolla, CA, and Life Technologies, Rockville, MD). To create the localization vector, a location construction of the PDE9 gene is added to a backbone of the vector described above. The PDE9 gene localization constructs of the present invention have at least one region of homology with the PDE9 gene. To elaborate the homology regions with the PDE9 gene, a PDE9 cDNA or genomic sequence is used as a basis to produce primers for PCR. 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: page 4967, 1991; Eckert and Kunkel 1: page 17, 1991; U.S. Patent No. 4,683,202). The genomic sequence is obtained from a library of genomic clones or from a preparation of genomic DNA, preferably from the animal species to be localized to alter the PDE9 gene. A PDE9 cDNA sequence can be used to prepare a PDE9 localization vector (e.g., GenBank® NM008804 (murine) or GenBank® NM002606 (human)). Preferably, the localization constructs of the present invention also include an exogenous nucleotide sequence that encodes a positive marker protein. The stable expression of a positive marker after the integration of the vector confers a characteristic that can be identified in the cells, ideally, without compromising cell viability. Therefore, in the case of a substitution vector, the marker gene is located between two regions of flanking homology, so that it is integrated into the PDE9 gene after the double cross-linking event in such a way that the marker gene it is located for expression after integration. It is preferred that the positive marker protein be a selectable protein; the stable expression of said protein in a cell confers a phenotypic characteristic that can be selected, that is, the characteristic enhances the survival of the cell under conditions that would otherwise be lethal. Therefore, by imposing the selectable condition, cells that stably express the sequence of the vector encoding the positive selectable marker of the other cells that have not successfully integrated the vector sequence can be isolated based on the viability. Examples of positive selectable marker proteins (and their selection agents) include neo (G418 or kanamycin), hyg (hygromycin), hisD (histidinol), gpt (xanthine), ble (bleomycin), and hprt ( hypoxanthine) (see, for example, Capecchi and Thomas, U.S. Patent No. 5,464,764, and Capecchi, Science 244: pages 1288-1292, 1989). Other positive labels that can also be used as an alternative to a selectable marker include reporter proteins such as β-galactosidase, firefly luciferase, or green fluorescent protein (see, for example, Current Protocols in Cytometry, Unit 9.5 , and Current Protocols in Molecular Biology _ Unit 9.6, John Wiley & amp;; Sons, New York, NY, 2000). The positive selection step described above does not distinguish between cells that have integrated the vector by homologous recombination located at the locus of the PDE9 gene versus non-homologous, random integration of the vector sequence at any position on the chromosome. Therefore, when a substitution vector is used for homologous recombination to make the non-human mammals and genetically modified animal cells of the present invention, it is also preferred to include a nucleotide sequence encoding a selectable marker protein negative. The expression of a negative selectable marker causes a cell expressing the marker to lose viability when exposed to a particular agent (ie, the marker protein becomes lethal to the cell under certain selectable conditions). Examples of negative selectable markers (and their lethal agents) include the herpes simplex virus thymidine kinase (ganciclovir or 1,2-deoxy-2-fluoro-ad-arabinofuranos) -5-yodouracol. ), Hprt (6-thioguanidine or 6-thioxanthine), and diphtheria toxin, ricin toxin, and cytosine deaminase (5-fluorocytosine). The nucleotide sequence encoding the negative selectable marker is located outside the two regions of homology of the substitution vector. Once this position is determined, the cells will only stably integrate and express the negative selectable marker if the Integration occurs by non-homologous, random recombination; the homologous recombination between the PDE9 gene and the two homology regions in the localization construct excludes the sequence encoding the negative selectable marker of the integration. Therefore, by imposing the negative condition, cells that have integrated the non-homologous recombination localization vector lose viability. The above-described combination of positive and negative selectable markers in a localization construct used to prepare the non-human mammals and genetically modified animal cells of the present invention is preferred because a series of positive and negative selection stages can be designed. to select more efficiently only those cells that have undergone vector integration by homologous recombination, and therefore, have a potentially altered PDE9 gene. Additional examples of positive-negative selection schemes, selectable markers, and location constructions are described, for example, in U.S. Pat. No. 5,464,764, WO 94/06908, U.S. Pat. No. 5,859,312, and the publication of Valancius and Smithies, Mol. Cell. Biol. 11: page 1402, 1991. For a marker protein to be stably expressed upon integration of the vector, the localization vector can be designed so that the sequence encoding the marker is operably linked to the endogenous PDE9 gene promoter after vector integration. Then, expression of the vector is driven by the PDE9 gene promoter in cells that normally express the PDE9 gene. Alternatively, each marker in the vector localization construct may contain its own promoter that directs expression independent of the promoter of the PDE9 gene. This latter scheme has the advantage of allowing the expression of markers in cells that typically do not express the PDE9 gene (Smith and Berg, Cold Spring Harbor Symp. Quant. Biol. 49: page 171, 1984; Sedivy and Sharp, Proc. Acad. Sci. (USA) 86: page 227, 1989, Thomas and Capecchi, Cell 51: page 503, 1987). Exogenous promoters that can be used to direct expression of the marker gene include cell-specific or stage-specific promoters, constitutive promoters, and promoters that can be induced or regulated. Non-limiting examples of these promoters include the herpes simplex thymidine kinase promoter, the promoter / enhancer of cytomegalovirus (CMV), the promoters of SV40, the promoter of PGK, PMC1-neo, promoter of metallothionein, the late promoter of adenovirus, the promoter of vaccinia virus 7.5 K, the promoter of beta- avian globin, histone promoters (eg, mouse histone H3-614), beta-actin promoter, neuron-specific enolase, muscle actin promoter, and 35S promoter of cauliflower mosaic virus (see in general , Sambrook et al., Molecular Cloning, Vols. I-III, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Current Protocols in Molecular Biology, John Wiley &; Sons, New York, NY, 2000; Stratagene, La Jolla, CA). To confirm whether the cells have integrated the vector sequence into the locus of the PDE9 gene localized when the non-human mammals and genetically modified animal cells of the present invention are prepared, primers or genomic probes that are specific for the event of integration of the desired vector, combined with the polymerase chain reaction (PCR) or Southern blot analysis to identify the presence of the integration of the desired vector at the locus of the PDE9 gene (Erlich et al., Science 252: pages 1643 at 1651, 1991, Zimmer and Gruss, Nature 338: page 150, 1989, Mouellic et al., Proc. Nati, Acad. Sci. (USA) 87: page 4712, 1990, and Shesely et al., Proc. Nati. Acad. Sci. (USA) 88: page 4294, 1991).

Gene capture Another method available for inserting a foreign nucleic acid sequence at the locus of the PDE9 gene to alter the PDE9 gene, based on the present disclosure, is gene capture. This method takes advantage of the cellular machinery present in all the cells of mammals that splices the exons in the mRNA, to insert a sequence that encodes the gene capture vector in a gene in a random way. Once inserted, the gene capture vector creates a mutation that can alter the captured PDE9 gene. In contrast to homologous recombination, this system by mutagenesis creates largely random mutations. Therefore, to obtain a genetically modified cell containing an altered PDE9 gene, cells containing this particular mutation must be identified and selected from a mixture of cells containing random mutations in a variety of genes. Gene capture systems and vectors have been described for use in murine cells and other types of genetically modified cells (see, for example, Alien et al., Nature 333: pages 852 855, 1988; Bellen et al., Genes Dev. 3: pages 1288 to 1300, 1989, Bier et al., Genes Dev 3: pages 1273 to 1287, 1989, Bonnerot et al., J. Virol 66: pages 4982 to 4991, 1992, Brenner et al., Proc. Nat. Acad. Sci. USA 86: pages 5517 to 5521, 1989, Chang et al., Virology 193: pages 737 to 747, 1993, Friedrich and Soriano, Methods Enzymol 225: pages 681 to 701, 1993; Soriano, Genes Dev. 5: pages 1513 to 1523, 1991, Goff, Methods Enzymol 152: pages 469 to 481, 1987, Gossler et al., Science 244: pages 463 to 465, 1989, Hope, Develop. 399 to 408, 1991, Kerr et al., Cold Spring Harb. Symp.Quant. Biol. 2: pages 767 to 776, 1989, Reddy et al., J. Virol. 65: pages 1507 to 1515, 1991; Reddy et al. al., Proc. Nati. Acad. Sci. USA 89: page 6721 to 6725, 1992; Skarnes et al., Genes Dev. 6: pages 903 to 918, 1992; von Melchner and Ruley, J. Virol. 63: pages 3227 to 3233, 1989; and Yoshida et al., Transgen. Res. 4: pages 277 to 287, 1995).

Individual mutant cell lines containing an altered PDE9 gene are identified in a population of mutated cells using, for example, reverse transcription (RT) and PCR to identify a mutation in a sequence of the PDE9 gene. This procedure can be rationalized by mixing clones. For example, to find a single clone containing an altered PDE9 gene, RT-PCR is performed using a primer anchored in the gene capture vector and the other primer located in the sequence of the PDE9 gene. A positive result of the RT-PCR indicates that the vector sequence is encoded in the transcription of the PDE9 gene, indicating that the PDE9 gene has been altered by an integration event by gene capture (see, for example, Sands et al. al., WO 98/14614, U.S. Patent No. 6,080,576).

Genetic alterations of temporary, spatial and induced PDE9s In some embodiments of the present invention, a functional alteration of the endogenous PDE9 gene occurs at specific stages of the cell cycle or development (temporal alteration) or in specific cell types (alteration). space). In other embodiments, alteration of the PDE9 gene can be induced when certain conditions are present. A recombinase suppression system, such as a Cre-Lox system, can be used to activate or inactivate the PDE9 gene at a specific stage of development, in a particular tissue or cell type, or at particular environmental conditions. In general, methods using Cre-Lox technology are performed as described by Torres and Kuhn, Laboratory Protocols for Conditional Gene Targeting, Oxford University Press, 1997. A methodology similar to that described by the Cre-Lox system can also be used. using the FLP-FRT system. Additional guidance is given regarding the use of recombinase suppression systems for genes that are conditionally altered by homologous recombination or viral insertion, for example, in the U.S. Patent. No. 5,626,159; Patent of E.U.A. No. 5,527,695; Patent of E.U.A. No. 5,434,066; WO 98/29533; Patent of E.U.A. No. 6,228,639; Orban et al., Proc. Nat. Acad. Sci. E.U.A. 89: pages 6861 to 6865, 1992; O'Gorman et al., Science 251: pages 1351-1355, 1991; Sauer et al., Nucleic Acids Research 17: pages 147 to 161, 1989; Barinaga, Science 265: pages 26 to 28,1994; and Akagi et al., Nucleic Acids Res. 25: pages 1766 to 1773, 1997. More than one recombinase system can be used to genetically modify a non-human mammal or animal cell of the present invention. When homologous recombination is used to alter the PDE9 gene in a temporal, spatial or inducible manner, using a recombinase system, such as the Cre-Lox system, a part of the region encoding the PDE9 gene is replaced by a localization construct comprising the region encoding the PDE9 gene flanked by the loxP sites. Non-human mammals and animal cells carrying this genetic modification contain a PDE9 gene flanked by functional loxP. The temporal, spatial or inducible aspect of the alteration of the PDE9 gene is produced by the expression standard of an additional transgene, a transgene of Cre recombinase, which is expressed in the non-human mammal or animal cell controlled by the spatially regulated, temporarily regulated or inducible promoter desired, respectively. A Cre recombinase localizes the loxP sites for recombination. Therefore, when the expression of Cre is activated, the LoxP sites are subjected to recombination to suppress the coding sequence of the interposed PDE9 gene, resulting in a functional alteration of the PDE9 gene (Rajewski et al., J. Clin. Invest. 98: pages 600 to 603, 1996; St.-Onge et al., Nucleic Acids Res. 24: pages 3875 to 3877, 1996; Agah et al., J. Clin. Invest. 100: pages 169 to 179 , 1997, Brocard et al., Proc. Nati, Acad. Sci. USA 94: pages 14559 to 14563, 1997, Feil et al., Proc. Nati, Acad. Sci. USA 93: pages 10887 to 10890, 1996; Kuhn et al., Science 269: pages 1427 to 1429, 1995). A cell containing both a Cre recombinase transgene and the PDE9 gene flanked by loxP can be generated by standard transgenic techniques, or in the case of non-human mammals genetically modified, by crossing genetically modified non-human mammals, in which one parent contains a PDE9 gene flanked by loxP and the other contains a Cre recombinase transgene controlled by the desired promoter. Additional guidance is found regarding the use of specific recombinase and promoter systems for the temporal, spatial or conditional alteration of the PDE9 gene, for example, in Sauer, Meth. Enz. 225: pages 890 to 900,1993; Gu et al., Science 265: pages 103 to 106, 1994; Araki et al., J. Biochem. 122: pages 977 to 982, 1997; Dymecki, Proc. Nati Acad. Sci. 93: pages 6191 to 6196, 1996; and Meyers et al., Nature Genetics 18: pages 136 to 141, 1998. An alteration that can be induced from the PDE9 gene using a binary system sensitive to tetracycline can also be achieved (Gossen and Bujard, Proc. Nati. Acad. Sci. USA 89: pages 5547 to 5551, 1992). This system involves genetically modifying a cell to introduce a Tet promoter into the regulatory element of the endogenous PDE9 gene and a transgene expressing a repressor that can be controlled with tetracycline (TetR). In said cell, the administration of tetracycline activates the TetR, which, in turn, inhibits the expression of the PDE9 gene, and therefore, alters the PDE9 gene (St.-Onge et al., Nucleic Acids Res. 24: pages 3875 to 3877, 1996; U.S. Patent No. 5,922,927). The systems described above for the temporal, spatial and inducible alterations of the PDE9 gene can also be adopted when gene capture is used as a method for genetic modification, as described, for example, in WO 98 / 29533 and US Patent No. 6,288,639, to create the non-human mammals and genetically modified animal cells of the present invention.

Creation of non-human mammals and genetically modified animal cells The methods described above for genetic modification can be used to alter a PDE9 gene in almost any type of somatic cell or mother obtained from an animal, to create the genetically modified animal cells of the animal. present invention. The genetically modified animal cells of the present invention include, but are not limited to, mammalian cells, including human cells, and avian cells. These cells can be obtained by genetically engineering any line of animal cells, such as culture-adapted, tumorigenic or transformed cell lines, genetically engineered differentiated ES cells, or can be isolated from a genetically modified non-human mammal, which carries the genetic modification of PDE9 desired. The cells may be heterozygous or homozygous for the altered PDE9 gene. To obtain cells that are homozygous for the alteration of the PDE9 (- / -) gene, direct and sequential localization of both alleles can be performed. This procedure can be facilitated by recycling a marker that can be selected positive. According to this scheme, the nucleotide sequence encoding the positive selectable marker is deleted after altering an allele using the Cre-LoxP system. Therefore, the same vector can be used in a subsequent round of localization to alter the second allele of the PDE9 gene (Abuin and Bradley, Mol Cell. Biol. 16: pages 1851 to 1856, 1996; Sedivy et al., TIG 15: pages 88 to 90, 1999, Cruz et al., Proc. Nati, Acad. Sci. (USA) 88: pages 7170 to 7174,1991, Mortensen et al., Proc. Nati. Acad. Sci. ) 88: pages 7036 to 7040, 1991; Riele et al., Nature (London) 348: pages 649 to 651, 1990). An alternative strategy to obtain ES cells that are PDE9 - / - is the homogenotization of cells from a population of cells that is heterozygous for the alteration of the PDE9 gene (PDE9 +/-). The method uses a scheme in which localized PDE9 +/- clones are selected that express a drug resistance marker that can be 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 alteration of the PDE9 gene (Mortensen et al., Mol. Cell. Biol. 12: pages 2391 to 2395, 1992). In addition, genetically modified animal cells can be obtained from non-human PDE9 - / - genetically modified mammals that are created by mating with non-human mammals that are PDE9 +/- in germline cells, as discussed in more detail below . After genetic modification of the desired cell or cell line, the locus of the PDE9 gene can be confirmed as the site of the modification by PCR analysis according to the standard Southern blot or PCR methods known in the art (see, for example, U.S. Patent No. 4,683,202; and Erlich et al., Science 252: page 1643, 1991). Further verification of the functional alteration of the PDE9 gene can also be performed if the levels of messenger RNA (mRNA) of the PDE9 gene and / or PDE9 polypeptide levels are reduced in cells that normally express the PDE9 gene. Measurements of mRNA levels of the PDE9 gene can be obtained using RT-PCR, Northern blot analysis, or by in situ hybridization. The quantification of PDE9 polypeptide levels produced by the cells can be performed, for example, by standard immunoassay methods known in the art. Such immunoassays include, but are not limited to, competitive and non-competitive assay systems using techniques such as RIA (radioimmunoassays), ELISA (enzyme-linked immunosorbent assays)., sandwich assays, immunoradiometric assays, precipitation-diffusion gel reactions, immunodiffusion assays, immunoassays in situ (using, for example, colloidal gold, enzymatic or radioisotope labels), Western blots, gel analysis dimensions, precipitation reactions, immunofluorescence assays, protein A assays and immunoelectrophoresis assays. Preferred genetically modified animal cells of the present invention are embryonic stem cells (ES) and ES-type cells. These cells are obtained from the preimplantation of embryos and blasts of different species, such as mice (Evans et al., Nature 129: pages 154 to 156, 1981; Martin, Proc. Nati Acad. Sci., USA, 78: pages 7634 to 7638, 1981), pigs and sheep (Notanianni et al., J. Reprod. Fert. Suppl., 43: pages 255 to 260, 1991; Campbell et al., Nature 380: pages 64 to 68,1996) and primates, including humans (Thomson et al., U.S. Patent No. 5,843,780, Thomson et al., Science 282: pages 1145 to 1147, 1995, and Thomson et al., Proc. Nati. Acad. Sci. USA 92: pages 7844 to 7848, 1995). The success of gene alteration mediated by homologous recombination in human ES cells has been described (Zwaka and Thomson, Nature Biotech, 21: pages 319 to 321, 2003). These types of cells are pluripotent, that is, under appropriate conditions, they differ in a wide variety of cell types obtained from the three embryonic germ layers: ectoderm, mesoderm and endoderm. Depending on the culture conditions, a sample of ES cells can be grown 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. Targeted differentiation is performed by including growth factors or specific matrix components under culture conditions, as described in more detail, for example, Keller et al., Curr. Opin. Cell Biol. 7: pages 862 to 869, 1995; Li et al., Curr. Biol. 8: page 971, 1998; Klug et al., J. Clin. Invest. 98: pages 216 to 224, 1996; Lieschke et al., Exp. Hematol. 23: pages 328 to 334, 1995; Yamane et al., Blood 90: pages 3516 to 3523, 1997; and Hirashima et al., Blood 93: pages 1253 to 1263, 1999. The particular embryonic stem cell line that is used for genetic modification is not critical; interspecific murine ES cell lines include AB-1 (McMahon and Bradley, Cell 62: pages 1073 to 1085, 1990), E14 (Hooper et al., Nature 326: pages 292 to 295, 1987), D3 (Doetschman et al., J. Embryol, Exp. Morph 87: pages 27 to 45, 1985), CCE (Robertson et al, Nature 323: pages 445 to 448, 1986), RW4 (Genome Systems, St. Louis, MO), and DBA / 1 lacJ (Roach et al., Exp. Cell Res. 221: pages 520 to 525, 1995); an exemplary human ES cell line is that of H1.1 cells (Zwaka and Thomson, Nature Biotech, 21: pages 319 to 321, 2003). ES cells from genetically modified murine can be used to generate genetically modified mice according to published procedures (Robertson, 1987, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Ed. EJ Robertson, Oxford: IRL Press, pp. 71 to 112 , 1987; Zjilstra et al., Nature 342: pages 435 to 438, 1989; and Schwartzberg et al., Science 246: pages 799 to 803, 1989). After confirming that the ES cells contain the desired functional alteration of the PDE9 gene, then these ES cells are injected into the appropriate blastocyst hosts to generate chimeric mice according to the methods known in the art (Capecchi, Trends Genet. 70, 1989). The particular mouse blasts used in the present invention are not critical. Examples of such blastocysts include those derived from C57BL6 mice, C57BL6 Albino mice, Swiss exogenous breeding mice, CFLP mice and MFI mice. Alternatively, ES cells can be interposed between tetraploid embryos in aggregation wells (Nagy et al., Proc. Nati Acad.Sci. E.U.A. 90: pages 8424 to 8428, 1993). Then the embryos or embryos containing the genetically modified ES cells are implanted in pseudopregnant female mice and allowed to develop in the uterus (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, EJ Robertson, ed., IRL Press, Washington, DC, 1987). The offspring born to the host mothers can be selected to identify those that are chimeric for the alteration of the PDE9 gene. In general, said proge contains some cells derived from the ES cells of the genetically modified donor as well as other cells derived from the original blastocyst. In these circumstances, the offspring can be selected initially according to the color of the mosaic coat, when a strategy of color selection of the coat has been used, to distinguish the cells derived from the donor ES cell from the other cells of the blastocyst. Alternatively, DNA from the tail tissue of the offspring can be used to identify mice that contain genetically modified cells.

Mating of chimeric mice containing the alteration of the PDE9 gene in germline cells produces a progeny that has the alteration of the PDE9 gene in all germline cells and somatic cells. Mice that are heterozygous for the alteration of the PDE9 gene can be crossed to produce homozygotes (see, for example, U.S. Patent No. 5,557,032 and U.S. Patent No. 5,532,158). An alternative to the ES cell technology described above for transferring a genetic modification of a cell to a whole animal is to use nuclear transfer. This method can be used to make other non-human mammals genetically modified in addition to mice, for example sheep (McCreath et al., Nature 29: pages 1066 to 1069, 2000; Campbell et al., Nature 389: pages 64 to 66, 1996; and Schnieke et al., Science 278: pages 2130 to 2133, 1997) and calves (Cibelli et al., Science 280: pages 1256 to 1258, 1998). Briefly, somatic cells (e.g., fibroblasts) or pluripotent stem cells (e.g., ES-type cells) are selected as nuclear donors and genetically modified to contain a functional alteration of the PDE9 gene. When inserting a DNA vector into a somatic cell to mutate the PDE9 gene, it is preferred to use a marker without promoter in the vector, such that integration of the vector into the PDE9 gene results in the expression of the controlled marker by the promoter of the PDE9 gene (Sedivy and Dutriaux, TIG 15: pages 88 to 90, 1999; McCreath et al., Nature 29: pages 1066 to 1069, 2000). The nuclei of the donor cells that have the appropriate PDE9 gene alteration are then transferred to parthenogenetic or fertilized oocytes that are enucleated (Campbell et al., Nature, 380: page 64, 1996, Wilmut et al., Nature 385: page 810, 1997). The embryos are reconstructed, cultured to develop at the morula / blastocyst stage, and transferred to host mothers to completely complete development in the uterus. The present invention also encompasses the progeny of genetically modified non-human mammals and genetically modified animal cells. Although the progeny are heterozygous or homozygous for the genetic modification that alters the PDE9 gene, they may not be genetically identical to non-human mammals and parental animal cells due to mutations or environmental influences, in addition to the original genetic alteration of the gene of PDE9, which may occur in successive generations. The cells of a genetically modified non-human animal can be isolated from tissues or organs using techniques known to those skilled in the art. In one embodiment, the genetically modified cells of the present invention are immortalized. According to this embodiment, cells can be immortalized by genetic design of the telomerase gene, an oncogene, for example, mos or v-src, or a gene that inhibits apoptosis, for example, bcl-2, in cells . Alternatively, the cells can be immortalized by fusion with a hybridization partner using techniques known to one skilled in the art.

"Humanized" nonhuman animal and mammalian cells Non-human mammals and genetically modified animal (non-human) cells of the present invention that contain an altered endogenous PDE9 gene can be further modified to express the sequence of human PDE9 (referred to as present memory, "humanized"). A preferred method for humanizing cells involves replacing the endogenous PDE9 sequence with a nucleic acid sequence encoding the sequence of human PDE9 (Jakobsson et al., Proc. Nati, Acad. Sci. EUA 96: pages 7220 to 7225, 1999) by homologous recombination. The vectors are similar to those traditionally used as location vectors with respect to the 5 'and 3' homology arms and the positive / negative selection schemes. However, the vectors also include the sequence which, after recombination, replaces the endogenous sequence with the sequence encoding human PDE9, or makes base pair changes, substitutions of exons or codon substitutions that modify the endogenous sequence for encode human PDE9. Once the homologous recombinants have been identified, any sequences based on selection (eg, neo) can be suppressed using site-directed recombination mediated by Cre or Flp (Dymecki, Proc. Nati. Acad. Sci. 93: pages 6191 to 6196, 1996).

When replacing the human PDE9 sequence with the endogenous sequence, it is preferred that these changes are introduced directly downstream of the endogenous translation initiation site. This position retains the endogenous spatial and temporal expression patterns of the PDE9 gene. The human sequence can be the full length human cDNA sequence with a polyA tail attached to the 3 'end for proper processing, or the entire genome sequence (Shiao et al., Transgenic Res. 8: pages 295-302, 1999 ). Further guidance regarding these methods of genetic modification of non-human cells and mammals is found to replace the expression of an endogenous gene with its human equivalent, for example, in Sullivan et al., J. Biol. Chem. 272: pages 17972 a 17980, 1997, Reaume et al., J. Biol. Chem. 271: pages 23380 to 23388, 1996, and Scott et al., US Patent. 5,777,194). Another method for creating such "humanized" organisms is a two-step procedure involving the alteration of the endogenous gene followed by the introduction of a transgene encoding the human sequence, by pronuclear microinjection in knock-out embryos.

Uses of non-human mammals and genetically modified animal cells The function of PDE9 and the therapeutic importance can be further elucidated by further investigation of the phenotype of non-human mammals and animal cells PDE9 - / - of the present invention, as illustrated, for example, in the following Examples. For example, non-human mammals and genetically modified PDE9 - / - animal cells can be used to determine whether PDE9 plays a role in making or preventing the development of symptoms or phenotypes in certain disease models, for example, obesity, feeding, cardiovascular disorders, insulin resistance syndrome, hypertension, and / or type 2 diabetes. Whether a symptom or phenotype is different in a non-human mammal or animal cell PDE9 - / - compared to a non-human mammal or animal cell wild (PDE9 + / +) or PDE9 +/-, then the PDE9 polypeptide plays a role in the regulation of functions associated with the symptom or phenotype. Examples of assays that can be used to evaluate the function of PDE9 include comparing PDE9 - / - mice with wild mice in terms of body weight, body fat, blood pressure, glucose / insulin metabolism (eg, glucose in isolated tissues, alterations in the activity of glycogen metabolism enzymes, alterations in glycogen levels in the liver or muscle, and / or alterations in body composition), and changes in the activity or state of phosphorylation of components in the insulin signaling pathway. In addition, under certain circumstances in which an agent has been identified as a PDE9 agonist or antagonist (for example, the agent significantly modifies one or more of the PDE9 polypeptide activities when the agent is administered to a non-human mammal or animal cell PDE9 + / + or PDE9 +/-), non-human mammals and genetically modified animal cells PDE9 - / - of the present invention are useful for characterizing any other effects produced by the agent in addition to those known to result from the ) PDE9 agonism (ie, non-human mammals and animal cells can be used as negative controls). For example, if the administration of the agent produces an effect in a non-human mammal or animal cell PDE9 + / + that is known to be associated with PDE9 polypeptide activity, then it can be determined whether the agent exerts this effect solely or mainly through of the modulation of PDE9 by the administration of the agent to a corresponding non-human mammal or animal cell PDE9 - / -. If this effect is absent, or is significantly reduced, in the non-human mammal or animal cell PDE9 - / -, then the effect is mediated, at least in part, by PDE9. However, if the non-human mammal or animal cell PDE9 - / - exhibits the effect to a degree that can be compared to the non-human mammal or animal cell PDE9 + / + or PDE9 +/-, then the effect is mediated by a path that does not imply PDE9 signaling. Furthermore, if it is suspected that an agent can exert an effect predominantly by means of a PDE9 pathway, then non-human mammals and animal cells PDE9 - / - are useful as negative controls to test this hypothesis. If the agent is actually acting through PDE9, then non-human mammals and animal cells PDE9 - / -, after administration of the agent, should not demonstrate the same effect observed in non-human mammals and animal cells PDE9 + / +. The non-human mammals and genetically modified animal cells of the present invention can also be used to identify genes whose expression is regulated differently in non-human mammals and animal cells PDE9 - / - or PDE9 +/- in relation to their respective wild controls . Techniques known to those skilled in the art can be used to identify such genes based on the present disclosure. For example, DNA matrices can be used to identify genes whose expression is regulated differently in PDE9 +/- or PDE9 - / - mice to compensate for a deficiency in PDE9 expression. DNA matrices are known to those skilled in the art (see, for example, Aigner et al., Arthritis and Rheumatism 44: pages 2777 to 2789, 2001; Patent of E.U.A. No. 5,965,352; Schena et al., Science 270: pages 467 to 470, 1995; Schena et al., Proc. Nati Acad. Sci. E.U.A. 93: pages 10614 to 10619, 1996; DeRisi et al., Nature Genetics 14: pages 457 to 460, 1996; Shalon et al., Genome Res. 6: pages 639 to 645, 1996; and Schena et al., Proc. Nati Acad. Sci. (E.U.A.) 93: pages 10539 to 11286, 1995; Patent of E.U.A. No. 5,474,796; Patent of E.U.A. No. 5,605,662; WO 95/25116; WO 95/35505; Heller et al., Proc. Nati Acad. Sci. 94: pages 2150 to 2155, 1997). A chemical coupling method and an ink jet device can be used to synthesize the elements of the matrix on a surface of a substrate. An analogous matrix to a spot or slot transfer can be used to arrange and join elements to the surface of a substrate using the thermal, UV, chemical or mechanical bonding methods. A typical matrix can be prepared manually or using the available methods and machines, and contain any suitable number of elements. After hybridization, the unhybridized probes are separated and a scanner is used to determine fluorescence levels and patterns. The degree of complementarity and relative abundance of each probe that hybridizes with an element on the microarray can be evaluated by analyzing the scanned images. The elements of a full length cDNA microarray may comprise expressed sequence tags (ESTs) or fragments thereof. Fragments suitable for hybridization can be selected using software known in the art, such as the LASERGENE software (DNASTAR). Full-length cDNAs, ESTs or fragments thereof, corresponding to one of the nucleotide sequences of the present invention, or randomly selected from a cDNA library related to the present invention, are arranged on a suitable substrate, for example, a glass slide. The cDNA is fixed to the slide using, for example, ultraviolet crosslinking followed by thermal and chemical treatments and subsequent drying. Fluorescent probes are prepared and used to hybridize the elements on the substrate. The substrate is analyzed by methods known in the art, for example, by scanning and analyzing microarray images. In addition, the non-human mammals and genetically modified animal cells of the present invention can also be used to identify proteins whose expression profile or post-translational modification is altered in non-human mammals and animal cells PDE9 +/- or PDE9 - / - in relation to their respective wild controls. The techniques known to those skilled in the art can be used to identify such proteins based on the present disclosure. For example, proteomic assays can be used to identify proteins whose expression profile or post-translational modification is altered in PDE9 +/- or PDE9 - / - mice to compensate for a deficiency in PDE9 expression. Proteomic assays are known to those skilled in the art (see, for example, Conrads et al., Biochem. Biophys. Res. Commun. 290: pages 896 to 890, 2002; Dongre et al., Biopolymers 60: pages 206 to 211, 2001, Van Eyk, Curr Opin, Mol Ther 3: pages 546 to 553, 2001, Colé et al., Electrophoresis 21: pages 1772 to 1781, 2000, Araki et al., Electrophoresis 21: pages 180 to 1889, 2000).

EXAMPLES 1. Preparation of the PDE9 localization vector A localization vector construct was designed according to the scheme shown in Figure 1. The construct contained two homologous arms with the genomic sequence of murine PDE9: an arm of 5 'homology of 0, 9 kb and a 3 'homology arm of 4.3 kb. Between these arms a LacZ-Neo construction was interposed. The DNA containing the localization construct was inserted into ES R1 cells by electroporation (Deng et al., Dev. Biol. 185: pages 42 to 54.1997; Udy et al., Exp. Cell Res. 231: pages 296 a 301, 1997). After homologous recombination, the base pairs 142 to 175 of the sequence coding for the PDE9 cDNA shown in Figure 2 (the base pairs 142 to 175 are underlined) of the endogenous gene were deleted and replaced by the LacZ Neo cassette. ES cells that were resistant to neomycin were analyzed by Southern blotting to confirm the alteration of a PDE9 gene. Then, these localized ES cells were used to generate chimeric mice by injecting the cells into blastocysts and implanting the blastocysts in pseudopregnant female mice (Capecchi et al., Trends Genet, 5: page 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, EJ Robertson, ed., IRL Press, Washington, DC, 1987). Then, the chimeric mice were bred with C57BL / 6 mice (Jackson Laboratories, Bar Harbor, ME) to create heterozygous PDE9 +/- F1, which in turn were bred to produce homozygous PDE9 - / - F2 mice (Charles River Laboratories, Wimington , MA). Functional alteration of the PDE9 gene in heterozygotes and homozygotes was confirmed by PCR and Southern blot analysis. 2. Effect of alteration of the PDE9 gene on body weight, body composition and metabolites Methods The male and female mice PDE9 KO (- / -), previously described and the controls of the same bait (+ / +) wild, were allowed to acclimate for at least a week before starting the study and were given free access to water and food for D11 mice (Purina, Brentwood, MO). Male and female mice (17-19 weeks of age) were divided into four experimental groups with two to five mice per cage. A group of control mice of each genus remained with the D11 mouse food and the rest of the groups of each genus were switched to a diet composed of 58% fat in kcal (D12331 Rodent Diet, Research Diets, Inc., New Brunswick , NJ) during the study period of 6 weeks. Body weight was determined on day 0 and followed weekly. The adipose deposit mass was analyzed on day 0 and at the end of the study, as described below in more detail.

On day 1, glucose was determined in the plasma by retro-orbital blood samples. 25 μl of blood was added to 100 μl of 0.025 percent heparinized saline in microtubes (Denville Scientific, Inc., Metuchen, NJ). The tubes were centrifuged in the highest position in a Beckman Microfuge 12 device for 2 minutes. Plasma was collected to determine glucose and triglycerides in the plasma, as described in more detail below. During the course of the study, body weight and feed intake were evaluated, and blood samples were taken at approximately 8 o'clock in the morning for glucose and triglyceride measurements in the plasma, as described in more detail below. On the morning of the last day of the study, blood samples were taken from the retro-orbital sinus to determine glucose and triglycerides in the plasma. The mice were then sacrificed and approximately one milliliter of blood was collected in Microtainer® plasma separator tubes with lithium heparin (Becton-Dickinson, Inc., Franklin Lakes, NJ). The tubes were centrifuged in a Beckman Microfuge 12 device at the maximum position for five minutes. Plasma was collected in 1.5 ml Eppendorf tubes, they were instantly frozen in liquid nitrogen, and stored at a temperature of -80 ° C until the levels of insulin, fructosamine or cGMP were analyzed. Glucose, triglycerides and fructosamine in the plasma were measured using the Roche / Hitachi 912 clinical chemistry analyzer (Roche Diagnostics Corp., Indianapolis, IN). The cGMP in the plasma was measured using the BioTrak ™ enzyme-immunoassay system (Amersham, Piscataway, NJ). Insulin in the plasma was evaluated by a similar technique using the Mercodia insulin ELISA kit supplied by ALPCO (Uppsala, Sweden). All the tests were carried out according to the instructions of each manufacturer. The quantification of the mass of adipose deposits was made five days before the end of the study. To evaluate the mass of adipose deposits, 360 ° fluoroscopic images of the mice were obtained using a commercially available micro-computerized tomography (CT) system (MicroCAT®, ImTek Inc., Oak Ridge, TN) with a CCD / screen detector of phosphorus of high resolution. The scanner consisted of a 36 mm / 36 mm cylindrical / long field field view with a spatial resolution of less than 50 μM. The X-ray source was directed at 40 keV with the anode current set at 0.4 mA. The anesthetized mice were placed in a mouse bed radically in an anatomically correct supine position, the caudal end closest to the micro-CT with the rostral end held 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 scanning acquisition zone at the level of the iliac crest of each mouse. Once the correct alignment was secured, each animal was scanned. Each scan consisted of 196 individual projections with an exposure time of 250 μs / projection; the total image acquisition time was approximately 12 minutes with a resolution of 145 μM. The reconstruction of the image, by means of which the 196 projections acquired in the micro-scan of the mouse were manipulated to produce two-dimensional cross-sectional images of the mouse, was carried out using the MicroCAT® reconstruction, visualization and analysis software (ImTek Inc., Oak Ridge, TN) (Paulus et al., Neoplasia 2: pages 62 to 70, 2000). Two groups of reconstructed images were generated by scanning for each mouse, to determine the mass of the individual fat deposits. The first group of six reconstructed images provided a montage to analyze the mass of the inguinal and epididymal adipose tissue deposit. The second reconstruction group consisted of nine cuts, determined by intervertebral and middle vertebral signals, and were used to determine the mass of the retroperitoneal and mesenteric adipose tissue deposit. To analyze the images, the reconstructed bitmap images were converted into TIFF images. Subsequently, the TIFF images were analyzed and the mass of the fat deposits was determined using Scion Image for Windows® (Scion Corporation, Frederick MD). Demarcation lines were placed separating individual fat deposits using a brush tool (pixel size # 3) and the total pixel counts of each adipose region were determined by the Scion Image software. A higher and lower pixel intensity threshold was chosen, in this study, it was determined that a look-up table (LUT) between 115-187 was optimal for capturing the adipose deposit. The average number of pixels between each cut was calculated ((cut + cut +?) / 2). The total number of pixels, which represented the individual fat deposits, was calculated by multiplying the average number of pixels between each cut by the average number of pixels of each cut. Finally, the pixel count was converted to the deposit mass with the following equation: Tank mass (mg) = 0.000915 g / μl x 0.000757 μl / voxel x 1000 mg / g x voxel count. The first factor corrects the specific gravity of glyceryl trioleate, representative of the density of the main storage form of lipids in adipose tissue, ie, triglycerides. The second factor is the volume per pixel and the third factor converts the resulting mass into units of mg.

Results The alteration of PDE9 resulted in a decrease in body weight gain and lower body weight in a high-fat diet in KO mice both males and females, compared to their wild-type counterparts (Figure 3). Female mice also showed a 6% decrease in body length. Along with the decrease in body weight, male and female KO mice also showed lower fat mass in different adipose deposits (Figs 4A and 4B). In male KO mice, significant decreases were observed in the retriperitoneal and mesenteric adipose deposits; in female KO mice, significant decreases in inguinal, gonadal and retriperitoneal deposits were observed. In comparison, in female mice fed a standard diet, no differences in body weight were observed between the KO and wild type mice (Fig. 5) and the tendency to the lower fat mass was significant only in the gonadal adipose deposit (Fig. Figs 6A and 6B). With respect to the metabolites in the plasma after the high-fat diet, the female KO mice showed higher cGMP, lower glucose and lower insulin (Table 1); male KO mice showed a tendency toward higher cGMP and a tendency towards lower glucose (Table 1).

TABLE 1 Metabolites in the plasma after a 6-week high-fat diet 3. Effect of Pharmacological Inhibition of PDE9 on Body Weight, Body Composition and Food Ingestion in OP / O¿ >mice Methods Ob / ob female mice obtained from Jackson Laboratories (Bar Harbor, ME) were used at 6 to 10 weeks of age. The mice were housed five per cage and were given free access to water, and initially to D11 mouse food. After a one-week acclimation period, mice were switched to a powder diet (Mouse Breeder / Auto-JL K20 mouse food, PMI Feeds, Inc., St. Louis, MO) for three days and allowed to adapt to the diet before beginning the dosing period of PDE9 inhibitor. The PDE9 inhibitor compound (Compound A) was administered in powdered mice food that was custom milled (Research Diets, Inc., New Brunswick, NJ) as a compound / meal mixture; the compounds were mixed with the food to achieve a consumption of the specified doses in the range of 1-200 mg / kg / day. In addition to a control group without a compound, a group consuming darglitazone (1 mg / kg / day) as a positive control was also included. Mice were randomly assigned to groups of ten with five mice per cage. Body weight was determined on day 0 and then weekly. On day 1, retro-orbital blood samples were obtained and plasma glucose was determined as previously described. On the final day of the study, blood samples were taken for measurements of glucose, triglycerides, insulin, and cGMP, as previously described.

Results The following results represent the results of several separate studies using the same protocol described above. Figure 7 shows a lower body weight increase in ob / ob mice fed 100 mg / kg / day of PDE9 Compound A inhibitor, compared to mice fed a control diet without compound or a diet treated with darglitazone. Compound A caused a dose-dependent effect after 2 and 4 days of treatment, both in terms of reduction in normal body weight gain (Figure 8A) and also in terms of reduction in food intake (Figure 8B). The effect of PDE9 on food intake could be transient because no effect was observed on food ingestion in the later stages of the study (Figure 9) with the intermediate dose of 100 mg / kg / day. The intermediate dose of 100 mg / kg / day of Compound A also resulted in lower glucose, triglycerides and fructosamine. The representative results are shown in Figure 10, Figure 11 and Figure 12, respectively. Both examples show that producing a decrease in the activity of PDE9 is an effective method for reducing body weight and / or body fat, and can be used, for example, to treat animal patients who are overweight, obese, or suffer from a Eating disorder, and can be used in animal species for feeding to produce more lean meat.

Claims (19)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of a phosphodiesterase 9 (PDE9) inhibitor, to prepare a medicament for treating an animal to reduce body fat.

2. The use claimed in Claim 1, wherein said mammal is overweight.

3. The use claimed in Claim 1, wherein said mammal is obese.

4. The use claimed in Claim 1, wherein said PDE9 inhibitor is a selective inhibitor of PDE9.

5. The use of a PDE9 inhibitor, to prepare a medicament for treating an animal of a feeding disorder.

6. The use claimed in Claim 5, wherein said PDE9 inhibitor is a selective inhibitor of PDE9.

7. A genetically modified mouse characterized because it is homozygous for the alteration of the PDE9 gene, and wherein said mouse, after a high-fat diet of six weeks, has a lower body weight or lower fat mass in an adipose deposit, compared to a wild mouse after a six-week high-fat diet.

8. - The mouse according to claim 7, further characterized in that said mouse expresses an exogenous reporter gene controlled by the regulatory sequences of said PDE9 gene.

9. The mouse according to claim 7, further characterized in that said mouse does not have PDE9 activity that can be detected.

10. A modified genetically modified mammalian cell, characterized in that it is homozygous for the alteration of the PDE9 gene, and wherein said cell, or a cell of the progeny derived from said cell, does not exhibit PDE9 polypeptide activity that can be detected , wherein said cell or cell of the progeny would present PDE9 polypeptide activity if said homozygous alteration is absent.

11. The genetically modified mammalian cell according to claim 10, further characterized in that said cell is an embryonic stem cell (ES).

12. The genetically modified cell according to claim 11, further characterized in that said cell is a murine ES cell.

13. The genetically modified cell according to claim 11, further characterized in that said cell is a human ES cell.

14. A method for producing a mouse as claimed in claim 7, the method is characterized in that it comprises: (a) introducing a DNA sequence into a mouse ES cell, wherein the DNA sequence comprises a of gene localization PDE9, which, after recombination with the PDE9 gene, alters the PDE9 gene; (b) selecting a mouse ES cell whose genome comprises an alteration of the PDE9 gene; (c) introducing an ES cell selected in step (b) into a blastocyst or mouse morulae; (d) transplanting the blastocyte or morulae of step (c) to a host host mouse; (e) develop the blastocyst or morulae transplanted to the end to produce a chimeric mouse; and (f) obtaining a homozygous mouse for the alteration of the PDE11 gene by reproducing the chimeric mice of step (e) and mice heterozygous for the alteration of PDE9; wherein said mouse homozygous for the alteration of the PDE9 gene, after a high-fat diet of six weeks, has lower body weight or lower fat mass in an adipose deposit, compared with a wild mouse after a high-fat diet of six weeks.

15. An isolated nucleic acid molecule comprising a PDE9 gene localization construct, wherein, after recombination with the PDE9 gene, said construct alters the PDE9 gene.

16. The use of a PDE9 inhibitor, to prepare a medicament to treat a mammal to reduce the consumption of food.

17. The use claimed in claim 16, wherein said mammal is overweight.

18. - The use claimed in claim 16, wherein said mammal is obese.

19. The use claimed in claim 16, wherein said PDE9 inhibitor is a selective inhibitor of PDE9.