EP1597386A1 - Outils de diagnostics et therapeutique pour maladies associees a la phosphodiesterase 2a humaine (pde2a) - Google Patents

Outils de diagnostics et therapeutique pour maladies associees a la phosphodiesterase 2a humaine (pde2a)

Info

Publication number
EP1597386A1
EP1597386A1 EP03810957A EP03810957A EP1597386A1 EP 1597386 A1 EP1597386 A1 EP 1597386A1 EP 03810957 A EP03810957 A EP 03810957A EP 03810957 A EP03810957 A EP 03810957A EP 1597386 A1 EP1597386 A1 EP 1597386A1
Authority
EP
European Patent Office
Prior art keywords
pde2a
diseases
disorders
polypeptide
disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03810957A
Other languages
German (de)
English (en)
Inventor
Stefan Golz
Ulf Brüggemeier
Holger Summer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer AG
Original Assignee
Bayer Healthcare AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer Healthcare AG filed Critical Bayer Healthcare AG
Priority to EP03810957A priority Critical patent/EP1597386A1/fr
Publication of EP1597386A1 publication Critical patent/EP1597386A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention is in the field of molecular biology, more particularly, the present invention relates to nucleic acid sequences and amino acid sequences of a human PDE2a and its regulation for the treatment of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in mammals.
  • PDE2a is a member of the enzyme family of phosphodiesterases (PDEs) [Rosman et al. (1997), Sadhu K. et al. (1999), EP1097719, US 6015677, US 5776752]. PDEs catalyze the hydrolyzation of 3', 5' cyclic nucleotides. That results in the formation of the respective nucleoside 5' monophosphates.
  • the cyclic nucleotides cAMP and cGMP serve as crucial second messengers in a number of cellular signaling pathways.
  • the PDEs as well as the guanylyl and adenylyl cyclases, which synthesize the cyclic nucleotides, are important cellular components to regulate the concentration of cyclic nucleotides and, thus, to regulate the signal transduction pathways. Because of their central role in regulating second messenger levels PDEs have been considered chemotherapeutic targets and have been worked on extensively.
  • PDE 1-11 Several families of PDEs have been identified. The nomenclature system includes first a number that indicates the PDE family. To date, eleven families (PDE 1-11) are known which are classified by: (i) primary structure; (ii) substrate preference; (iii) response to different modulators; (iv) sensitivity to specific inhibitors; and (v) modes of regulation [Loughney and Ferguson, (1996)]. The number indicating the family is followed by a capital letter, indicating a distinct gene, and the capital letter followed by a second number, indicating a specific splice variant or a specific transcript that utilizes a unique transcription initiation site.
  • PDEs show of the following structural features:
  • the amino terminal regions of the various PDEs are highly variable and include other family specific determinants and diverse regulatory motifs such as: (i) calmodulin binding sites (PDEl); (ii) non-catalytic cyclic GMP binding sites (PDE2, PDE5, PDE6); (iii) membrane targeting sites (PDE4); (iv) hydrophobic membrane association sites (PDE3); and (v) phosphorylation sites for either the calmodulin-dependent kinase LI (PDEl), the cAMP-dependent kinase (PDEl, PDE3, PDE4), or the cGMP dependent kinase (PDE5) [Beavo, (1995); Manganiello, et al. (1995); Conti, et al. (1995)].
  • PDEl calmodulin binding sites
  • PDE2, PDE5, PDE6 non-catalytic cyclic GMP binding sites
  • PDE4 membrane targeting sites
  • PDE3 hydrophobic membrane association sites
  • PDE3
  • PDEl calcium-calmodulin dependent.
  • the group is composed of at least three genes with several splicing variants [Kakkar, R. et al. (1999)]; PDE1A and PDE1B preferentially hydrolyze cGMP while PDE1C is dualspecific, it exhibits a high affinity for both cAMP and cGMP.
  • PDE Is have been shown to be expressed in lung, heart and brain.
  • the PDE2 family is characterized as being specifically stimulated by cGMP
  • PDE2 species have been found in cerebellum, neocortex, heart, kidney, lung, pulmonary artery, and skeletal muscle [Sadhu, K. et al. (1999)]. Only one gene has been identified, PDE2A.
  • the respective PDE2A protein is specifically inhibited by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA).
  • EHNA erythro-9-(2-hydroxy-3-nonyl)adenine
  • PDE3A and PDE3B Two genes have been identified in the PDE3 family, PDE3A and PDE3B, both having high affinity for both cAMP and cGMP, although the N m a x for cGMP hydrolysis is low enough that cGMP functions as a competitive inhibitor for cAMP hydrolysis.
  • Enzymes in the PDE3 family are specifically inhibited by cGMP.
  • PDE3 enzymes are specifically inhibited by milrinone and enoximone [Loughney and Ferguson, supra].
  • PDE4s are specific for cAMP hydrolysis.
  • the family is comprised of four genes, PDE4A, PDE4B, PDE4C, and PDE4D.
  • the genes give rise to multiple splice variants and are expressed in airway smooth muscle, the vascular endothelium, and all inflammatory cells.
  • the enzymes can be activated by cAMP-dependent phosphorylation.
  • Members of this family are specifically inhibited by the anti- depressant drug rolipram.
  • PDE5 is highly selective for cGMP [Turko, IN. et al. (1998)]. Members of PDE5 family bind cGMP at non-catalytic sites [McAllister-Lucas, L.M. (1995)]. CGMP binding at non-catalytic sides has been suggested to be important for phosphorylation by cGMP-dependent protein kinase. PDE5 is highly expressed in vascular smooth muscle, platelets, lung, and kidney. Only one gene, PDE5 A, has been identified.
  • PDE6s the photoreceptor enzymes specifically hydrolyze cGMP [Loughney and Ferguson, supra]. PDE6s possess 2 regulatory high affinity cGMP binding sides.
  • Genes include PDE6A and PDE6B (the protein products of which dimerize and bind two copies of a smaller ⁇ inhibitory subunit to form rod PDE), in addition to PDE6C which associates with three smaller proteins to form cone PDE.
  • PDE7 effects cAMP hydrolysis but, in contrast to the PDE4 family, is not inhibited by rolipram [Loughney and Ferguson, supra].
  • PDE7A Only one gene, PDE7A, has been identified.
  • PDE7A gives rise to multiple splice variants.
  • PDE7 mRNA can be found in several tissues but PDE7 protein expression appears to be restricted [Han, P. et al. (1997); Perry, M.J. and G.A. Higgs (1998)]. Not much is known about the physiological function of PDE7.
  • the PDE8 family is closely related to the the PDE4 family. PDE8s have been shown to hydrolyze both cAMP and cGMP and are insensitive to inhibitors specific for PDEs 1-5. PDE8s are found in thyroid gland, testis, eye, liver, skeletal muscle, heart, kidney, ovary, and brain.
  • the PDE9 family preferentially hydrolyzes cAMP and is not sensitive to inhibition by rolipram, a PDE4-specific inhibitor, or isobutyl methyl xanthine (IBMX), a nonspecific PDE inhibitor.
  • PDE9 expression has been demonstrated in kidney, liver, lung, brain, spleen, and small intestine.
  • PDE9 is also refened to as PDE8, but is distinct from PDE8 mentioned above. To date, two genes have been identified in the PDE9 family.
  • PDE 10 family members hydrolyze both cAMP and cGMP.
  • PDE 10s show expression in brain, thyroid and testis. [Soderling, S.H. et al. (1999); Fujishige, K. et al. (1999); Loughney, K. et al (1999)] .
  • PDE11 splice variants exhibit different regulatory sequences in the N-terminal region. This suggests the possibility of differential regulation of PDEl Is [Hetman JM, Robas N, Baxendale R, Fidock M, Phillips SC, Soderling SH, Beavo JA (2000)].
  • PDE4-specific inhibitor rolipram mentioned above as an anti-depressant, inhibits lipopolysaccharide-induced expression of TNF- ⁇ , and has been effective in treating multiple sclerosis in an animal model.
  • Other PDE4- specific inhibitors are being investigated for use as anti-inflammatory therapeutics, and efficacy in attenuating the late asthmatic response to allergen challenge has been demonstrated [Harbinson, et al. (1997)].
  • Inhibitors specific for the PDE3 family have been approved for treatment of congestive heart failure.
  • PDE5 inhibitors such as
  • Sildenafil are in use for treatment of penile erectile dysfunction [Tenett, N. et al. (1996)].
  • PDE5-inhibitors are under investigation as agents for cardiovascular therapy [Perry, M.J. and G.A. Higgs (1998)].
  • PDEs cyclic nucleotide levels have been suggested to influence proliferation of different cell types [Conti et al. (1995)]. For example, growth of the prostatic carcinoma cell lines DU145 and LNCaP was inhibited by cAMP derivatives and PDE inhibitors [Bang, Y.J. et al. (1994)]. Furthermore, PDEs have been implemented to additional cancers.
  • Non-specific inhibitors such as theophylline and pentoxifyUine, are cunently used in the treatment of respiratory and vascular disorders, respectively.
  • cAMP and cGMP play a central role in intracellular second messenger signaling.
  • the value as pharmaceutical targets has been proven for several PDEs.
  • Selective inhibitors have been developed as therapeutic agents for diseases such as cancer, heart failure, depression and sexual disfunction.
  • the identification of further disease implications of PDE species and their splice variants may lead to the development of specific inhibitors or modulators, or suggest new utilities for known compounds affecting PDEs. That in rum will provide additional pharmacological approaches to treat diseases and conditions in which alterations in cyclic nucleotide pathways are involved.
  • This diseases may include, but are not limited to, infections such as bacterial, fungal, protozoan, and viral infections, particularly those caused by HIV viruses, cancers, allergies including asthma, cardiovascular diseases including acute heart failure, hypotension, hypertension, angina pectoris, myocardial infarction, hematological diseases, genito-urinary diseases including urinary incontinence and benign prostate hyperplasia, osteoporosis, and peripheral and central nervous system disorders including pain, Alzheimer's disease and Parkinson's disease.
  • infections such as bacterial, fungal, protozoan, and viral infections, particularly those caused by HIV viruses, cancers, allergies including asthma, cardiovascular diseases including acute heart failure, hypotension, hypertension, angina pectoris, myocardial infarction, hematological diseases, genito-urinary diseases including urinary incontinence and benign prostate hyperplasia, osteoporosis, and peripheral and central nervous system disorders including pain, Alzheimer's disease and Parkinson's disease.
  • TaqMan is a recently developed technique, in which the release of a fluorescent reporter dye from a hybridisation probe in real-time during a polymerase chain reaction (PCR) is proportional to the accumulation of the PCR product. Quantification is based on the early, linear part of the reaction, and by determining the threshold cycle (CT), at which fluorescence above background is first detected.
  • CT threshold cycle
  • Gene expression technologies may be useful in several areas of drug discovery and development, such as target identification, lead optimization, and identification of mechanisms of action.
  • the TaqMan technology can be used to compare differences between expression profiles of normal tissue and diseased tissue.
  • Expression profiling has been used in identifying genes, which are up- or downregulated in a variety of diseases.
  • An interesting application of expression profiling is temporal monitoring of changes in gene expression during disease progression and drag treatment or in patients versus healthy individuals.
  • the premise in this approach is that changes in pattern of gene expression in response to physiological or environmental stimuli (e.g., drugs) may serve as indirect clues about disease-causing genes or drug targets.
  • the effects of drags with established efficacy on global gene expression patterns may provide a guidepost, or a genetic signature, against wliich a new drug candidate can be compared.
  • the nucleotide sequence of PDE2a is accessible in public databases by the accession number NM_002599 and is given in SEQ LD NO:l.
  • PDE2a is depicted in SEQ LD NO:2. Rosman et al. (1997) cloned a cDNA conesponding to human PDE2A.
  • the PDE2A gene encodes a 941 amino acid polypeptide with a predicted molecular mass of 106 kD.
  • the protein sequence is 90% identical to bovine and rat PDE2A sequences.
  • Northern blot analysis showed that PDE2A was expressed as a 4.2-kb mRNA in heart, placenta, lung, skeletal muscle, kidney and pancreas, with greatest expression in brain. Expression studies revealed that PDE2A hydrolyzes cAMP and cGMP and is inhibited by the PDE2A-specific inhibitor.
  • PDE2A immunostaining was also absent from luminal endothelial cells of large vessels, such as aorta, pulmonary, and renal arteries, but was present in the endothelial cells of the vasa vasorum. PDE2A immunostaining was detected in the endothelial cells of a variety of microvessels, including those in renal and cardiac interstitial spaces, renal glomerulus, skin, brain, and liver.
  • PDE2A is published in EP 1097719, US 6015677, US 5776752.
  • PDE2A shows the highest homology (31%) to the human PDEl 1 A4 as shown in example 1.
  • the invention relates to novel disease associations of PDE2a polypeptides and polynucleotides.
  • the invention also relates to novel methods of screening for therapeutic agents for the treatment of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal.
  • the invention also relates to pharmaceutical compositions for the treatment of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal comprising a PDE2a polypeptide, a PDE2a polynucleotide, or regulators of PDE2a or modulators of PDE2a activity.
  • the invention further comprises methods of diagnosing hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal.
  • Fig. 1 shows the nucleotide sequence of a PDE2A polynucleotide (SEQ LD NO: 1).
  • Fig. 2 shows the amino acid sequence of a PDE2A polypeptide (SEQ ID NO:2).
  • Fig. 3 shows the nucleotide sequence of a primer useful for the invention (SEQ LD NO:3).
  • Fig. 4 shows the nucleotide sequence of a primer useful for the invention (SEQ LD
  • Fig. 5 shows a nucleotide sequence useful as a probe to detect proteins of the invention (SEQ LD NO:5).
  • oligonucleotide is a stretch of nucleotide residues which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal, or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides.
  • Probes may be derived from naturally occurring or recombinant single- or double- stranded nucleic acids or may be chemically synthesized. They are useful in detecting the presence of identical or similar sequences. Such probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Nucleic acid probes may be used in southern, northern or in situ hybridizations to determine whether DNA or RNA encoding a certain protein is present in a cell type, tissue, or organ.
  • a “fragment of a polynucleotide” is a nucleic acid that comprises all or any part of a given nucleotide molecule, the fragment having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb.
  • Reporter molecules are radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents which associate with a particular nucleotide or amino acid sequence, thereby establishing the presence of a certain sequence, or allowing for the quantification of a certain sequence.
  • Chimeric molecules may be constructed by introducing all or part of the nucleotide sequence of this invention into a vector containing additional nucleic acid sequence which might be expected to change any one or several of the following PDE2a characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.
  • PDE2a polypeptide refers to those forms, fragments, or domains of a PDE2a polypeptide which retain the biological and/or antigenic activity of a PDE2a polypeptide.
  • Naturally occurring PDE2a polypeptide refers to a polypeptide produced by cells which have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including but not limited to acetylation, carboxylation, giycosylation, phosphorylation, lipidation and acylation.
  • Derivative refers to polypeptides which have been chemically modified by techniques such as ubiquitination, labeling (see above), pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.
  • Constant amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
  • a “signal sequence” or “leader sequence” can be used, when desired, to direct the polypeptide through a membrane of a cell. Such a sequence may be naturally present - li ⁇
  • oligopeptide is a short stretch of a ino acid residues and may be expressed from an oligonucleotide.
  • Ohgopeptides comprise a stretch of amino acid residues of at least 3, 5, 10 amino acids and at most 10, 15, 25 amino acids, typically of at least 9 to 13 arnino acids, and of sufficient length to display biological and/or antigenic activity.
  • inhibitor is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists.
  • Standard expression is a quantitative or qualitative measurement for comparison. It is based on a statistically appropriate number of normal samples and is created to use as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles.
  • Animal as used herein may be defined to include human, domestic (e.g., cats, dogs, etc.), agricultural (e.g., cows, horses, sheep, etc.) or test species (e.g., mouse, rat, rabbit, etc.).
  • domestic e.g., cats, dogs, etc.
  • agricultural e.g., cows, horses, sheep, etc.
  • test species e.g., mouse, rat, rabbit, etc.
  • PDE2a polynucleotide within the meaning of the invention, shall be understood as being a nucleic acid molecule selected from a group consisting of
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ LD NO: 2,
  • nucleic acid molecules comprising the sequence of SEQ D NO: 1,
  • nucleic acid molecules having the sequence of SEQ LD NO: 1 (iii) nucleic acid molecules having the sequence of SEQ LD NO: 1 , (iv) nucleic acid molecules the complementary strand of wliich hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii); and
  • nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code;
  • polypeptide encoded by said nucleic acid molecule has PDE2a activity.
  • PDE2a polypeptide within the meaning of the invention, shall be understood as being a polypeptide selected from a group consisting of
  • polypeptides comprising the sequence of SEQ LD NO: 2
  • polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii);
  • polypeptide has PDE2a activity.
  • nucleotide sequences encoding a PDE2a have numerous applications in techniques Icnown to those skilled in the art of molecular biology.
  • nucleotide sequences encoding a PDE2a disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art.
  • nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are cunently known, e.g., the triplet genetic code, specific base pair interactions, etc.
  • nucleotide sequences which encode a PDE2a, its derivatives or its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occirrring PDE2a polynucleotide under stringent conditions, it may be advantageous to produce nucleotide sequences encoding PDE2a polypeptides or its derivatives possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • Nucleotide sequences encoding a PDE2a polypeptide may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA techniques.
  • Useful nucleotide sequences for joining to PDE2a polynucleotides include an assortment of cloning vectors such as plasmids, cosmids, lambda phage derivatives, phagemids, and the like.
  • Vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, etc. In general, vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for one or more host cell systems.
  • Another aspect of the subject invention is to provide for PDE2a-specific hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding PDE2a. Such probes may also be used for the detection of similar PDE encoding sequences and should preferably show at least 40% nucleotide identity to PDE2a polynucleotides.
  • the hybridization probes of the subject invention may be derived from the nucleotide sequence presented as SEQ LD NO: 1 or from genomic sequences including promoter, enhancers or introns of the native gene. Hybridization probes may be labelled by a variety of reporter molecules using techniques well known in the art.
  • the invention relates to nucleic acid sequences that hybridize with such
  • PDE2a encoding nucleic acid sequences under stringent conditions.
  • Stringent conditions refers to conditions that allow for the hybridization of substantially related nucleic acid sequences. For instance, such conditions will generally allow hybridization of sequence with at least about 85% sequence identity, preferably with at least about 90% sequence identity, more preferably with at least about 95% sequence identity. Hybridization conditions and probes can be adjusted in well-characterized ways to achieve selective hybridization of human-derived probes. Stringent conditions, within the meaning of the invention are 65°C in a buffer containing 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7 % (w/v) SDS. Nucleic acid molecules that will hybridize to PDE2a polynucleotides under stringent conditions can be identified functionally.
  • examples of the uses for hybridization probes include: histochemical uses such as identifying tissues that express PDE2a; measuring mRNA levels, for instance to identify a sample's tissue type or to identify cells that express abnormal levels of PDE2a; and detecting polymorphisms of PDE2a.
  • PCR provides additional uses for oligonucleotides based upon the nucleotide sequence which encodes PDE2a.
  • probes used in PCR may be of recombinant origin, chemically synthesized, or a mixture of both.
  • Oligomers may comprise discrete nucleotide sequences employed under optimized conditions for identification of PDE2a in specific tissues or diagnostic use. The same two oligomers, a nested set of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for identification of closely related DNAs or RNAs.
  • PCR primers i.e., preparations of primers that are heterogeneous at given sequence locations, can be designed to amplify nucleic acid sequences that are highly homologous to, but not identical with PDE2a.
  • Strategies are now available that allow for only one of the primers to be required to specifically hybridize with a known sequence.
  • appropriate nucleic acid primers can be ligated to the nucleic acid sought to be amplified to provide the hybridization partner for one of the primers. In this way, only one of the primers need be based on the sequence of the nucleic acid sought to be amplified.
  • PCR methods for amplifying nucleic acid will utilize at least two primers.
  • One of these primers will be capable of hybridizing to a first sfrand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first direction.
  • the other will be capable of hybridizing the reciprocal sequence of the first strand (if the sequence to be amplified is single stranded, this sequence will initially be hypothetical, but will be synthesized in the first amplification cycle) and of priming nucleic acid synthesis from that strand in the direction opposite the first direction and towards the site of hybridization for the first primer.
  • Conditions for conducting such amplifications particularly under prefened stringent hybridization conditions, are well known.
  • RNA polymerase as T7 or SP6 RNA polymerase and the appropriate reporter molecules.
  • nucleic acid sequence can be inserted into any of the many available DNA vectors and their respective host cells using techniques which are well Icnown in the art.
  • synthetic chemistry may be used to introduce mutations into the nucleotide sequence. Alternately, a portion of sequence in which a mutation is desired can be synthesized and recombined with longer portion of an existing genomic or recombinant sequence.
  • PDE2a polynucleotides may be used to produce a purified oligo-or polypeptide using well known methods of recombinant DNA technology.
  • the oligopeptide may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which the nucleotide sequence was derived or from a different species. Advantages of producing an oligonucleotide by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures.
  • Quantitative determinations of nucleic acids An important step in the molecular genetic analysis of human disease is often the enumeration of the copy number of a nucleis acid or the relative expression of a gene in particular tissues.
  • Chromosome-based techniques such as comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH) facilitate efforts to cytogenetically localize genomic regions that are altered in tumor cells. Regions of genomic alteration can be nanowed further using loss of heterozygosity analysis (LOH), in which disease DNA is analyzed and compared with normal DNA for the loss of a heterozygous polymorphic marker.
  • LOH heterozygosity analysis
  • the first experiments used restriction fragment length polymorphisms (RFLPs) [Johnson, (1989)], or hyper- variable minisatellite DNA [Barnes, 2000].
  • RFLPs restriction fragment length polymorphisms
  • RFLPs restriction fragment length polymorphisms
  • Barnes, 2000 hyper- variable minisatellite DNA
  • LOH has been performed primarily using PCR amplification of microsatellite markers and electrophoresis of the radio labelled [Jeffreys, (1985)] or fluorescently labelled PCR products [Weber,
  • a gene sequence contained in all samples at relatively constant quantity is typically utilized for sample amplification efficiency normalization.
  • This approach suffers from several drawbacks.
  • the method requires that each sample has equal input amounts of the nucleic acid and that the amplification efficiency between samples is identical until the time of analysis.
  • Another method called quantitative competitive (QC)-PCR relies on the inclusion of an internal control competitor in each reaction [Piatak, (1993), BioTechniques].
  • the efficiency of each reaction is normalized to the internal competitor.
  • a known amount of internal competitor is typically added to each sample.
  • the unknown target PCR product is compared with the known competitor PCR product to obtain relative quantitation.
  • a difficulty with this general approach lies in developing an internal control that amplifies with the same efficiency than the target molecule.
  • Fluorogenic nuclease assays are a real time quantitation method that uses a probe to monitor formation of amplification product.
  • the basis for this method of monitoring the formation of amplification product is to measure continuously PCR product accumulation using a dual-labelled fluorogenic oligonucleotide probe, an approach frequently refened to in the literature simply as the "TaqMan method” [Piatak,(1993), Science; Heid, (1996); Gibson, (1996); Holland. (1991)].
  • the probe used in such assays is typically a short (about 20-25 bases) oligonucleotide that is labeled with two different fluorescent dyes.
  • the 5' terminus of the probe is attached to a reporter dye and the 3' terminus is attached to a quenching dye, although the dyes could be attached at other locations on the probe as well.
  • the probe is designed to have at least substantial sequence complementarity with the probe binding site. Upstream and downstream PCR primers which bind to flanking regions of the locus are added to the reaction mixture. When the probe is intact, energy transfer between the two fluorophors occurs and the quencher quenches emission from the reporter.
  • the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase such as Taq polymerase, thereby releasing the reporter from the oligonucleotide-quencher and resulting in an increase of reporter emission intensity wliich can be measured by an appropriate detector.
  • a nucleic acid polymerase such as Taq polymerase
  • One detector which is specifically adapted for measuring fluorescence emissions such as those created during a fluorogenic assay is the ABI 7700 or 4700 HT manufactured by Applied Biosystems, Inc. in Foster City, Calif.
  • the ABI 7700 uses fiber optics connected with each well in a 96-or 384 well PCR tube anangement.
  • the instrument includes a laser for exciting the labels and is capable of measuring the fluorescence spectra intensity from each tube with continuous monitoring during PCR amplification. Each tube is re-examined every 8.5 seconds.
  • Computer software provided with the instrument is capable of recording the fluorescence intensity of reporter and quencher over the course of the amplification. The recorded values will then be used to calculate the increase in normalized reporter emission intensity on a continuous basis. The increase in emission intensity is plotted versus time, i.e., the number of amplification cycles, to produce a continuous measure of amplification.
  • the amplification plot is examined at a point during the log phase of product accumulation. This is accomplished by assigning a fluorescence threshold intensity above background and determining the point at which each amplification plot crosses the threshold (defined as the threshold cycle number or Ct). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube. Assuming that each reaction functions at 100% PCR efficiency, a difference of one Ct represents a two-fold difference in the amount of starting template.
  • the fluorescence value can be used in conjunction with a standard curve to determine the amount of amplification product present.
  • amplification product (wliich is double stranded) binds dye molecules in solution to form a complex.
  • dyes it is possible to distinguish between dye molecules free in solution and dye molecules bound to amplification product.
  • certain dyes fluoresce only when bound to amplification product. Examples of dyes which can be used in methods of this general type include, but are not limited to, Syber Green.TM. and Pico Green from Molecular Probes, Inc.
  • These detection methods involve some alteration to the structure or conformation of a probe hybridized to the locus between the amplification primer pair.
  • the alteration is caused by the template-dependent extension catalyzed by a nucleic acid polymerase during the amplification process.
  • the alteration generates a detectable signal which is an indirect measure of the amount of amplification product formed.
  • some methods involve the degradation or digestion of the probe during the extension reaction. These methods are a consequence of the 5'-3' nuclease activity associated with some nucleic acid polymerases. Polymerases having this activity cleave mononucleotides or small oligonucleotides from an oligonucleotide probe annealed to its complementary sequence located within the locus.
  • the 3' end of the upstream primer provides the initial binding site for the nucleic acid polymerase.
  • the nucleic acid polymerase displaces a portion of the 5' end of the probe and through its nuclease activity cleaves mononucleotides or oligonucleotides from the probe.
  • the upstream primer and the probe can be designed such that they anneal to the complementary strand in close proximity to one another. In fact, the 3' end of the upstream primer and the 5' end of the probe may abut one another. In this situation, extension of the upstream primer is not necessary in order for the nucleic acid polymerase to begin cleaving the probe. In the case in which mtervening nucleotides separate the upstream primer and the probe, extension of the primer is necessary before the nucleic acid polymerase encounters the 5' end of the probe.
  • the 5'-3' exonuclease activity of the nucleic acid polymerase begins cleaving mononucleotides or oligonucleotides from the 5' end of the probe. Digestion of the probe continues until the remaining portion of the probe dissociates from the complementary strand.
  • the two end sections can hybridize with each other to form a hairpin loop.
  • the reporter and quencher dye are in sufficiently close proximity that fluorescence from the reporter dye is effectively quenched by the quencher dye.
  • Hybridized probe in contrast, results in a linearized conformation in which the extent of quenching is decreased.
  • the labeled probe is selected so that its sequence is substantially complementary to a segment of the test locus or a reference locus. As indicated above, the nucleic acid site to which the probe binds should be located between the primer binding sites for the upstream and downstream amplification primers. Primers
  • the primers used in the amplification are selected so as to be capable of hybridizing to sequences at flanking regions of the locus being amplified.
  • the primers are chosen to have at least substantial complementarity with the different strands of the nucleic acid being amplified.
  • the primers are selected in such that they flank the probe, i.e. are located upstream and downstream of the probe.
  • the primer must have sufficient length so that it is capable of priming the synthesis of extension products in the presence of an agent for polymerization.
  • the length and composition of the primer depends on many parameters, including, for example, the temperature at which the annealing reaction is conducted, proximity of the probe binding site to that of the primer, relative concentrations of the primer and probe and the particular nucleic acid composition of the probe.
  • the primer includes
  • the length of the primer may be more or less depending on the complexity of the primer binding site and the factors listed above.
  • the labels used for labeling the probes or primers of the cunent invention and which can provide the signal conesponding to the quantity of amplification product can take a variety of forms.
  • a fluorescent signal is one signal which can be measured.
  • measurements may also be made, for example, by monitoring radioactivity, colorimetry, absorption, magnetic parameters, or enzymatic activity.
  • labels which can be employed include, but are not limited to, fluorophors, chromophores, radioactive isotopes, electron dense reagents, enzymes, and ligands having specific binding partners (e.g., biotin-avidin).
  • a number of labels useful for attachment to probes or primers are commercially available including fluorescein and various fluorescein derivatives such as FAM, HEX, TET and JOE (all which are available from Applied Biosystems, Foster City, Calif.); lucifer yellow, and coumarin derivatives.
  • Labels may be attached to the probe or primer using a variety of techniques and can be attached at the 5' end, and/or the 3' end and/or at an internal nucleotide.
  • the label can also be attached to spacer arms of various sizes which are attached to the probe or primer. These spacer arms are useful for obtaining a desired distance between multiple labels attached to the probe or primer.
  • a single label may be utilized; whereas, in other instances, such as with the 5' fluorogenic nuclease assays for example, two or more labels are attached to the probe.
  • the probe includes multiple labels, it is generally advisable to maintain spacing between the labels wliich is sufficient to permit separation of the labels during digestion of the probe through the 5'-3' nuclease activity of the nucleic acid polymerase.
  • a number of diseases are associated with changes in the copy number of a certain gene.
  • the real-time PCR method can be used to determine if the patient has copy number alterations which are known to be linked with diseases that are associated with the symptoms the patient has.
  • Fusion proteins are useful for generating antibodies against PDE2a polypeptides and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of PDE2a polypeptides. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a PDE2a fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment can comprise at least 54, 75, 100, 125, 139, 150, 175, 200, 225, 250, 275, 300, 325 or 350 contiguous amino acids of SEQ LD NO: 2 or of a biologically active variant, such as those described above.
  • the first polypeptide segment also can comprise full-length PDE2a.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include, but are not limited to ⁇ -galactosidase, ⁇ -glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chlorarnphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV- G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex vims (HSV) BP16 protein fusions.
  • a fusion protein also can be engineered to contain a cleavage site located adjacent to the PDE2a.
  • a naturally occurring PDE2a polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated PDE2a polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise PDE2a nucleotide sequences. Isolated polynucleotides are in preparations wliich are free or at least 70, 80, or 90% free of other molecules.
  • PDE2a cDNA molecules can be made with standard molecular biology techniques, using PDE2a mRNA as a template. PDE2a cDNA molecules can thereafter be replicated using molecular biology techniques known in the art. An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
  • PCR-based methods can be used to extend nucleic acid sequences encoding human PDE2a, for example to detect upstream sequences of PDE2a gene such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus. Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region.
  • Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate equipment and software (e.g., GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • PDE2a can be obtained, for example, by purification from human cells, by expression of PDE2a polynucleotides, or by direct chemical synthesis.
  • PDE2a can be purified from any human cell which expresses the enzyme, including those which have been transfected with expression constructs which express PDE2a.
  • a purified PDE2a is separated from other compounds which normally associate with PDE2a in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • PDE2a polynucleotides can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding PDE2a and appropriate transcriptional and franslational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding PDE2a. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculo virus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic vims, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculo virus)
  • plant cell systems transformed with virus expression vectors e.g., cauliflower mosaic virus, CaMV
  • control elements or regulatory sequences are those non-translated regions of the vector - enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRLPT phagemid (Sfratagene, LaJolla, Calif.) or pSPORTl plasmid (Life
  • the baculoviras polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding PDE2a, vectors based on SN40 or EBN can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected.
  • vectors wliich direct high level expression of fusion proteins that are readily purified can be used.
  • Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRLPT (Sfratagene).
  • BLUESCRLPT Sfratagene
  • a sequence encoding PDE2a can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ - galactosidase so that a hybrid protein is produced.
  • pLN vectors or pGEX vectors also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione- agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • sequences encoding PDE2a can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used. These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.
  • An insect system also can be used to express PDE2a.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • Sequences encoding PDE2a can be cloned into a non-essential region of the vims, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • Successful insertion of PDE2a will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which PDE2a can be expressed.
  • a number of viral-based expression systems can be used to express PDE2a in mammalian host cells.
  • sequences encoding PDE2a can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing PDE2a in infected host cells [Engelhard, 1994)].
  • transcription enhancers such as the Rous sarcoma vims (RSN) enhancer, can be used to increase expression in mammalian host cells.
  • RSN Rous sarcoma vims
  • HACs Human artificial chromosomes
  • HACs also can be used to deliver larger fragments of D ⁇ A than can be contained and expressed in a plasmid.
  • HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding PDE2a. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding PDE2a, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals including the ATG initiation codon
  • the initiation codon should be in the conect reading frame to ensure translation of the entire insert.
  • Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic.
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed PDE2a in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, giycosylation, phosphorylation, lipidation, and acylation.
  • Post- translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate conect insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the
  • ATCC American Type Culture Collection
  • Stable expression is prefened for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express PDE2a can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced PDE2a sequences.
  • Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems can be used to recover transformed cell lines.
  • genes wliich can be employed in tk ⁇ or aprf cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate [Lowy, (1980)]
  • npt confers resistance to the aminoglycosides, neomycin and G-418 [Wigler, (1980)]
  • als and pat confer resistance to cHorsulfuron and phosphinotricin acetyltransferase, respectively [Colbere-Garapin, 1981]. Additional selectable genes have been described.
  • trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine.
  • Nisible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify fransformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system
  • marker gene expression suggests that a PDE2a polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding PDE2a is inserted within a marker gene sequence, transformed cells containing sequences which encode PDE2a can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding PDE2a under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of PDE2a polynucleotide.
  • host cells which contain a PDE2a polynucleotide and which express PDE2a can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • these procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding PDE2a can be detected by DNA-DNA or DNA- RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding PDE2a.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected
  • a variety of protocols for detecting and measuring the expression of PDE2a, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two- site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on PDE2a can be used, or a competitive binding assay can be employed.
  • a wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding PDE2a include oligolabeling, nick translation, end- labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding PDE2a can be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6.
  • reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as subsfrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with PDE2a polynucleotides can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing PDE2a polynucleotides can be designed to contain signal sequences which direct secretion of soluble PDE2a through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound PDE2a.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and PDE2a also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing PDE2a and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The stidine residues facilitate purification by LMAC (immobilized metal ion affinity chromatography) Maddox, (1983)], while the enterokinase cleavage site provides a means for purifying PDE2a from the fusion protein [Porath, (1992)].
  • LMAC immobilized metal ion affinity chromatography
  • Sequences encoding PDE2a can be synthesized, in whole or in part, using chemical methods well known in the art.
  • PDE2a itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of PDE2a can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography.
  • the composition of a synthetic PDE2a can be confirmed by amino acid analysis or sequencing. Additionally, any portion of the amino acid sequence of PDE2a can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein. Production of Altered Polypeptides
  • codons prefened by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences refened to herein can be engineered using methods generally known in the art to alter PDE2a polynucleotides for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter giycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Any type of antibody known in the art can be generated to bind specifically to an epitope of PDE2a.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of PDE2a.
  • Fab fragment antigen binding protein
  • F(ab') 2 fragment antigen binding protein
  • Fv fragment antigen binding protein
  • epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acid.
  • An antibody which specifically binds to an epitope of PDE2a can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the PDE2a immunogen.
  • an antibody which specifically binds to PDE2a provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies wliich specifically bind to PDE2a do not detect other proteins in immunochemical assays and can immunoprecipitate PDE2a from solution.
  • PDE2a can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, PDE2a can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response.
  • a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • mineral gels e.g., aluminum hydroxide
  • surface active substances e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Gueri ⁇
  • Corynebacterium parvum are especially useful.
  • Monoclonal antibodies which specifically bind to PDE2a can be prepared using any technique wliich provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique [Roberge, (1995)].
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
  • Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues wliich differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • Antibodies which specifically bind to PDE2a can contain antigen bmding sites which are either partially or fully humanized, as disclosed in
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to PDE2a.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries.
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template.
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tefravalent, bispecific single-chain antibodies is taught.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology.
  • Antibodies which specifically bind to PDE2a also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents. Other types of antibodies can be constracted and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins wliich are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which PDE2a is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of PDE2a gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkyl- phosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
  • Modifications of PDE2a gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the PDE2a gene.
  • Ohgonucleotides derived from the transcription initiation site e.g., between positions -10 and +10 from the start site, are prefened.
  • inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a PDE2a polynucleotide, each separated by a sfretch of contiguous nucleotides which are not complementary to adjacent PDE2a nucleotides, can provide sufficient targeting specificity for PDE2a mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non- complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular PDE2a polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a PDE2a polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • intemucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • These modified oligonucleotides can be prepared by methods well known in the art. Ribozymes
  • Ribozymes are RNA molecules with catalytic activity [Uhlmann, (1987)]. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a PDE2a polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from a PDE2a polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art.
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.
  • ribozyme cleavage sites within a PDE2a RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between
  • ribonucleotides conesponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable.
  • Suitability of candidate PDE2a RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • the nucleotide sequences shown in SEQ ID NO: 1 and its complement provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target.
  • the hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease PDE2a expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding
  • DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells (U.S. 5,641,673). Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • transcriptional regulatory elements such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal
  • Regulators as used herein refer to compounds that affect the activity of PDE2a in vivo and/or in vitro. Regulators can be agonists and antagonists of PDE2a polypeptide and can be compounds that exhert their effect on the PDE2a activity via the enzymatic activity, expression, post-franslational modifications or by other means.
  • Agonists of PDE2a are molecules which, when bound to PDE2a, increase or prolong the activity of PDE2a.
  • Agonists of PDE2a include proteins, nucleic acids, carbohydrates, small molecules, or any other molecule which activate PDE2a.
  • Antagonists of PDE2a are molecules which, when bound to PDE2a, decrease the amount or the duration of the activity of PDE2a.
  • Antagonists include proteins, nucleic acids, carbohydrates, antibodies, small molecules, or any other molecule which decrease the activity of PDE2a.
  • modulate refers to a change in the activity of PDE2a polypeptide. For example, modulation may cause an increase or a decrease in enzymatic activity, binding characteristics, or any other biological, functional, or immunological properties of PDE2a.
  • the terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigenic determinant or epitope). For example, if an antibody is specific for epitope "A" the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • the invention provides methods (also refened to herein as "screening assays") for identifying compounds which can be used for the treatment of hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases.
  • the methods entail the identification of candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other molecules) wliich bind to PDE2a and/or have a stimulatory or inhibitory effect on the biological activity of PDE2a or its expression and then determining which of these compounds have an effect on symptoms or diseases regarding the hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito- urological disorders and inflammation diseases in an in vivo assay.
  • candidate or test compounds or agents e.g., peptides, peptidomimetics, small molecules or other molecules
  • Candidate or test compounds or agents which bind to PDE2a and/or have a stimulatory or inhibitory effect on the activity or the expression of PDE2a are identified either in assays that employ cells which express PDE2a (cell-based assays) or in assays with isolated PDE2a (cell-free assays).
  • the various assays can employ a variety of variants of PDE2a (e.g., full-length PDE2a, a biologically active fragment of PDE2a, or a fusion protein which includes all or a portion of PDE2a).
  • PDE2a can be derived from any suitable mammalian species (e.g., human PDE2a, rat PDE2a or murine PDE2a).
  • the assay can be a binding assay entailing direct or mdirect measurement of the bmding of a test compound or a known PDE2a ligand to
  • the assay can also be an activity assay entailing direct or indirect measurement of the activity of PDE2a.
  • the assay can also be an expression assay entailing direct or indirect measurement of the expression of PDE2a mRNA or PDE2a protein.
  • the various screening assays are combined with an in vivo assay entailing measuring the effect of the test compound on the symptoms of hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases.
  • the present invention includes biochemical, cell free assays that allow the identification of inhibitors and agonists of PDEs suitable as lead structures for pharmacological drug development.
  • Such assays involve contacting a form of PDE2a (e.g., full-length PDE2a, a biologically active fragment of PDE2a, or a fusion protein comprising all or a portion of PDE2a) with a test compound and determining the ability of the test compound to act as an antagonist (preferably) or an agonist of the enzymatic activity of PDE2a.
  • the assay includes monitoring the PDE activity of PDE2a by measuring the conversion of either cAMP or cGMP to its nucleoside monophosphate after contacting PDE2a with a test compound.
  • cAMP and cGMP levels can be measured by the use of the tritium containing compounds 3 HcAMP and 3 HcGMP as described in [Hansen, R.S., and Beavo, J.A., PNAS USA1982;79: 2788-92].
  • SPA microtiter plate-based scintillation proximity assay
  • the phosphodiesterase activity of the recombinant protein can be assayed using a commercially available SPA kit (Amersham Pharmacia).
  • the PDE enzyme hydrolyzes cyclic nucleotides, e.g. cAMP and cGMP to their linear counterparts.
  • the SPA assay utilizes the tritiated cyclic nucleotides [ 3 H]cAMP or [ 3 H]cGMP, and is based upon the selective interaction of the tritiated non cyclic product with the SPA beads whereas the cyclic substrates are not effectively binding. Radiolabelled product bound to the scintillation beads generates light that can be analyzed in a scintillation counter.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of PDE2a.
  • Such assays can employ full-length PDE2a, a biologically active fragment of PDE2a, or a fusion protein which includes all or a portion of PDE2a.
  • the test compound can be obtained by any suitable means, e.g., from conventional compound libraries.
  • Determining the ability of the test compound to modulate the activity of PDE2a can be accomphshed, for example, by determining the ability of PDE2a to bind to or interact with a target molecule.
  • the target molecule can be a molecule with which PDE2a binds or interacts with in nature.
  • the target molecule can be a component of a signal transduction pathway which facilitates fransduction of an extracellular signal.
  • the target PDE2a molecule can be, for example, a second intracellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with PDE2a.
  • Determining the ability of PDE2a to bind to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca 2+ , diacylglycerol, IP 3 , etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response.
  • a reporter gene e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • PDE2a or a PDE2a target molecule
  • binding of a test compound to PDE2a it may be desirable to immobilize PDE2a (or a PDE2a target molecule) to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to PDE2a (or a PDE2a target molecule) to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to PDE2a (or a PDE2a target molecule) to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to
  • PDE2a or interaction of PDE2a with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants.
  • vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase (GST) fusion proteins or glutathione-S- transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St.
  • the test compound or the test compound and either the non- adsorbed target protein or PDE2a are then combined with the test compound or the test compound and either the non- adsorbed target protein or PDE2a, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • conditions conducive to complex formation e.g., at physiological conditions for salt and pH.
  • the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of binding or activity of PDE2a can be determined using standard techniques.
  • PDE2a or its target molecule can be immobilized utilizing conjugation of biotin and sfreptavidin.
  • Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, 111.), and immobilized in the wells of streptavidin- coated plates (Pierce Chemical).
  • antibodies reactive with PDE2a or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with PDE2a or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with PDE2a or target molecule.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564.
  • large numbers of different small test compounds are synthesized on a solid subsfrate, such as plastic pins or some other surface.
  • the test compounds are reacted with PDE2a, or fragments thereof, and washed.
  • Bound PDE2a is then detected by methods well Icnown in the art.
  • Purified PDE2a can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non- neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • the screening assay can also involve monitoring the expression of PDE2a.
  • regulators of expression of PDE2a can be identified in a method in which a cell is contacted with a candidate compound and the expression of PDE2a protein or mRNA in the cell is determined. The level of expression of PDE2a protein or mRNA the presence of the candidate compound is compared to the level of expression of PDE2a protein or mRNA in the absence of the candidate compound. The candidate compound can then be identified as a regulator of expression of
  • PDE2a based on this comparison. For example, when expression of PDE2a protein or mRNA protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of PDE2a protein or mRNA expression. Alternatively, when expression of PDE2a protein or mRNA is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of PDE2a protein or rnRNA expression.
  • the level of PDE2a protein or mRNA expression in the cells can be determined by methods described below.
  • the test compound is preferably a small molecule which binds to and occupies the active site of PDE2a polypeptide, thereby making the ligand binding site inaccessible to substrate such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • Potential ligands which bind to a polypeptide of the invention include, but are not limited to, the natural ligands of known PDE2a PDEs and analogues or derivatives thereof.
  • either the test compound or the PDE2a polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound which is bound to PDE2a polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • binding of a test compound to a PDE2a polypeptide can be determined without labeling either of the interactants.
  • a microphysiometer can be used to detect binding of a test compound with a PDE2a polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • a microphysiometer is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and PDE2a [Haseloff, (1988)].
  • Determiriing the abihty of a test compound to bind to PDE2a also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) [McConnell, (1992); Sjolander, (1991)].
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcoreTM). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • a PDE2a-like polypeptide can be used as a
  • bait protein in a two-hybrid assay or three-hybrid assay [Szabo, (1995); U.S. 5,283,317), to identify other proteins which bind to or interact with PDE2a and modulate its activity.
  • the two-hybrid system is based on the modular nature of most franscription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding PDE2a can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey" or "sample” can be fused to a polynucleotide that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the franscription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with PDE2a.
  • a reporter gene e.g., LacZ
  • either the PDE2a (or polynucleotide) or the test compound can be bound to a solid support.
  • suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • any method known in the art can be used to attach PDE2a-like polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covending linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an anay, so that the location of individual test compounds can be tracked. Binding of a test compound to PDE2a (or a polynucleotide encoding for PDE2a) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • PDE2a is a fusion protein comprising a domain that allows binding of PDE2a to a solid support.
  • glutathione-S-fransferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed PDE2a; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • PDE2a or a polynucleotide encoding PDE2a
  • a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated PDE2a (or a polynucleotide encoding biotinylated PDE2a) or test compounds can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin- coated plates (Pierce Chemical).
  • biotinylation kit Pierce Chemicals, Rockford, 111.
  • streptavidin- coated plates Piereptavidin- coated plates
  • antibodies which specifically bind to PDE2a, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of PDE2a can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to PDE2a polypeptide or test compound, enzyme- linked assays which rely on detecting an activity of PDE2a polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Screening for test compounds wliich bind to a PDE2a polypeptide or polynucleotide also can be carried out in an intact cell.
  • Any cell which comprises a PDE2a polypeptide or polynucleotide can be used in a cell-based assay system.
  • a PDE2a polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to PDE2a or a polynucleotide encoding PDE2a is determined as described above. Functional Assays
  • Test compounds can be tested for the ability to increase or decrease PDE2a activity of a PDE2a polypeptide.
  • the PDE2a activity can be measured, for example, using methods described in the specific examples, below.
  • PDE2a activity can be measured after contacting either a purified PDE2a or an intact cell with a test compound.
  • a test compound which decreases PDE2a activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing PDE2a activity.
  • a test compound which increases PDE2a activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing PDE2a activity.
  • test compounds which increase or decrease PDE2a gene expression are identified.
  • the term "conelates with expression of a polynucleotide” indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding PDE2a, by northern analysis or realtime PCR is indicative of the presence of nucleic acids encoding PDE2a in a sample, and thereby conelates with expression of the transcript from the polynucleotide encoding PDE2a.
  • microanay refers to an anay of distinct polynucleotides or oligonucleotides anayed on a substrate, such as paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • a PDE2a polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of PDE2a polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a regulator of expression based on this comparison.
  • test compound when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of PDE2a mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of PDE2a polynucleotide can be determined, for example, using a variety of techniques Icnown in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labelled amino acids into PDE2a.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell which expresses PDE2a polynucleotide can be used in a cell-based assay system.
  • the PDE2a polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line can be used.
  • test compounds for use in the screening assays of the invention can be obtained from any suitable source, e.g., conventional compound libraries.
  • the test compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds [Lam, (1997)]. Examples of methods for the synthesis of molecular libraries can be found in the art. Libraries of compounds may be presented in solution or on beads, bacteria, spores, plasmids or phage.
  • Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate PDE2a expression or activity. Having identified such a compound or composition, the active sites or regions are identified. Such sites might typically be the enzymatic active site, regulator binding sites, or ligand binding sites.
  • the active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found.
  • the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular stracture. On the other hand, solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of stracture determination can be used to obtain partial or complete geometric structures.
  • the geometric structures may be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure determined.
  • the methods of computer based numerical modeling can be used to complete the structure or improve its accuracy.
  • Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields Icnown in physical chemistry.
  • the incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
  • candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential PDE2a modulating compounds.
  • these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand.
  • the composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modeling methods described above applied to the new composition.
  • the altered stracture is then compared to the active site structure of the compound to determine if an improved fit or interaction results. Ln this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.
  • PDE2a is expressed in various human tissues.
  • Neurology CNS disorders include disorders of the central nervous system as well as disorders of the peripheral nervous system.
  • CNS disorders include, but are not limited to brain injuries, cerebrovascular diseases and their consequences, Parkinson's disease, corticobasal degeneration, motor neuron disease, dementia, including ALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke, post-traumatic brain injury, and small-vessel cerebrovascular disease.
  • Dementias such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia and Parkinsonism linked to chromosome 17, frontotemporal dementias, including Pick's disease, progressive nuclear palsy, corticobasal degeneration, Huntington's disease, thalamic degeneration, Creutzfeld- Jakob dementia, HIN dementia, schizophrenia with dementia, and Korsakoff s psychosis, within the meaning of the definition are also considered to be C ⁇ S disorders.
  • cognitive-related disorders such as mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, vascular cognitive impairment, attention deficit disorders, attention deficit hyperactivity disorders, and memory disturbances in children with learning disabilities are also considered to be C ⁇ S disorders.
  • Pain within the meaning of this definition, is also considered to be a C ⁇ S disorder. Pain can be associated with C ⁇ S disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord
  • ⁇ on-cenfral neuropathic pain includes that associated with post mastectomy pain, phantom feeling, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIN/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia.
  • RSD reflex sympathetic dystrophy
  • Headache pain for example, migraine with aura, migraine without aura, and other migraine disorders
  • episodic and chronic tension-type headache tension-type like headache, cluster headache, and chronic paroxysmal hemicrania are also CNS disorders.
  • Visceral pain such as pancreatits, intestinal cystitis, dysmenonhea, irritable Bowel syndrome, Crohn's disease, biliary colic, ureteral colic, myocardial infarction and pain syndromes of the pelvic cavity, e.g., vulvodynia, orchialgia, urethral syndrome and protatodynia are also CNS disorders.
  • a disorder of the nervous system are acute pain, for example postoperative pain, and pain after trauma.
  • PDE2A is highly expressed in various brain tissues such as fetal brain, brain, Alzheimer brain, cerebral cortex, Alzheimer cerebral cortex, frontal lobe, Alzheimer brain frontal lobe, occipital lobe, parietal lobe, temporal lobe, precenfral gyms and neuroblastoma LMR32 cells.
  • the expression in the above mentioned tissues suggests an association of PDE2A with nervous system diseases.
  • PDE2A can be used to treat or to diagnose diseases of the nervous system.
  • Heart failure is defined as a pathophysiological state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failures such as high-output and low-output, acute and chronic, right- sided or left-sided, systolic or diastolic, independent of the underlying cause.
  • Myocardial infarction (MI) is generally caused by an abmpt decrease in coronary- blood flow that follows a thrombotic occlusion of a coronary artery previously nanowed by arteriosclerosis.
  • ME prophylaxis primary and secondary prevention is included as well as the acute treatment of MI and the prevention of complications.
  • Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen.
  • This group of diseases includes stable angina, unstable angina and asymptomatic ischemia.
  • Anhythmias include all forms of atrial and ventricular tachyanhythmias, atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation, as well as bradycardic forms of arrhythmias.
  • Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension, renal, endocrine, neurogenic, others.
  • the genes may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications arising from cardiovascular diseases.
  • Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders.
  • PAOD peripheral arterial occlusive disease
  • acute arterial thrombosis and embolism inflammatory vascular disorders
  • Raynaud's phenomenon Raynaud's phenomenon
  • Atherosclerosis is a cardiovascular disease in which the vessel wall is remodeled, compromising the lumen of the vessel.
  • the atherosclerotic remodeling process involves accumulation of cells, both smooth muscle cells and monocyte/macrophage inflammatory cells, in the intima of the vessel wall. These cells take up lipid, likely from the circulation, to form a mature atherosclerotic lesion.
  • the formation of these lesions is a chronic process, occurring over decades of an adult human life, the majority of the morbidity associated with atherosclerosis occurs when a lesion ruptures, releasing thrombogenic debris that rapidly occludes the artery. When such an acute event occurs in the coronary artery, myocardial infarction can ensue, and in the worst case, can result in death.
  • the formation of the atherosclerotic lesion can be considered to occur in five overlapping stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition.
  • stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition.
  • Each of these processes can be shown to occur in man and in animal models of atherosclerosis, but the relative contribution of each to the pathology and clinical significance of the lesion is unclear.
  • Cardiovascular diseases include but are not limited to disorders of the heart and the vascular system like congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular anhythmias, hypertensive vascular diseases, peripheral vascular diseases, and atherosclerosis.
  • the risk to develop atherosclerosis and coronary artery or carotid artery disease (and thus the risk of having a heart attack or stroke) increases with the total cholesterol level increasing. Nevertheless, extremely low cholesterol levels may not be healthy.
  • hyperlipidemia abnormally high levels of fats (cholesterol, triglycerides, or both) in the blood, may be caused by family history of hyperlipidemia), obesity, a high-fat diet, lack of exercise, moderate to high alcohol consumption, cigarette smoking, poorly controlled diabetes, and an underactive thyroid gland), hereditary hyperlipidemias (type I hyperlipoproteinemia (familial hyperchylomicronemia), type LI hyperlipoproteinemia (familial hypercholesterolemia), type HI hyperlipoproteinemia, type IN hyperlipoproteinemia, or type N hyperlipoproteinemia), hypolipoproteinemia, hpidoses (caused by abnormalities in the enzymes that metabolize fats), Gaucher's disease, ⁇ iemann-Pick disease, Fabry's disease, Whyan's disease, cerebrotendinous xanthomatosis, sitosterolemia, Refsum's disease, or Tay-Sachs disease.
  • hyperlipidemia abnormally high levels of fats (cholesterol,
  • Kidney disorders may lead to hypertension or hypotension. Examples for kidney problems possibly leading to hypertension are renal artery stenosis, pyelonephritis, glomerulonephritis, kidney tumors, polycistic kidney disease, injury to the kidney, or radiation therapy affecting the kidney. Excessive urination may lead to hypotension.
  • the human PDE2A is highly expressed in the following cardiovascular related tissues: fetal heart, heart, pericardium, heart atrium (right), heart atrium (left), heart ventricle (left), interventricular septum. Expression in the above mentioned tissues demonstrates that the human PDE2A or rnR ⁇ A can be utilized to diagnose of cardiovascular diseases. Additionally the activity of the human PDE2A can be modulated to treat cardiovascular diseases.
  • Hematological disorders comprise diseases of the blood and all its constituents as well as diseases of organs and tissues involved in the generation or degradation of all the constituents of the blood. They include but are not limited to 1) Anemias, 2) Myeloproliferative Disorders, 3) Hemonhagic Disorders, 4) Leukopenia, 5) Eosinophilic Disorders, 6) Leukemias, 7) Lymphomas, 8) Plasma Cell Dyscrasias, 9) Disorders of the Spleen in the course of hematological disorders. Disorders according to 1) include, but are not limited to anemias due to defective or deficient hem synthesis, deficient erythropoiesis.
  • Disorders according to 2) include, but are not limited to polycythemia vera, tumor-associated erythrocytosis, myelofibrosis, thrombocythemia.
  • Disorders according to 3) include, but are not limited to vasculitis, thrombocytopenia, heparin-induced thrombocytopenia, thrombotic thrombocytopenic purpura, hemolytic-uremic syndrome, hereditary and acquired disorders of platelet function, hereditary coagulation disorders.
  • Disorders according to 4) include, but are not limited to neutropenia, lymphocytopenia.
  • Disorders according to 5) include, but are not limited to hypereosinophilia, idiopathic hypereosinophilic syndrome.
  • Disorders according to 6) include, but are not limited to acute myeloic leukemia, acute lymphoblastic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome.
  • Disorders according to 7) include, but are not limited to Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, mycosis fungoides cutaneous T-cell lymphoma.
  • Disorders according to 8) include, but are not limited to multiple myeloma, macroglobulinemia, heavy chain diseases.
  • PDE2A is highly expressed in spleen, spleen liver cinhosis and liver liver cinhosis.
  • PDE2A can be regulated in order to treat or to diagnose hematological disorders.
  • Gastrointestinal diseases comprise primary or secondary, acute or chronic diseases of the organs of the gastrointestinal tract which may be acquired or inherited, benign or malignant or metaplastic, and which may affect the organs of the gastrointestinal tract or the body as a whole. They comprise but are not limited to 1) disorders of the esophagus like achalasia, vigoruos achalasia, dysphagia, cricopharyngeal incoordination, pre-esophageal dysphagia, diffuse esophageal spasm, globus sensation, Ba ⁇ ett's metaplasia, gasfroesophageal reflux, 2) disorders of the stomach and duodenum like functional dyspepsia, inflammation of the gastric mucosa, gastritis, stress gastritis, chronic erosive gastritis, atrophy of gastric glands, metaplasia of gastric tissues, gastric ulcers, duodenal ulcers, neoplasms of the stomach, 3) disorders of the pan
  • Liver diseases comprise primary or secondary, acute or chronic diseases or injury of the liver wliich may be acquired or inherited, benign or malignant, and which may affect the liver or the body as a whole. They comprise but are not limited to disorders of the bilirubin metabolism, jaundice, syndroms of Gilbert's, Crigler- ⁇ ajjar, Dubin- Johnson and Rotor; intrahepatic cholestasis, hepatomegaly, portal hypertension, ascites, Budd-Chiari syndrome, portal-systemic encephalopathy, fatty liver, steatosis, Reye's syndrome, liver diseases due to alcohol, alcoholic hepatitis or cinhosis, fibrosis and cinhosis, fibrosis and cinhosis of the liver due to inborn enors of metabolism or exogenous substances, storage diseases, syndromes of Gaucher's, Zellweger's, Wilson's - disease, acute or chronic hepatitis, viral hepatitis
  • the human PDE2A is highly expressed in the following tissues of the gastroenterological system: rectum, liver, liver liver cinhosis.
  • the expression in the above mentioned tissues and in particular the differential expression between diseased tissue liver liver cinhosis and healthy tissue liver demonstrates that the human PDE2A or mRNA can be utilized to diagnose of gastroenterological disorders. Additionally the activity of the human PDE2A can be modulated to treat gastroenterological disorders.
  • the skin serves several functions. It's an multi-layered organ system that builds an effective protective cover and regulates body temperature, senses painful and pleasant stimuli, keeps substances from entering the body, and provides a shield from the sun's harmful effects. Skin color, texture, and folds help mark people as individuals. Thus, skin disorders or diseases often have important consequences for physical and mental health. Skin disorders include, but are not limited to the conditions described in the following.
  • Itching is a sensation that instinctively demands scratching, wliich may be caused by a skin condition or a systemic diseas.
  • Superficial Skin Disorders affect the uppermost layer of the skin, the stratum comeum or the keratin layer, and it consists of many layers of flattened, dead cells and acts as a barrier to protect the underlying tissue from injury and infection.
  • Disorders of the superficial skin layers involve the sfratum comeum and deeper layers of the epidermis. Examples of superficial skin disorders are provided in the following.
  • ichthyosis Dry skin often occurs in people past middle age, severe dry skin (ichthyosis) results from an inherited scaling disease, such as ichthyosis vulgaris or epidermolytic hyperkeratosis. Ichthyosis also results from nonhereditary disorders, such as leprosy, underactive thyroid, lymphoma, AIDS, and sarcoidosis.
  • Keratosis pilaris is a common disorder in which dead cells shed from the upper layer of skin and form plugs that fill the openings of hair follicles.
  • a callus is an area on the stratum comeum or keratin layer, that becomes abnormally thick in response to repeated rubbing.
  • a com is a pea-sized, thickened area of keratin that occurs on the feet.
  • Psoriasis is a chronic, recurring disease recognizable by silvery scaling bumps and various-sized plaques (raised patches). An abnormally high rate of growth and turnover of skin cells causes the scaling.
  • Pityriasis rosea is a mild disease that causes scaly, rose-colored, inflamed skin.
  • Pityriasis rosea is possibly caused by an infectious agent, although none has been identified.
  • Lichen planus a recurring itchy disease, starts as a rash of small discrete bumps that then combine and become rough, scaly plaques (raised patches).
  • Dermatitis is an inflammation of the upper layers of the skin, causing blisters, redness, swelling, oozing, scabbing, scaling, and usually itching.
  • dermatitis forms of dermatitis are contact dermatitis, or chronic dermatitis of the hands and feet, e.g. Pompholyx.
  • dermatitic disorders are atopic dermatitis, sebonheic dermatitis, nummular dermatitis, generalized exfohative dermatitis, stasis dermatitis, or localized scratch dermatitis (lichen simplex chronicus, neurodermatitis).
  • Drug rashes are side effects of medications, mainly allergic reactions to medications.
  • Toxic epidermal necrolysis is a life-threatening skin disease in which the top layer of the skin peels off in sheets. This condition can be caused by a reaction to a drug, or by some other serious disease.
  • Erythema multiforme often caused by herpes simplex is a disorder characterized by patches of red, raised skin that often look like targets and usually are distributed symmetrically over the body.
  • Erythema nodosum is an inflammatory disorder that produces tender red bumps (nodules) under the skin, most often over the sliins but occasionally on the arms and other areas.
  • Granuloma annulare is a chronic skin condition of unknown cause in which small, firm, raised bumps form a ring with normal or slightly sunken skin in the center.
  • Some skin disorders are characterized as blistering diseases.
  • Three autoimmune diseases pemphigus, bullous pemphigoid, and dermatitis herpetiformis are among the most serious.
  • Pemphigus is an uncommon, sometimes fatal, disease in which blisters (buUae) of varying sizes break out on the skin, the lining of the mouth, and other mucous membranes.
  • Bullous pemphigoid is an autoimmune disease that causes blistering.
  • Dermatitis he etiformis is an autoimmune disease in which clusters of intensely itchy, small blisters and hive-like swellings break out and persist.
  • proteins in wheat, rye, barley, and oat products activate the immune system, which attacks parts of the skin and somehow causes the rash and itching.
  • Sweating disorders also belong to skin disorders.
  • Prickly heat is an itchy skin rash caused by trapped sweat.
  • Excessive sweating may affect the entire surface of the skin, but often it's limited to the palms, soles, armpits, or groin.
  • the affected area is often pink or bluish white, and in severe cases the skin may be cracked, scaly, and soft, especially on the feet.
  • Sebaceous gland disorders can affect the sebaceous glands.
  • the sebaceous glands which secrete oil onto the skin, lie in the dermis, the skin layer just below the surface layer (epidermis).
  • Sebaceous gland disorders include acne, rosacea, perioral dermatitis, and sebaceous cysts.
  • Acne is a common skin condition in which the skin pores become clogged, leading to pimples and inflamed, infected abscesses (collections of pus). Acne tends to develop in teenagers.
  • Rosacea is a persistent skin disorder that produces redness, tiny pimples, and broken blood vessels, usually on the central area of the face.
  • Perioral dermatitis is a red, often bumpy rash around the mouth and on the chin.
  • a sebaceous cyst (keratinous cyst) is a slow-growing bump containing dead skin, skin excretions, and other skin particles. These cysts may be small and can appear anywhere.
  • Hair disorders also are skin disorders. Hair disorders include excessive hairiness, baldness, and ingrown beard hairs.
  • Bacterial skin infections can range in seriousness from minor acne to a life-threatening condition, such as staphylococcal scalded skin syndrome.
  • the most common bacterial skin infections are caused by
  • Staphylococcus and Streptococcus are for example diabetes, AIDS or skin leasons.
  • Impetigo is a skin infection, caused by Staphylococcus or Streptococcus, leading to the formation of small pus-filled blisters (pustules).
  • FoUiculitis is an inflammation of the hair follicles caused by infection with Staphylococcus. The infection damages the hairs, which can be easily pulled out.
  • Boils are large, tender, swollen, raised areas caused by staphylococcal infection around hair follicles.
  • Carbuncles are clusters of boils that result in extensive sloughing of skin and scar formation. Carbuncles develop and heal more slowly than single boils and may lead to fever and fatigue. Erysipelas is a skin infection caused by Streptococcus. A shiny, red, slightly swollen, tender rash develops, often with small bhsters. Lymph nodes around the infected area may become enlarged and painful.
  • Cellulitis is a spreading infection in, and sometimes beneath, the deep layers of the skin. Cellulitis most often results from a streptococcal infection or a staphylococcal infection. However, many other bacteria can also cause cellulitis.
  • Paronychia is an infection around the edge of a fingernail or toenail. Paronychia can be caused by many different bacteria, including Pseudomonas and Proteus, and by fungi, such as Candida.
  • Staphylococcal scalded skin syndrome is a widespread skin infection that can lead to toxic shock syndrome, in wliich the skin peels off as though burned. Certain types of staphylococci produce a toxic substance that causes the top layer of skin (epidermis) to split from the rest of the skin.
  • Erythrasma is an infection of the top layers of the skin by the bacterium Corynebacterium minutissimum.
  • Skin infections are often caused by fungi. Fungi that infect the skin (dermatophytes) live only in the dead, topmost layer (stratum comeum) and don't penetrate deeper. Some fungal infections cause no symptoms or produce only a small amount of irritation, scaling, and redness. Other fungal infections cause itching, swelling, bhsters, and severe scaling.
  • Ringworm is a fungal skin infection caused by several different fungi and generally classified by its location on the body.
  • Athlete's foot foot ringworm, caused by either Trichophyton or
  • Epidermophyton can be caused by a variety of fungi and yeasts
  • scalp ringworm caused by Trichophyton or Microsporum
  • nail ringworm and body ringworm (caused by Trichophyton).
  • Candida infection is an infection by the yeast Candida.
  • Candida usually infects the skin and mucous membranes, such as the lining of the mouth and vagina. Rarely, it invades deeper tissues as well as the blood, causing life- threatening systemic candidiasis.
  • the following types of Candida infections can be distinguished: Infections in skinfolds (intertriginous infections), vaginal and penile Candida infections (vulvovaginitis), thrash, Perleche (candida infection at the comers of the mouth), candidal paronychia (candida growing in the nail beds, produces painful swelling and pus).
  • Tinea versicolor is a fungal infection that causes white to light brown patches on the skin.
  • the skin can also be affected by parasites, mainly tiny insects or worms.
  • Scabies is a mite infestation that produces tiny reddish pimples and severe itching. Scabies is caused by the itch mite Sarcoptes scabiei.
  • Lice infestation causes intense itching and can affect almost any area of the skin. Head lice and pubic lice are two different species.
  • Creeping eruption (cutaneous larva migrans) is a hookworm infection transmitted from warm, moist soil to exposed skin. The infection is caused by a hookworm that normally inhabits dogs and cats.
  • Warts are caused by the papillomaviras
  • cold sores are caused by the herpes simplex virus.
  • Another important group of viruses that infect the skin belongs to the poxviras family. Chickenpox remains a common childhood infection.
  • a poxvirus also causes molluscum contagiosum, which is an infection of the skin by a poxvirus that causes skin-colored, smooth, waxy bumps.
  • UVB ultraviolet B
  • Drags can cause skin photosensitivity reactions which can occur after only a few minutes of sun exposure. These reactions include redness, peeling, hives, blisters, and thickened, scaling patches (photosensitivity).
  • Pigment Disorders Some skin disorders are characterized as Pigment Disorders.
  • Albinism is a rare, inherited disorder in which no melanin is formed.
  • Vitiligo is a condition in which a loss of melanocytes results in smooth, whitish patches of skin, which may occur after unusual physical trauma and tends to occur with certain other diseases, including Addison's disease, diabetes, pernicious anemia, and thyroid disease.
  • Tinea versicolor is a fungal infection of the skin that sometimes results in hyperpigmentation.
  • Melasma appears on the face (usually the forehead, cheeks, temples, and jaws) as a roughly symmetric group of dark brown patches of pigmentation that are often clearly delineated. Skin growths, wliich are abnormal accumulations of different types of cells, may be present at birth or develop later. Noncancerous (benign) growth and cancerous (malignant) growth types are distinguished.
  • Moles are small, usually dark, skin growths that develop from pigment- producing cells in the skin (melanocytes). Most moles are harmless. However, noncancerous moles can develop into malignant melanoma.
  • Skin tags are soft, small, flesh-colored or slightly darker skin flaps that appear mostly on the neck, in the armpits, or in the groin.
  • Lipomas are soft deposits of fatty material that grow under the skin, causing round or oval lumps.
  • Angiomas are collections of abnormally dense blood or lymph vessels that are usually located in and below the skin and that cause red or purple discolorations.
  • angiomas examples include port-wine stains, strawberry marks, cavernous hemangiomas, spider angiomas, and lymphangiomas.
  • Pyogenic granulomas are scarlet, brown, or blue-black slightly raised areas caused by increased growth of capillaries (the smallest blood vessels) and swelling of the surrounding tissue.
  • Sebonheic keratoses (sometimes called sebonheic warts) are flesh-colored, brown, or black growths that can appear anywhere on the skin.
  • Dermatofibromas are small, red-to-brown bumps (nodules) that result from an accumulation of fibroblasts, the cells that populate the soft tissue under the skin. Keratoacanthomas are round, firm, usually flesh-colored growths that have an unusual central crater containing a pasty material.
  • Keloids are smooth, shiny, slightly pink, often dome-shaped, proliferative growths of fibrous tissue that form over areas of injury or over surgical wounds.
  • Skin cancer is the most common form of cancer, but most types of skin cancers are curable.
  • Basal cell carcinoma is a cancer that originates in the lowest layer of the epidermis.
  • Squamous cell carcinoma is cancer that originates in the middle layer of the epidermis.
  • Bowen's disease is a form of squamous cell carcinoma that's confined to the epidermis and hasn't yet invaded the underlying dermis.
  • Melanoma is a cancer that originates in the pigment-producing cells of the skin (melanocytes).
  • Kaposi's sarcoma is a cancer that originates in the blood vessels, usually of the skin.
  • Paget's disease is a rare type of skin cancer that looks like an inflamed, reddened patch of skin (dermatitis); it originates in glands in or under the skin.
  • the human PDE2A is highly expressed in the following dermatological tissues: skin.
  • skin tissues demonstrates that the human PDE2A or mRNA can be utilized to diagnose of dermatological diseases. Additionally the activity of the human PDE2A can be modulated to treat those diseases.
  • the present invention provides for both prophylactic and therapeutic methods for hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases.
  • the regulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of PDE2a.
  • An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally- occurring cognate Hgand of the polypeptide, a peptide, a peptidomimetic, or any small molecule.
  • the agent stimulates one or more of the biological activities of PDE2a. Examples of such stimulatory agents include the active PDE2a and nucleic acid molecules encoding a portion of PDE2a.
  • the agent inhibits one or more of the biological activities of PDE2a. Examples of such inhibitory agents include antisense nucleic acid molecules and antibodies.
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by unwanted expression or activity of PDE2a or a protein in the PDE2a signaling pathway.
  • the method involves administering an agent like any agent identified or being identifiable by a screening assay as described herein, or combination of such agents that modulate say upregulate or downregulate the expression or activity of PDE2a or of any protein in the PDE2a signaling pathway.
  • the method involves administering a regulator of PDE2a as therapy to compensate for reduced or undesirably low expression or activity of PDE2a or a protein in the PDE2a signaling pathway.
  • Stimulation of activity or expression of PDE2a is desirable in situations in which enzymatic activity or expression is abnormally low and in which increased activity is likely to have a beneficial effect. Conversely, inhibition of enzymatic activity or expression of PDE2a is desirable in situations in which activity or expression of PDE2a is abnormally high and in which decreasing its activity is likely to have a beneficial effect.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • the invention includes pharmaceutical compositions comprising a regulator of PDE2a expression or activity (and or a regulator of the activity or expression of a protein in the PDE2a signaling pathway) as well as methods for preparing such compositions by combining one or more such regulators and a pharmaceutically acceptable carrier. Also within the invention are pharmaceutical compositions comprising a regulator identified using the screening assays of the invention packaged with instructions for use. For regulators that are antagonists of PDE2a activity or which reduce PDE2a expression, the instructions would specify use of the pharmaceutical composition for treatment of hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases.
  • the instructions would specify use of the pharmaceutical composition for treatment of hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases.
  • An inhibitor of PDE2a may be produced using methods which are generally known in the art. Lh particular, purified PDE2a may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind PDE2a. Antibodies to PDE2a may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies like those which inhibit dimer formation are especially prefened for therapeutic use.
  • the polynucleotides encoding PDE2a may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding PDE2a may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding PDE2a.
  • complementary molecules or fragments may be used to modulate PDE2a activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding PDE2a.
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well Icnown to those skilled in the art can be used to construct vectors which will express nucleic acid sequence complementary to the polynucleotides of the gene encoding
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition containing PDE2a in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of PDE2a, antibodies to PDE2a, and mimetics, agonists, antagonists, or inhibitors of PDE2a.
  • the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EMTM (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof.
  • a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, tbimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent wliich delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the prefened methods of preparation are vacuum drying and freeze- ing which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch
  • a lubricant such as magnesium stearate or sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or sac
  • the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, mcluding implants and microencapsulated delivery systems.
  • a controlled release formulation such as a controlled release formulation, mcluding implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the freatment of individuals.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • compositions which include an antagonist of PDE2a activity, a compound wliich reduces expression of PDE2a, or a compound which reduces expression or activity of a protein in the PDE2a signaling pathway or any combination thereof
  • the instructions for adniimstration will specify use of the composition for hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases.
  • compositions which include an agonist of PDE2a activity, a compound which increases expression of PDE2a, or a compound which increases expression or activity of a protein in the PDE2a signaling pathway or any combination thereof
  • the instructions for administration will specify use of the composition for hematological and cardiovascular diseases, disorders of the peripheral and central nervous system, COPD, asthma, genito-urological disorders and inflammation diseases.
  • antibodies which specifically bind PDE2a may be used for the diagnosis of disorders characterized by the expression of PDE2a, or in assays to monitor patients being treated with PDE2a or agonists, antagonists, and inhibitors of PDE2a.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for PDE2a include methods which utilize the antibody and a label to detect PDE2a in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent joining with a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • a variety of protocols for measuring PDE2a are Icnown in the art and provide a basis for diagnosing altered or abnormal levels of PDE2a expression.
  • Normal or standard values for PDE2a expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to PDE2a under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, preferably by photometric means. Quantities of PDE2a expressed in subject samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • the polynucleotides encoding PDE2a may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of PDE2a may be conelated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of PDE2a, and to monitor regulation of PDE2a levels during therapeutic intervention.
  • Polynucleotide sequences encoding PDE2a may be used for the diagnosis of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases associated with expression of PDE2a.
  • the polynucleotide sequences encoding PDE2a may be used in Southern, Northern, or dot-blot analysis, or other membrane- based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; and in microanays utilizing fluids or tissues from patient biopsies to detect altered PDE2a expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding PDE2a may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding PDE2a may be labelled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value.
  • nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding PDE2a in the sample indicates the presence of the associated disorder.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding PDE2a, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases PDE2a activity relative to PDE2a activity which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED50 (the dose therapeutically effective in
  • LD 50 the dose lethal to 50% of the population
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions which exhibit large therapeutic indices are prefened. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ⁇ D 5 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment.
  • Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect.
  • Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 micrograms to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, fransferrin-polycation- mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun", and DEAE- or calcium phosphate-mediated transfection.
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of PDE2a gene or the activity of PDE2a by at least about 10, preferably about 50, more preferably about 75, 90, or 100 % relative to the absence of the reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of PDE2a gene or the activity of PDE2a can be assessed using methods well known in the art, such as hybridization of nucleotide probes to PDE2a-specific mRNA, quantitative RT-PCR, immunologic detection of PDE2a, or measurement of PDE2a activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergisti- cally to effect the freatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • Nucleic acid molecules of the invention are those nucleic acid molecules which are contained in a group of nucleic acid molecules consisting of (i) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, (ii) nucleic acid molecules comprising the sequence of SEQ LD NO: 1, (iii) nucleic acid molecules having the sequence of SEQ ID NO: 1, (iv)nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii); and (v) nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code, wherein the polypeptide encoded by said nucleic acid molecule has PDE2a activity.
  • Polypeptides of the invention are those polypeptides which are contained in a group of polypeptides consisting of (i) polypeptides having the sequence of SEQ JD NO: 2, (ii) polypeptides comprising the sequence of SEQ JD NO: 2, (iii) polypeptides encoded by nucleic acid molecules of the invention and (iv) polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii), wherein said purified polypeptide has PDE2a activity.
  • An object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal comprising the steps of (i) contacting a test compound with a PDE2a polypeptide, (ii) detect binding of said test compound to said PDE2a polypeptide.
  • a test compound with a PDE2a polypeptide
  • detect binding of said test compound to said PDE2a polypeptide are identified potential therapeutic agents for such a disease.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal comprising the steps of (i) determining the activity of a PDE2a polypeptide at a certain concentration of a test compound or in the absence of said test compound, (ii) deterrnining the activity of said polypeptide at a different concentration of said test compound.
  • compounds that lead to a difference in the activity of the PDE2a polypeptide in (i) and (ii) are identified potential therapeutic agents for such a disease.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal comprising the steps of (i) determining the activity of a PDE2a polypeptide at a certain concentration of a test compound, (ii) deterrnining the activity of a PDE2a polypeptide at the presence of a compound known to be a regulator of a PDE2a polypeptide.
  • PDE2a polypeptide in (i) as compared to compounds used in (ii) are identified potential therapeutic agents for such a disease.
  • Another objects of the invention are methods of the above, wherein the compound is coupled to a detectable label.
  • Other objects of the invention are methods of the above, wherein the test compound displaces a ligand which is first bound to the polypeptide.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal comprising the steps of (i) contacting a test compound with a PDE2a polynucleotide, (ii) detect binding of said test compound to said PDE2a polynucleotide.
  • Compounds that, e.g., bind to the PDE2a polynucleotide are potential therapeutic agents for the treatment of such diseases.
  • Another object of the invention is the method of the above, wherein the nucleic acid molecule is RNA.
  • Another object of the invention is a method of the above, wherein the contacting step is in or at the surface of a cell.
  • Another object of the invention is a method of the above, wherein the contacting step is in a cell-free system.
  • Another object of the invention is a method of the above, wherein the polynucleotide is coupled to a detectable label.
  • Another object of the invention is a method of the above, wherein the test compound is coupled to a detectable label.
  • Another object of the invention is a method of diagnosing a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal comprising the steps of (i) determining the amount of a PDE2a polynucleotide in a sample taken from said mammal, (ii) determining the amount of PDE2a polynucleotide in healthy and/or diseased mammal.
  • a disease is diagnosed, e.g., if there is a substantial similarity in the amount of PDE2a polynucleotide in said test mammal as compared to a diseased mammal.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal comprising a therapeutic agent which binds to a PDE2a polypeptide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal comprising a therapeutic agent which regulates the activity of a PDE2a polypeptide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal comprising a therapeutic agent which regulates the activity of a PDE2a polypeptide, wherein said therapeutic agent is (i) a small molecule, (ii) an RNA molecule, (iii) an antisense oligonucleotide, (iv) a polypeptide, (v) an antibody, or (vi) a ribozyme.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal comprising a PDE2a polynucleotide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal comprising a PDE2a polypeptide.
  • Another object of the invention is the use of regulators of a PDE2a for the preparation of a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal.
  • Another object of the invention is a method for the preparation of a pharmaceutical composition useful for the treatment of a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal comprising the steps of (i) identifying a regulator of PDE2a, (ii) determining whether said regulator ameliorates the symptoms of a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases in a mammal; and (iii) combining of said regulator with an acceptable pharmaceutical carrier.
  • Another object of the invention is the use of a regulator of PDE2a for the regulation of PDE2a activity in a mammal having a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and cenfral nervous system, dermatological diseases, cardiovascular disease and gastroenterological diseases.
  • the degree of homology can readily be calculated by known methods. Prefened methods to determine homology are designed to give the largest match between the sequences tested. Methods to determine homology are codified in publicly available computer programs such as BestFit, BLASTP, BLASTN, and FASTA. The BLAST programs are publicly available from NCBI and other sources in the internet.
  • PDE2A3 PDE2A3 mRNA, complete eds [Homo sapiens]
  • RNA contamination For relative quantitation of the mRNA distribution of PDE2a, total RNA from each cell or tissue source was first reverse transcribed. 85 ⁇ g of total RNA was reverse transcribed using 1 ⁇ mole random hexamer primers, 0.5 mM each of dATP, dCTP, dGTP and dTTP (Qiagen, Hilden, Germany), 3000 U RnaseQut (Invitrogen, Groningen, Netherlands) in a final volume of 680 ⁇ l. The first strand synthesis buffer and Omniscript reverse transcriptase (2 u/ ⁇ l) were from (Qiagen, Hilden, Germany). The reaction was incubated at 37°C for 90 minutes and cooled on ice. The volume was adjusted to 6800 ⁇ l with water, yielding a final concentration of 12.5 ng/ ⁇ l of starting RNA.
  • the CT (threshold cycle) value is calculated as described in the "Quantitative determination of nucleic acids" section.
  • the CF-value (factor for threshold cycle conection) is calculated as follows :
  • PCR reactions were set up to quantitate the housekeeping genes (HKG) for each cDNA sample.
  • CTu KG - alues were calculated as described in the "Quantitative determination of nucleic acids" section.
  • CTn KG - n -mean value (CTHK GI -value + CTHK G2 - value +... + CT ⁇ KG - n -value) / n
  • CTpan ⁇ ei mean value (CT mean value of all HKG in all tested cDNAs)
  • CTcDNA-n CT value of the tested gene for the cDNA n
  • CF cDN A- n detection factor for cDNA n
  • CT cor -cDNA- n co ⁇ ected CT value for a gene on cDNA n
  • Table 1 Relative expression ofPDE2a in various human tissues.
  • HUVEC cells 461 skin 666 adrenal gland 292 thyroid 106 thyroid tumor 224 pancreas 13 pancreas liver cirrhosis 256 esophagus 161 esophagus tumor 18 stomach 132 stomach tumor 76 colon 119 colon tumor 40 small intestine 58 ileum 155 ileum tumor 11 ileum chronic inflammation 87 rectum 699 salivary gland 1 fetal liver 81 liver 96 liver liver cirrhosis 832 liver tumor 136
  • HeLa cells 35 placenta 52 uterus 122 uterus tumor 7 ovary 119 ovary tumor 267 breast 1226 breast tumor 244
  • MDA MB 231 cells (breast tumor) 169 mammary gland 261 prostate 17 prostate BPH 32 bladder 201 ureter 131 penis 99 corpus cavernosum 139 fetal kidney 1499 kidney 280 kidney tumor 36
  • Oligonucleotides, cDNA or genomic fragments comprising the antisense sfrand of a polynucleotide coding for PDE2a are used either in vitro or in vivo to inhibit translation of the mRNA.
  • antisense molecules can be designed at various locations along the nucleotide sequences.
  • the gene of interest is effectively turned off.
  • the function of the gene is ascertained by observing behavior at the intracellular, cellular, tissue or organismal level (e.g., lethality, loss of differentiated function, changes in morphology, etc.).
  • modifications of gene expression is obtained by designing antisense sequences to infron regions, promoter/enhancer elements, or even to transacting regulatory genes.
  • Expression of PDE2a is accomplished by subcloning the cDNAs into appropriate expression vectors and transfecting the vectors into expression hosts such as, e.g., E. coli.
  • the vector is engineered such that it contains a promoter for ⁇ -galactosidase, upstream of the cloning site, followed by sequence containing the amino-terminal Methionine and the subsequent seven residues of ⁇ -galactosidase.
  • an engineered bacteriophage promoter useful for artificial priming and transcription and for providing a number of unique endonuclease restriction sites for cloning.
  • Induction of the isolated, transfected bacterial strain with Isopropyl- ⁇ -D-thio- galactopyranoside (LPTG) using standard methods produces a fusion protein conesponding to the first seven residues of ⁇ -galactosidase, about 15 residues of "linker", and the peptide encoded within the cDNA. Since cDNA clone inserts are generated by an essentially random process, there is probability of 33% that the included cDNA will lie in the conect reading frame for proper translation.
  • the cDNA is not in the proper reading frame, it is obtained by deletion or insertion of the appropriate number of bases using well known methods mcluding in vitro mutagenesis, digestion with exonuclease LTJ or mung bean nuclease, or the inclusion of an oligonucleotide linker of appropriate length.
  • the PDE2a cDNA is shuttled into other vectors known to be useful for expression of proteins in specific hosts.
  • Oligonucleotide primers containing cloning sites as well as a segment of DNA (about 25 bases) sufficient to hybridize to stretches at both ends of the target cDNA is synthesized chemically by standard methods. These primers are then used to amplify the desired gene segment by PCR. The resulting gene segment is digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternately, similar gene segments are produced by digestion of the cDNA with appropriate restriction enzymes. Using appropriate primers, segments of coding sequence from more than one gene are ligated together and cloned in appropriate vectors. It is possible to optimize expression by construction of such chimeric sequences.
  • Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells., insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae and bacterial cells such as E. coli.
  • a useful expression vector also includes an origin of replication to allow propagation in bacteria, and a selectable marker such as the ⁇ -lactamase antibiotic resistance gene to allow plasmid selection in bacteria.
  • the vector may include a second selectable marker such as the neomycin phosphofransferase gene to allow selection in transfected eukaryotic host cells.
  • Vectors for use in eukaryotic expression hosts require RNA processing elements such as 3' polyadenylation sequences if such are not part of the cDNA of interest.
  • the vector contains promoters or enhancers which increase gene expression.
  • promoters are host specific and include MMTV, SV40, and metallothionine promoters for CHO cells; tip, lac, tac and T7 promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGH promoters for yeast.
  • Transcription enhancers such as the rous sarcoma vims enhancer, are used in mammalian host cells. Once homogeneous cultures of recombinant cells are obtained through standard culture methods, large quantities of recombinantly produced PDE2a are recovered from the conditioned medium and analyzed using chromatographic methods known in the art.
  • PDE2a can be cloned into the expression vector pcDNA3, as exemplified herein.
  • This product can be used to fransform, for example, HEK293 or COS by methodology standard in the art. Specifically, for example, using Lipofectamine (Gibco BRL catolog no. 18324-020) mediated gene transfer.
  • PDE2a is expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine- tryptophan modules that allow purification on immobilized metals [Appa Rao, 1997] and the domain utilized in the FLAGS extension/affinity purification system
  • cleavable linker sequence such as Factor Xa or enterokinase (Invifrogen, Groningen, The Netherlands) between the purification domain and the PDE2a sequence is useful to facilitate expression of PDE2a.
  • PDE2a is generated using the baculoviras expression system BAC-TO-BAC (GIBCO BRL) based on Autographa californica nuclear polyhedrosis virus (AcNPN) infection o ⁇ Spodopterafrugiperda insect cells (Sf9 cells).
  • BAC-TO-BAC baculoviras expression system BAC-TO-BAC
  • AcNPN Autographa californica nuclear polyhedrosis virus
  • Sf9 cells o ⁇ Spodopterafrugiperda insect cells
  • cD ⁇ A encoding PDE is cloned into either the donor plasmid pFASTBACl or pFASTBAC-HT which contain a mini-Tn7 transposition element.
  • the recombinant plasmid is transformed into DHIOBAC competent cells wliich contain the parent bacmid bMO ⁇ 14272 (AcNPV infectious DNA) and a helper plasmid.
  • the mini-Tn7 element on the pFASTBAC donor can transpose to the attTn7 attachment site on the bacmid thus introducing the PDE gene into the viral genome. Colonies containing recombinant bacmids are identified by dismption of the lacZ gene.
  • the PDE/bacmid construct can then be isolated and infected into insect cells (Sf9 cells) resulting in the production of infectious recombinant baculoviras particles and expression of either unfused recombinant enzyme (pFastbacl) or PDE2a-His fusion protein
  • PDE2a is confirmed by coomassie staining after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting onto a PVDF membrane of an unstained SDS-PAGE.
  • SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis
  • the PDE-His fusion protein is detected due to the interaction between the Ni-NTA HRP conjugate and the His-tag which is fused to PDE2a.
  • denatured protein from reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein is used to immunize mice or rabbits using standard protocols; about 100 ⁇ g are adequate for immunization of a mouse, while up to 1 mg might be used to immunize a rabbit.
  • the denatured protein is radioiodinated and used to screen potential murine B-cell hybridomas for those which produce antibody. This procedure requires only small quantities of protein, such that 20 mg is sufficient for labeling and screening of several thousand clones.
  • the amino acid sequence of an appropriate PDE2a domain is analyzed to determine regions of high antigenicity.
  • Oligopeptides comprising appropriate hydrophilic regions are synthesized and used in suitable immunization protocols to raise antibodies.
  • the optimal amino acid sequences for immunization are usually at the C-terminus, the N- terminus and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation.
  • selected peptides typically, about 15 residues in length, are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH; Sigma, St. Louis, MO) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester, MBS. If necessary, a cysteine is introduced at the N-terminus of the peptide to permit coupling to KLH.
  • KLH keyhole limpet hemocyanin
  • Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant.
  • the resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG.
  • Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with labeled PDE2a to identify those fusions producing the monoclonal antibody with the desired specificity.
  • wells of plates FAST; Becton-Dickinson, Palo Alto, CA
  • affinity purified, specific rabbit anti-mouse (or suitable antispecies 1 g) antibodies at 10 mg/ml.
  • the coated wells are blocked with 1% bovine serum albun in, (BSA), washed and incubated with supernatants from hybridomas. After washing the wells are incubated with labeled PDE2a at 1 mg/ml.
  • 10 8 M “1 preferably 10 9 to 10 10 M "1 or stronger, are typically made by standard procedures.
  • PDE2a antibodies are useful for investigating signal fransduction and the diagnosis of infectious or hereditary conditions which are characterized by differences in the amount or distribution of PDE2a or downstream products of an active signaling cascade.
  • Diagnostic tests for PDE2a include methods utilizing antibody and a label to detect PDE2a in human body fluids, membranes, cells, tissues or extracts of such.
  • the polypeptides and antibodies of the present invention are used with or without modification. Frequently, the polypeptides and antibodies are labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, subsfrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, chromogenic agents, magnetic particles and the like.
  • a variety of protocols for measuring soluble or membrane-bound PDE2a, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmuno- assay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmuno- assay
  • FACS fluorescent activated cell sorting
  • a two-site monoclonal- based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on PDE2a is prefened, but a competitive binding assay may be employed.
  • Native or recombinant PDE2a is purified by immunoaffinity chromatography using antibodies specific for PDE2a.
  • an immunoaffinity column is constracted by covalently coupling the anti-TRH antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia).
  • monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology).
  • Such immunoaffinity columns are utilized in the purification of PDE2a by preparing a fraction from cells containing PDE2a in a soluble form. This preparation is derived by solubilization of whole cells or of a subcellular fraction obtained via differential centrifugation (with or without addition of detergent) or by other methods well known in the art. Alternatively, soluble PDE2a containing a signal sequence is secreted in useful quantity into the medium in which the cells are grown. A soluble PDE2a-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PDE2a (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/protein binding (e.g., a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and
  • PDE2a is collected.
  • This invention is particularly useful for screening therapeutic compounds by using
  • PDE2a or fragments thereof in any of a variety of drug screening techniques.
  • the following example provides a system for drug screening measuring the phosphodiesterase activity.
  • the phosphodiesterase activity of the crude extracts is measured and confirmed that the PDE cDNA encodes a phosphodiesterase which is able to hydrolyze cAMP or cGMP or both.
  • the recombinant PDE-His fusion protein can be purified from the crude lysate by metal-affinity chromatography using Ni-NTA agarose. This allows the specific retention of the recombinant material (since this is fused to the His-tag) whilst the endogenous insect proteins are washed off. The recombinant material is then eluted by competition with imidazol.
  • the phosphodiesterase activity of the recombinant protein is assayed using a commercially available SPA (scintillation proximity assay) kit (Amersham Pharmacia).
  • the PDE enzyme hydrolyzes cyclic nucleotides, e.g. cAMP and cGMP to their linear counterparts.
  • the SPA assay utilizes the tritiated cyclic nucleotides [ H]cAMP or [ H]cGMP, and is based upon the selective interaction of the tritiated non cyclic product with the SPA beads whereas the cyclic substrates are not effectively binding. Radiolabelled product bound to the scintillation beads generates light that can be analyzed in a scintillation counter.
  • the goal of rational drag design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, agonists, antagonists, or inhibitors. Any of these examples are used to fashion drags which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo.
  • the three-dimensional stracture of a protein of interest, or of a protein-inhibitor complex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the stracture and to determine active site(s) of the molecule. Less often, useful information regarding the stracture of a polypeptide is gained by modeling based on the stracture of homologous proteins. In both cases, relevant structural information is used to design efficient inhibitors. Useful examples of rational drag design include molecules which have improved activity or stability or which act as inhibitors, agonists, or antagonists of native peptides.
  • a target-specific antibody selected by functional assay, as described above, and then to solve its crystal stracture.
  • This approach in principle, yields a pharmacore upon which subsequent drag design is based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a minor image of a minor image, the binding site of the anti-ids is expected to be an analog of the original receptor. The anti-id is then used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides then act as the pharmacore.
  • anti-ids anti-idiotypic antibodies
  • Labeled PDE2a is useful as a reagent for the purification of molecules with which it interacts.
  • PDE2a is covalently coupled to a chromatography column.
  • Cell-free extract derived from synovial cells or putative target cells is passed over the column, and molecules with appropriate affinity bind to PDE2a.
  • PDE2a-complex is recovered from the column, and the PDE2a-binding ligand disassociated and subjected to N-terminal protein sequencing. The amino acid sequence information is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
  • antibodies are raised against PDE2a, specifically monoclonal antibodies.
  • the monoclonal antibodies are screened to identify those which inhibit the binding of labeled PDE2a. These monoclonal antibodies are then used therapeutically.
  • Example 12 Use and Administration of Antibodies, Inhibitors, or Antagonists
  • LSTs Antibodies, inhibitors, or antagonists of PDE2a or other treatments and compunds that are limiters of signal transduction (LSTs), provide different effects when administered therapeutically.
  • LSTs are formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8, more preferably 6 to 8, although pH may vary according to the characteristics of the antibody, inhibitor, or antagonist being formulated and the condition to be treated. Characteristics of LSTs include solubility of the molecule, its half-life and antigenicity/immunogenicity. These and other characteristics aid in defining an effective carrier. Native human proteins are prefened as LSTs, but organic or synthetic molecules resulting from drug screens are equally effective in particular situations.
  • LSTs are delivered by known routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally a ⁇ t ⁇ iinistered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally a ⁇ t ⁇ iinistered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • the particular formulation, exact dosage, and route of administration is determined by the attending physician and varies according to each specific situation.
  • Such determinations are made by considering multiple variables such as the condition to be treated, the LST to be administered, and the pharmacokinetic profile of a particular LST. Additional factors which are taken into account include severity of the disease state, patient's age, weight, gender and diet, time and frequency of LST administration, possible combination with other drags, reaction sensitivities, and tolerance/response to therapy. Long acting LST formulations might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular LST.
  • Normal dosage amounts vary from 0.1 to 10 5 ⁇ g, up to a total dose of about 1 g, depending upon the route of adrninistration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.
  • Those skilled in the art employ different formulations for different LSTs.
  • Administration to cells such as nerve cells necessitates delivery in a manner different from that to other cells such as vascular endothelial cells. It is contemplated that abnormal signal transduction, trauma, or diseases which trigger PDE2a activity are treatable with LSTs.
  • Animal model systems which elucidate the physiological and behavioral roles of the PDE2a are produced by creating nonhuman transgenic animals in which the activity of the PDE2a is either increased or decreased, or the amino acid sequence of the expressed PDE2a is altered, by a variety of techniques.
  • Examples of these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a PDE2a, by microinjection, electroporation, retroviral transfection or other means well known to those skilled in the art, into appropriately fertilized embryos in order to produce a transgenic animal or 2) homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the stracture of these PDE2a sequences.
  • the technique of homologous recombination is well known in the art.
  • Microinjection adds genes to the genome, but does not remove them, and the technique is useful for producing an animal which expresses its own and added PDE2a, resulting in overexpression of the PDE2a.
  • transgenic animal One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as cesiumchloride M2 medium. DNA or cDNA encoding PDE2a is purified from a vector by methods well known to the one skilled in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the transgene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the transgene.
  • microinjection needle which may be made from capillary tubing using a piper puller
  • the egg to be injected is put in a depression slide.
  • the needle is inserted into the pronucleus of the egg, and the DNA solution is injected.
  • the injected egg is then transfened into the oviduct of a pseudopregnant mouse which is a mouse stimulated by the appropriate hormones in order to maintain false pregnancy, where it proceeds to the uterus, implants, and develops to term.
  • microinjection is not the only method for inserting DNA into the egg but is used here only for exemplary purposes.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne une PDE2a humaine qui est associée à des maladies hématologiques, des troubles du système nerveux central et périphérique, des maladies dermatologiques, des maladies cardiovasculaires et des maladies gastro-entérologiques. Cette invention concerne aussi des recherches d'identification de composés qui conviennent pour le traitement ou la prévention de maladies hématologiques, de troubles du système nerveux central et périphérique, de maladies dermatologiques, de maladies cardiovasculaires et de maladies gastro-entérologiques. Cette invention concerne enfin des composés qui se lient à la PDE2a et/ou qui activent ou inhibent l'activité de cette phosphodiestérase, ainsi que des compositions pharmaceutiques comprenant ces composés.
EP03810957A 2002-11-13 2003-10-31 Outils de diagnostics et therapeutique pour maladies associees a la phosphodiesterase 2a humaine (pde2a) Withdrawn EP1597386A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP03810957A EP1597386A1 (fr) 2002-11-13 2003-10-31 Outils de diagnostics et therapeutique pour maladies associees a la phosphodiesterase 2a humaine (pde2a)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP02025505 2002-11-13
EP02025505 2002-11-13
EP03810957A EP1597386A1 (fr) 2002-11-13 2003-10-31 Outils de diagnostics et therapeutique pour maladies associees a la phosphodiesterase 2a humaine (pde2a)
PCT/EP2003/012124 WO2004044234A1 (fr) 2002-11-13 2003-10-31 Outils de diagnostics et therapeutique pour maladies associees a la phosphodiesterase 2a humaine (pde2a)

Publications (1)

Publication Number Publication Date
EP1597386A1 true EP1597386A1 (fr) 2005-11-23

Family

ID=32309338

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03810957A Withdrawn EP1597386A1 (fr) 2002-11-13 2003-10-31 Outils de diagnostics et therapeutique pour maladies associees a la phosphodiesterase 2a humaine (pde2a)

Country Status (3)

Country Link
EP (1) EP1597386A1 (fr)
AU (1) AU2003276220A1 (fr)
WO (1) WO2004044234A1 (fr)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7758890B2 (en) 2001-06-23 2010-07-20 Lyotropic Therapeutics, Inc. Treatment using dantrolene
DK1759700T3 (da) * 2004-08-19 2009-10-12 Switch Biotech Llc Anvendelse af en PDE5-inhibitor til behandling og forebyggelse af hypopigmentlidelser
WO2006018088A1 (fr) * 2004-08-19 2006-02-23 Switch Biotech Ag Utilisation d'un inhibiteur pde5 aux fins de traitement et de prevention de troubles hypopigmentaires
DE102006048693A1 (de) * 2006-10-14 2008-04-17 Bayer Healthcare Ag Inhibition der PDE2A
WO2013161913A1 (fr) 2012-04-25 2013-10-31 武田薬品工業株式会社 Composé hétérocyclique azoté
EP2873669A4 (fr) 2012-07-13 2015-11-25 Takeda Pharmaceutical Composé hétérocyclique
EP2975031A4 (fr) 2013-03-14 2017-04-19 Takeda Pharmaceutical Company Limited Composé hétérocyclique
US10053468B2 (en) 2013-07-03 2018-08-21 Takeda Pharmaceutical Company Limited Heterocyclic compound
WO2015002230A1 (fr) 2013-07-03 2015-01-08 武田薬品工業株式会社 Composé amide
EP3134413B1 (fr) 2014-04-23 2019-09-11 Dart NeuroScience (Cayman) Ltd. Composés de [1,2,4]triazolo[1,5-a]pyrimidin-7-yle substitués utilisables en tant qu'inhibiteurs de pde2
WO2016073424A1 (fr) 2014-11-05 2016-05-12 Dart Neuroscience, Llc Composés 5-méthyl-[1, 2, 4]-triazolo-[1,5-a)-pyrimidin-2-amines substitués utilisés comme inhibiteurs de pde2
MX2019007803A (es) 2016-12-28 2019-08-29 Dart Neuroscience Llc Compuestos de pirazolopirimidinona sustituida como inhibidores de fosfodiesterasas 2 (pde2).

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL132366A0 (en) * 1998-10-15 2001-03-19 Cell Pathways Inc Methods for identifying compounds for inhibition of neoplastic lesions and pharmaceutical compositions containing such compounds
US6376225B1 (en) * 2001-01-05 2002-04-23 Pe Corporation (Ny) Isolated human phosphodiesterase proteins, nucleic acid molecules encoding human phosphodiesterase proteins, and uses thereof
US6465494B1 (en) * 2001-08-24 2002-10-15 Cell Pathways, Inc. Methods for treatment of cystic fibrosis

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004044234A1 *

Also Published As

Publication number Publication date
WO2004044234A1 (fr) 2004-05-27
AU2003276220A1 (en) 2004-06-03

Similar Documents

Publication Publication Date Title
EP1597386A1 (fr) Outils de diagnostics et therapeutique pour maladies associees a la phosphodiesterase 2a humaine (pde2a)
WO2004042389A2 (fr) Moyens diagnostiques et therapeutiques destines a des maladies associees a la phosphodiesterase 4d (pde4d) humaine
WO2004042390A2 (fr) Diagnostic et therapeutique de maladies associees a la phosphodiesterase 4b (pde4b) humaine
WO2004053495A1 (fr) Diagnostic et traitement de maladies associees a la phosphodiesterase 9a1(pde9a1)humaine
US20060166911A1 (en) Diagnostics and therapeutics for diseases associated with human phosphodiesterase 11a (pde11a)
US20060188882A1 (en) Diagnostics and therapeutics for diseases associated with phosphodi-esterase 1c (pde1c)
EP1604202A2 (fr) Moyens de diagnostic et therapeutiques pour des maladies associees a la phosphodiesterase 1a (pde1a)
WO2004044235A1 (fr) Procedes de diagnostic de maladies associees a la phosphodiesterase 7a1 (pde7a1) humaine et moyens therapeutiques correspondants
EP1426767A1 (fr) Diagnostic et traitement pour des maladies associées au phosphodiesterase 4c humain (PDE4C)
US20060110783A1 (en) Diagnostics and therapeutics for diseases associated with human phosphodiesterase 10a (pde10a)
EP1573333A1 (fr) AGENTS DE DIAGNOSTIC ET DE TRAITEMENT DE MALADIES ASSOCIEES A LA PHOSPHODIESTERASE HUMAINE 9A2 (PDE9a2)
WO2004044196A1 (fr) Procedes de diagnostic de maladies associees a la phosphodiesterase 7b (pde7b) humaine et moyens therapeutiques correspondants
WO2004053493A1 (fr) Diagnostic et traitement de maladies associees a la phosphodiesterase 9a4 (pde9a4) humaine
WO2004071377A2 (fr) Methodes diagnostiques et therapeutiques relatives a des etats pathologiques lies a la photodiesterase 3a (pde3a)
EP1573332A1 (fr) Diagnostic et traitement de maladies associees a la phosphodiesterase 9a3 (pde9a3) humaine
WO2006010516A1 (fr) Diagnostic et traitement therapeutique des maladies associees a la proteine de type methionine aminopeptidase 2 (metap2-like)
US20070202567A1 (en) Diagnostics And Therapeutics For Diseases Associated With Arginyl Aminopeptidase Rnpep (Rnpep)
WO2004044229A2 (fr) Procedes de diagnostic de maladies associees a la phosphodiesterase 7a2 (pde7a2) humaine et moyens therapeutiques correspondants
US20060281085A1 (en) Diagnostics and therapeutics for diseases associated with phosphodiesterase 1b (pde1b)
EP1604035A2 (fr) Diagnostics et medicaments pour des maladies associees a la phosphodiesterase 8a (pde8a) humaine
WO2004042076A2 (fr) Diagnostic et therapeutique de maladies associees a la phosphodiesterase 4a (pde4a) humaine
EP1604034A2 (fr) Diagnostics et traitements de maladies associees a la phosphodiesterase 11a1 humaine (pde11a1)
WO2004075812A2 (fr) Produits diagnostiques et therapeutiques pour des maladies associees a une phosphodiesterase 3b (pde3b)
WO2004082575A2 (fr) Diagnostics et traitements contre des maladies associees au phosphodiesterase 6b (pde6b)
EP1604033A2 (fr) Moyens diagnostiques et therapeutiques destines a des maladies associees a la phosphodiesterase 11a3 (pde11a3) humaine

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050824

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20080424

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20081105