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WO2004044235A1 - Procedes de diagnostic de maladies associees a la phosphodiesterase 7a1 (pde7a1) humaine et moyens therapeutiques correspondants - Google Patents

Procedes de diagnostic de maladies associees a la phosphodiesterase 7a1 (pde7a1) humaine et moyens therapeutiques correspondants Download PDF

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WO2004044235A1
WO2004044235A1 PCT/EP2003/012341 EP0312341W WO2004044235A1 WO 2004044235 A1 WO2004044235 A1 WO 2004044235A1 EP 0312341 W EP0312341 W EP 0312341W WO 2004044235 A1 WO2004044235 A1 WO 2004044235A1
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Prior art keywords
pde7al
diseases
polypeptide
disorders
disease
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Inventor
Stefan Golz
Ulf Brüggemeier
Holger Summer
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Bayer AG
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Bayer Healthcare AG
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Priority to EP03773703A priority Critical patent/EP1597385A1/fr
Priority to AU2003282087A priority patent/AU2003282087A1/en
Publication of WO2004044235A1 publication Critical patent/WO2004044235A1/fr
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    • 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
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • 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
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • 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 l ⁇ iown 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.
  • PDE1A and PDE1B preferentially hydrolyze cGMP while PDE1C is dualspecific, it exhibits a high affinity for both cAMP and cGMP.
  • PDE1C preferentially hydrolyzes cGMP while PDE1C is dualspecific, it exhibits a high affinity for both cAMP and cGMP.
  • In vitro experiments show regulation of some PDE1 species by phosphorylation, which decreases the affinity of the enzyme for calmodulin [Kakkar, (1999)].
  • PDEls have been shown to be expressed in lung, heart and brain.
  • 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 max 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 endotheliurn, 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.
  • 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.
  • 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)]
  • 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 turn will provide additional pharmacological approaches to treat diseases and conditions in which alterations in cyclic nucleotide pathways are involved.
  • 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 drug 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.
  • physiological or environmental stimuli e.g., drugs
  • the effects of drugs with established efficacy on global gene expression patterns may provide a guidepost, or a genetic signature, against which a new drug candidate can be compared.
  • Fig. 1 shows the nucleotide sequence of a PDE7A1 polynucleotide (SEQ LD NO:l).
  • Fig. 2 shows the amino acid sequence of a PDE7A1 polypeptide (SEQ TD NO:2).
  • Fig. 5 shows a nucleotide sequence useful as a probe to detect proteins of the invention (SEQ J-D NO:5).
  • Naturally occurring PDE7al 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, glycosylation, phosphorylation, lipidation and acylation.
  • oligopeptide is a short stretch of amino acid residues and may be expressed from an oligonucleotide. Oligopeptides 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 amino 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.
  • polypeptide encoded by said nucleic acid molecule has PDE7al activity.
  • PDE7al polypeptide within the meaning of the invention, shall be understood as being a polypeptide selected from a group consisting of
  • Another aspect of the subject invention is to provide for PDE7al -specific hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding PDE7al. Such probes may also be used for the detection of similar PDE encoding sequences and should preferably show at least 40% nucleotide identity to PDE7al 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.
  • 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.
  • PDE7al cD ⁇ A molecules can be made with standard molecular biology techniques, using PDE7al mR ⁇ A as a template. PDE7al cD ⁇ A 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.
  • Narious PCR-based methods can be used to extend nucleic acid sequences encoding human PDE7al, for example to detect upstream sequences of PDE7al gene such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus. Genomic D ⁇ A 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 R ⁇ A polymerase and sequenced using reverse transcriptase.
  • PDE7al 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 PDE7al and appropriate transcriptional and translational 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 PDE7al. 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, CaMN; tobacco mosaic virus, TMN) 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, CaM
  • Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic.
  • host cells which contain a PDE7al polynucleotide and which express
  • Nucleic acid amplification-based assays involve the use of ohgonucleotides selected from sequences encoding PDE7al to detect transformants which contain a PDE7al polynucleotide.
  • a variety of protocols for detecting and measuring the expression of PDE7al, 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 PDE7al can be used, or a competitive binding assay can be employed.
  • Host cells transformed with PDE7al 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 PDE7al polynucleotides can be designed to contain signal sequences which direct secretion of soluble PDE7al through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound PDE7al.
  • 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.).
  • PDE7al polynucleotides possessing non-naturally occurring codons For example, codons preferred 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.
  • PDE7al can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, PDE7al 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.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to PDE7al.
  • 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 tetravalent, 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 PDE7al 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 constructed and used therapeutically in methods of the invention.
  • chimeric antibodies can be constructed as disclosed in WO 93/03151.
  • Antisense ohgonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both.
  • Ohgonucleotides 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.
  • 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 PDE7al polynucleotide sequence.
  • Antisense ohgonucleotides can be modified without affecting their ability to hybridize to a PDE7al polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside 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 ohgonucleotides can be prepared by methods well known in the art.
  • 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.
  • 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.
  • the assay can be a binding assay entailing direct or indirect measurement of the binding of a test compound or a known PDE7al ligand to PDE7al.
  • the assay can also be an activity assay entailing direct or indirect measurement of the activity of PDE7al .
  • the assay can also be an expression assay entailing direct or indirect measurement of the expression of
  • glutathione-S-transferase (GST) fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or PDE7al, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • glutathione sepharose beads Sigma Chemical; St. Louis, Mo.
  • glutathione derivatized microtitre plates which are then combined with the test compound or the test compound and either the non-adsorbed target protein or PDE7al, and the mixture incubated under 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 PDE7al can be determined using standard techniques.
  • the screening assay can also involve monitoring the expression of PDE7al .
  • regulators of expression of PDE7al can be identified in a method in which a cell is contacted with a candidate compound and the expression of PDE7al protein or mRNA in the cell is determined. The level of expression of PDE7al protein or mRNA the presence of the candidate compound is compared to the level of expression of PDE7al protein or mRNA in the absence of the candidate compound. The candidate compound can then be identified as a regulator of expression of PDE7al based on this comparison.
  • reporter gene e.g., LacZ
  • a reporter gene e.g., LacZ
  • 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 withPDE7al.
  • PDE7al is a fusion protein comprising a domain that allows binding of PDE7al to a solid support.
  • glutathione-S-transferase 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 PDE7al; 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.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell which expresses PDE7al polynucleotide can be used in a cell-based assay system.
  • the PDE7al 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.
  • 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 structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In 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.
  • CNS disorders include disorders of the central nervous system as well as disorders of the peripheral nervous system.
  • CNS 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 CNS disorders.
  • Pain within the meaning of this definition, is also considered to be a CNS disorder. Pain can be associated with CNS 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 (e.g., infarct, hemorrhage, vascular malformation).
  • CNS 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 (e.g., infarct, hemorrhage, vascular malformation).
  • 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.
  • MI Myocardial infarction
  • 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.
  • 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 raptures, 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, recraitment 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.
  • 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 II hyperlipoproteinemia (familial hypercholesterolemia), type III hyperlipoproteinemia, type IN hyperlipoproteinemia, or type N hyperlipoproteinemia), hypolipoproteinemia, lipidoses (caused by abnormalities in the enzymes that metabolize fats), Gaucher's disease, ⁇ iemann-Pick disease, Fabry's disease, Wolman's disease, cerebrotendinous xanthomatosis, sitosterolemia, Refsum's disease, or Tay-Sachs disease.
  • hyperlipidemia abnormally high levels of fats (cholesterol, t
  • 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.
  • PDE7Alis highly expressed in different cardiovascular related tissues such as pericardium, heart atrium (right), heart atrium (left), heart apex, Purkinje fibers, interventricular septum and pulmonic valve. Expression in the above mentioned tissues suggests an association between PDE7A1 and cardiovascular diseases.
  • PDE7A1 can be regulated to treat or to diagnose 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) Hemorrhagic 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.
  • Cancerous cells and tissues may affect the body as a whole when causing paraneoplastic syndromes or if cancer occurs within a vital organ or tissue, normal function will be impaired or halted, with possible fatal results.
  • the ultimate involvement of a vital organ by cancer, either primary or metastatic, may lead to the death of the mammal affected. Cancer tends to spread, and the extent of its spread is usually related to an individual's chances of surviving the disease.
  • cancer cells divide at a higher rate than do normal cells, but the distinction between the growth of cancerous and normal tissues is not so much the rapidity of cell division in the former as it is the partial or complete loss of growth restraint in cancer cells and their failure to differentiate into a useful, limited tissue of the type that characterizes the functional equilibrium of growth of normal tissue.
  • Cancer tissues may express certain molecular receptors and probably are influenced by the host's susceptibility and immunity and it is known that certain cancers of the breast and prostate, for example, are considered dependent on specific hormones for their existence.
  • Cancer according to 2) develops in connective tissues, including fibrous tissues, adipose (fat) tissues, muscle, blood vessels, bone, and cartilage like e.g. osteogenic sarcoma; liposarcoma, fibrosarcoma, synovial sarcoma.
  • Cancer according to 3) is cancer that develops in both epithelial and connective tissue.
  • Cancer disease within the scope of this definition may be primary or secondary, whereby primary indicates that the cancer originated in the tissue where it is found rather than was established as a secondary site through metastasis from another lesion.
  • Cancers and tumor diseases within the scope of this definition may be benign or malign and may affect all anatomical structures of the body of a mammal.
  • Stimulation of activity or expression of PDE7al 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 PDE7al is desirable in situations in which activity or expression of PDE7al is abnormally high and in which decreasing its activity is likely to have a beneficial effect.
  • nucleic acid molecules also referred to herein as antibodies
  • the polynucleotides encoding PDE7al, or any fragment or complement thereof may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding PDE7al 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 PDE7al.
  • complementary molecules or fragments may be used to modulate PDE7al activity, or to achieve regulation of gene function.
  • sense or antisense ohgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding PDE7al .
  • 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.
  • 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.
  • 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, thimerosal, and the like.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the pu ⁇ ose of oral therapeutic administration, the active compound can be inco ⁇ orated 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.
  • 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, including implants and microencapsulated delivery systems.
  • a controlled release formulation including 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 Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • 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.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • 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.
  • compositions which include an agonist of PDE7al activity, a compound which increases expression of PDE7al, or a compound which increases expression or activity of a protein in the PDE7al 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 PDE7al may be used for the diagnosis of disorders characterized by the expression of PDE7al, or in assays to monitor patients being treated with PDE7al or agonists, antagonists, and inhibitors of PDE7al.
  • Antibodies useful for diagnostic pu ⁇ oses may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for PDE7al include methods which utilize the antibody and a label to detect PDE7al 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.
  • PDE7al A variety of protocols for measuring PDE7al, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of PDE7al expression.
  • Normal or standard values for PDE7al expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to PDE7al under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, preferably by photometric means. Quantities of PDE7al 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 PDE7al may be used for diagnostic pu ⁇ oses.
  • 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 PDE7al may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of PDE7al, and to monitor regulation of PDE7al levels during therapeutic intervention.
  • Polynucleotide sequences encoding PDE7al may be used for the diagnosis of hematological diseases, disorders of the peripheral and central nervous system, cardiovascular diseases, cancer diseases, respiratory disease and inflammation associated with expression of PDE7al.
  • the polynucleotide sequences encoding PDE7al 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 microarrays utilizing fluids or tissues from patient biopsies to detect altered PDE7al expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding PDE7al may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding PDE7al 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.
  • 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 PDE7al, 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. Determination of a Therapeutically Effective Dose
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases PDE7al activity relative to PDE7al 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.
  • the effectiveness of the mechanism chosen to decrease the level of expression of PDE7al gene or the activity of PDE7al can be assessed using methods well known in the art, such as hybridization of nucleotide probes to PDE7al -specific mRNA, quantitative RT-PCR, immunologic detection of PDE7al, or measurement of PDE7al 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 synergistically to effect the treatment 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.
  • Polypeptides of the invention are those polypeptides which are contained in a group of polypeptides consisting of (i) polypeptides having the sequence of SEQ LD NO: 2,
  • 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, cardiovascular diseases, cancer diseases, respiratory disease and inflammation in a mammal comprising the steps of (i) determining the activity of a PDE7al polypeptide at a certain concentration of a test compound or in the absence of said test compound, (ii) determining the activity of said polypeptide at a different concentration of said test compound.
  • compounds that lead to a difference in the activity of the PDE7al polypeptide in (i) and (ii) are identified potential therapeutic agents for such a disease.
  • 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 diagnosing a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, cardiovascular diseases, cancer diseases, respiratory disease and inflammation in a mammal comprising the steps of (i) determining the amount of a PDE7al polynucleotide in a sample taken from said mammal, (ii) determining the amount of PDE7al polynucleotide in healthy and/or diseased mammal.
  • a disease is diagnosed, e.g., if there is a substantial similarity in the amount of PDE7al 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, cardiovascular diseases, cancer diseases, respiratory disease and inflammation in a mammal comprising a therapeutic agent which regulates the activity of a PDE7al 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, cardiovascular diseases, cancer diseases, respiratory disease and inflammation in a mammal comprising a therapeutic agent which regulates the activity of a PDE7al 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, cardiovascular diseases, cancer diseases, respiratory disease and inflammation in a mammal comprising a PDE7al polypeptide.
  • Another object of the invention is the use of regulators of a PDE7al 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, cardiovascular diseases, cancer diseases, respiratory disease and inflammation 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, cardiovascular diseases, cancer diseases, respiratory disease and inflammation in a mammal comprising the steps of (i) identifying a regulator of PDE7al, (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, cardiovascular diseases, cancer diseases, respiratory disease and inflammation 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 PDE7al for the regulation of PDE7al activity in a mammal having a disease comprised in a group of diseases consisting of hematological diseases, disorders of the peripheral and central nervous system, cardiovascular diseases, cancer diseases, respiratory disease and inflammation.
  • 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.
  • 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 (Invifrogen, 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,
  • 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.
  • PDE7al were designed using the Perkin Elmer ABI Primer ExpressTM software and were synthesized by TibMolBiol (Berlin, Germany).
  • the PDE7al forward primer sequence was: Primerl (SEQ LD NO: 3).
  • the PDE7al reverse primer sequence was Primer2 (SEQ ID NO: 5).
  • Probel SEQ LD NO: 4
  • FAM carboxyfluorescein succinimidyl ester
  • TAMRA carboxytetramethyhhodamine
  • MgCl 2 200 nM of dATP, dCTP, dGTP, and dUTP, 0.025 U/ ⁇ l AmpliTaq GoldTM, 0.01 U/ ⁇ l AmpErase and Probel (SEQ LD NO: 4), PDE7al forward and reverse primers each at 200 nM, 200 nM PDE7al FAM/TAMRA-labelled probe, and 5 ⁇ l of template cDNA.
  • Thermal cycling parameters were 2 min at 50°C, followed by 10 min at 95°C, followed by 40 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min.
  • 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 :
  • CTn G -values were calculated as described in the "Quantitative determination of nucleic acids" section.
  • CFcDNA-n CTpannei-mean value - CTHKG- ⁇ - mean value
  • CT C DNA-n CT value of the tested gene for the cDNA n
  • CF CD NA-I CT cor -cD N
  • CT cor -cD N CT cor -cD N
  • Table 1 Relative expression ofPDElal in various human tissues.
  • HEP G2 cells 24 leukocytes (peripheral blood) 265
  • Alzheimer cerebral cortex 87 frontal lobe 108 Alzheimer brain frontal lobe 49 occipital lobe 61 parietal lobe 51 temporal lobe 74 precentral gyrus 57 postcentral gyrus 2 tonsilla cerebelli 94 vermis cerebelli 82 pons 53 substantia nigra 131 cerebral meninges 13 cerebral peduncles 50 corpus callosum 120 hippocampus 71 thalamus 33 dorsal root ganglia 12 spinal cord 41 neuroblastoma SK-N-MC cells 70 neuroblastoma SH-SY5Y cells 93 neuroblastoma IMR32 cells 164 glial tumor H4 cells 23 retina 19 fetal lung 161 fetal lung fibroblast IMR-90 cells 22 fetal lung fibroblast MRC-5 cells 47 lung 31 lung right upper lobe 87 lung right mid lobe 42 lung right lower lobe 97 lung lupus disease 61 lung tumor 63 lung COPD 9
  • MDA MB 231 cells (breast tumor) 10 mammary gland 89 prostate 45 prostate BPH 11 bladder 34 ureter 69 penis 21 corpus cavernosum 3 fetal kidney 65 kidney 27 kidney tumor 45 HEK 293 cells 56
  • Ohgonucleotides, cDNA or genomic fragments comprising the antisense strand of a polynucleotide coding for PDE7al are used either in vitro or in vivo to inhibit franslation 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 intron regions, promoter/enhancer elements, or even to transacting regulatory genes.
  • Expression of PDE7al 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.
  • IPTG Isopropyl- ⁇ -D-thio- galactopyranoside
  • 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 including in vitro mutagenesis, digestion with exonuclease III or mung bean nuclease, or the inclusion of an oligonucleotide linker of appropriate length.
  • the PDE7al 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 phosphotransferase 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, SN40, and metallothionine promoters for CHO cells; trp, 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 virus enhancer, are used in mammalian host cells. Once homogeneous cultures of recombinant cells are obtained through standard culture methods, large quantities of recombinantly produced PDE7al are recovered from the conditioned medium and analyzed using chromatographic methods known in the art.
  • PDE7al can be cloned into the expression vector pcDNA3, as exemplified herein.
  • This product can be used to transform, 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.
  • PDE7al 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 (Immunex Corp., Seattle, Washington).
  • the inclusion of a cleavable linker sequence such as Factor Xa or enterokinase (Invifrogen, Groningen, The Netherlands) between the purification domain and the PDE7al sequence is useful to facilitate expression of PDE7al.
  • the following example provides a method for purifying PDE7al .
  • PDE7al is generated using the baculovirus expression system BAC-TO-BAC (GIBCO BRL) based on Autographa californica nuclear polyhedrosis virus (AcNPV) infection of Spodoptera frugiperda insect cells (Sf9 cells).
  • cDNA 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 DH10BAC competent cells which contain the parent bacmid bMON14272 (AcNPV infectious D ⁇ A) 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 disruption 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 baculovirus particles and expression of either unfused recombinant enzyme (pFastbacl) or PDE7al-His fusion protein (pFastbacHT).
  • PDE7al is confirmed by coomassie staining after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting onto a PNDF 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 ⁇ i- ⁇ TA HRP conjugate and the His-tag which is fused to PDE7al .
  • 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 PDE7al 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 ⁇ - 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 finoc-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. Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant.
  • KLH keyhole limpet hemocyanin
  • 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 PDE7al 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 albumin, (BSA), washed and incubated with supernatants from hybridomas. After washing the wells are incubated with labeled PDE7al at 1 mg/ml.
  • BSA bovine serum albumin
  • Diagnostic tests for PDE7al include methods utilizing antibody and a label to detect PDE7al 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.
  • a wide variety of labels and conjugation techmques are known and have been reported extensively in both the scientific and patent literature.
  • a variety of protocols for measuring soluble or membrane-bound PDE7al, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non- interfering epitopes on PDE7al is prefened, but a competitive binding assay may be employed.
  • Example 8 Purification of Native PDE7al Using Specific Antibodies
  • Such in-ununoaffinity columns are utilized in the purification of PDE7al by preparing a fraction from cells containing PDE7al 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 PDE7al containing a signal sequence is secreted in useful quantity into the medium in which the cells are grown.
  • PDE7al 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 PDE7al is collected.
  • a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion e.g., a chaotrope such as urea or thiocyanate ion
  • 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 [ 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 goal of rational drug 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 drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo.
  • a target-specific antibody selected by functional assay, as described above, and then to solve its crystal structure.
  • This approach in principle, yields a pharmacore upon which subsequent drug 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 PDE7al is useful as a reagent for the purification of molecules with which it interacts.
  • PDE7al 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 PDE7al.
  • PDE7al -complex is recovered from the column, and the PDE7al -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.
  • Example 12 Use and Administration of Antibodies, Inhibitors, or Antagonists
  • LSTs are delivered by known routes of administration including but not limited to topical creams and gels; fransmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • routes of administration including but not limited to topical creams and gels; fransmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • the particular formulation, exact dosage, and route of adminisfration 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 adminisfration, possible combination with other drugs, 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 administration.
  • 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.
  • Adminisfration to cells such as nerve cells necessitates delivery in a manner different from that to other cells such as vascular endothelial cells.
  • Animal model systems which elucidate the physiological and behavioral roles of the PDE7al are produced by creating nonhuman transgenic animals in which the activity of the PDE7al is either increased or decreased, or the amino acid sequence of the expressed PDE7al is altered, by a variety of techniques.
  • these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a PDE7al, by microinjection, electroporation, retro viral 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 structure of these PDE7al sequences.
  • the technique of homologous recombination is well known in the art. It replaces the native gene with the inserted gene and hence is useful for producing an animal that cannot express native PDE7als but does express, for example, an inserted mutant PDE7al, which has replaced the native PDE7al in the animal's genome by recombination, resulting in underexpression of the transporter. 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 PDE7al , resulting in overexpression of the PDE7al .
  • 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 PDE7al 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.

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Abstract

L'invention concerne une phosphodiestérase 7A1 (PDE7a1) humaine qui est associée à des maladies hématologiques, troubles du système nerveux central et périphérique, maladies cardiovasculaires, maladies cancéreuses, maladies respiratoires ainsi qu'à l'inflammation. Cette invention se rapporte également à des techniques d'identification de composés servant à traiter ou prévenir des maladies hématologiques, troubles du système nerveux central et périphérique, maladies cardiovasculaires, maladies cancéreuses, maladies respiratoires et l'inflammation. La présente invention concerne en outre des composés qui se fixent sur ladite PDE7a1 et/ou stimulent ou inhibent son activité, ainsi que des compositions pharmaceutiques contenant ces composés.
PCT/EP2003/012341 2002-11-13 2003-11-05 Procedes de diagnostic de maladies associees a la phosphodiesterase 7a1 (pde7a1) humaine et moyens therapeutiques correspondants Ceased WO2004044235A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP03773703A EP1597385A1 (fr) 2002-11-13 2003-11-05 PROCEDES DE DIAGNOSTIC DE MALADIES ASSOCIEES A LA PHOSPHODIESTERASE 7A1 (PDE7a1) HUMAINE ET MOYENS THERAPEUTIQUES CORRESPONDANTS
AU2003282087A AU2003282087A1 (en) 2002-11-13 2003-11-05 DIAGNOSTICS AND THERAPEUTICS FOR DISEASES ASSOCIATED WITH HUMAN PHOSPHODIESTERASE 7A1 (PDE7a1)

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EP2019835A4 (fr) * 2006-05-19 2010-08-04 Topigen Pharmaceuticals Inc Oligonucléotides modifiant l'expression de phosphodiestérases
US8173796B2 (en) 2003-09-29 2012-05-08 Topigen Pharmaceutique Inc. Oligonucleotide compositions and methods for treating disease including inflammatory conditions
US8637528B2 (en) 2007-03-27 2014-01-28 Omeros Corporation Use of PDE7 inhibitors for the treatment of movement disorders

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8173796B2 (en) 2003-09-29 2012-05-08 Topigen Pharmaceutique Inc. Oligonucleotide compositions and methods for treating disease including inflammatory conditions
EP2019835A4 (fr) * 2006-05-19 2010-08-04 Topigen Pharmaceuticals Inc Oligonucléotides modifiant l'expression de phosphodiestérases
US7982028B2 (en) 2006-05-19 2011-07-19 Topigen Pharmaceuticals, Inc. Oligonucleotides affecting expression of phosphodiesterases
US8637528B2 (en) 2007-03-27 2014-01-28 Omeros Corporation Use of PDE7 inhibitors for the treatment of movement disorders
US9119822B2 (en) 2007-03-27 2015-09-01 Omeros Corporation Use of PDE7 inhibitors for the treatment of movement disorders

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