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EP1540332A1 - Procedes de recherche systematique de composes destines a l'activite de modulation des grk6 - Google Patents

Procedes de recherche systematique de composes destines a l'activite de modulation des grk6

Info

Publication number
EP1540332A1
EP1540332A1 EP03763111A EP03763111A EP1540332A1 EP 1540332 A1 EP1540332 A1 EP 1540332A1 EP 03763111 A EP03763111 A EP 03763111A EP 03763111 A EP03763111 A EP 03763111A EP 1540332 A1 EP1540332 A1 EP 1540332A1
Authority
EP
European Patent Office
Prior art keywords
grk6
gpcr
cell
mice
compound
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Withdrawn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
EP03763111A
Other languages
German (de)
English (en)
Inventor
Marc G. Caron
Raul R. Gainetdinov
Robert J. Lefkowitz
Richard T. Premont
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.)
Duke University
Original Assignee
Duke University
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Filing date
Publication date
Application filed by Duke University filed Critical Duke University
Publication of EP1540332A1 publication Critical patent/EP1540332A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
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    • A01K67/0276—Knock-out vertebrates
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
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    • G01—MEASURING; TESTING
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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    • A01K2227/10—Mammal
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the invention is in the field of identifying compounds that modulate GRK6 and their use in treating disease.
  • G protein-coupled receptors are cell surface proteins that translate hormone or ligand binding into intracellular signals. GPCRs are found in all animals, insects, and plants. GPCR signaling plays a pivotal role in regulating various physiological functions including phototransduction, olfaction, neurotransmission, vascular tone, cardiac output, digestion, pain, and fluid and electrolyte balance. Although they are involved in numerous physiological functions, GPCRs share a number of common structural features. They contain seven membrane domains bridged by alternating intracellular and extracellular loops and an intracellular carboxyl-terminal tail of variable length.
  • GPCRs have been implicated in a number of disease states, including, but not limited to: cardiac indications such as angina pectoris, essential hypertension, myocardial infarction, supraventricular and ventricular arrhythmias, congestive heart failure, atherosclerosis, renal failure, diabetes, respiratory indications such as asthma, chronic bronchitis, bronchospasm, emphysema, airway obstruction, upper respiratory indications such as rhinitis, seasonal allergies, inflammatory disease, inflammation in response to injury, rheumatoid arthritis, chronic inflammatory bowel disease, glaucoma, hypergastrinemia, gastrointestinal indications such as acid/peptic disorder, erosive esophagitis, gastrointestinal hypersecretion, mastocytosis, gastrointestinal reflux, peptic ulcer, Zollinger-Ellison syndrome, pain, obesity, bulimia nervosa, depression, obsessive-compulsive disorder, organ malformations (for example,
  • GPCRs G protein-coupled receptor kinases
  • arrestins bind activated GPCRs, including those that have been agonist-activated and especially those that have been phosphorylated by G protein-coupled receptor kinases (GRKs).
  • GRKs G protein-coupled receptor kinases
  • Multiple GRK enzymes are found in brain regions, but the relative physiological importance of each GRK to the function of any given neurotransmitter receptor was unclear, and no clear role for GRKs in drug abuse or addiction susceptibility had been demonstrated.
  • FIG. 1 illustrates that GRK6 is present in striatal neurons expressing DARPP-32.
  • Upper-left Immunofluorescence analysis reveals GRK6 immunoreactivity in the striatal neurons of WT mouse (+0.74 from bregma).
  • Upper-right Lack of GRK6 immunoreactivity in the striatal neurons of a mouse lacking a functional GRK6 gene (GRK6-KO mouse).
  • FIG. 2 shows the targeted inactivation of the mouse GRK6 gene.
  • Figure 2A is a schematic diagram of the wild type mouse GRK6 gene locus, the GRK6/lox targeting vector, the integrated targeting construct, and the Cre recombinase-deleted GRK6 locus (GRK6-KO).
  • GRK6 exons are shown as open boxes, and numbered from the first coding exon.
  • LoxP sites are shown as filled triangles, and the location of the Southern blot probe as a hatched box.
  • FIG. 2B shows the genotyping of targeted GRK6-KO mice.
  • the wild type and GRK6-KO loci were distinguished by triplex PCR amplification.
  • the WT GRK6 locus gives a 460 bp band while the GRK6-KO locus gives a 610 bp band, as indicated.
  • Figure 2C illustrates GRK6 protein expression by Western blotting. Membrane proteins from brainstem and striatum of wild type and GRK6-KO animals were subjected to immunoblotting using an anti-GRK6 antiserum.
  • GRK6-KO homozygote animals exhibit a loss of the 68-kDa immunoreactive band compared to wild type animals (Arrow).
  • Figure 3 illustrates the cocaine supersensitivity in GRK6 mutant mice.
  • GRK6 heterozygous and GRK6-KO mice demonstrate greater locomotor behavior than their wildtype littermates, and are significantly different from WT controls (p ⁇ 0.001 , two-way analysis of variance (ANOVA).
  • Figure 3C illustrates cocaine sensitization in GRK6-KO mice.
  • mice in the 90 min period after cocaine administration on days 1 and 7 are shown in the lower panel. **p ⁇ 0.01 ; *** p ⁇ 0.001 vs. WT littermates for the 1st day group (Student's t-test).
  • Figure 4 illustrates the enhanced locomotor effects of d-amphetamine and ⁇ - phenylethylamine in GRK6 mutant mice.
  • GRK6 heterozygous and GRK6-KO mice are significantly different from WT controls in responses to d-amphetamine. p ⁇ 0.001 , two-way ANOVA.
  • FIG. 5 shows analyses of presynaptic dopamine function in WT and GRK6-KO mice.
  • Figure 6 illustrates that alterations in GRK6 level modulate dopamine receptor coupling to G-proteins.
  • Figure 6A shows [ 35 S]GTP ⁇ S binding to striatal membranes from GRK6 mutant and wild type mice. Total [ 35 S]GTP ⁇ S binding is portrayed after subtracting unstimulated [ 35 S]GTP ⁇ S binding from each point. [ 35 S]GTP ⁇ S binding to striatal membranes was determined after stimulation with the D2 dopamine agonist quinpirole. Percent stimulated [ 35 S]GTP ⁇ S binding was calculated by dividing unstimulated [ 35 S]GTP ⁇ S binding into each agonist-stimulated point.
  • Figure 6B shows [ 35 S]GTP ⁇ S binding to HEK-293 cell membranes expressing the D3 dopamine receptor subtype (D2R) was determined after stimulation with dopamine. At least two independent experiments were performed in triplicate.
  • Figure 6C shows [ 35 S]GTP ⁇ S binding to HEK-293 cell membranes expressing the D3 dopamine receptor subtype (D3R) plus the G protein subunit Go- ⁇ was determined after stimulation with dopamine. At least two independent experiments were performed in triplicate. The same procedure was employed for data treatments. p ⁇ 0.001 , two-way ANOVA.
  • Figure 7 illustrates that the dopamine agonist effect is enhanced in dopamine-depleted GRK6-KO mice.
  • reserpine 5 mg/kg, i.p.
  • ⁇ -methyl-p-tyrosine 250 mg/kg, i.p.
  • GRK6-KO mice are significantly different from WT controls (p ⁇ 0.001 , two-way ANOVA).
  • Figure 8 illustrates that locomotion in DA-depleted wild type and GRK6-KO mice were restored by administration of apomorphine (0.2 mg/kg, s.c.)
  • Figure 9 illustrates that locomotion in DA-depleted wild type and GRK6-KO mice were restored by administration of apomorphine (0.5 mg/kg, s.c).
  • Figure 10 shows the nucleic acid and protein sequences of the present invention.
  • SEQ ID No: 1 is the nucleic acid sequence inserted into the pBS vector, as described in Figure 2.
  • SEQ ID No: 2 is the nucleic acid sequence of the Cre-deleted locus, as described in Figure 2.
  • SEQ ID Nos: 3, 4, and 5 are the nucleic acid sequences of the primers used to confirm the construction of the GRK6 deletion.
  • SEQ ID Nos: 6, 8, and 10 are the nucleic acid sequences of the mouse GRK6A, GRK6B, and GRK ⁇ c splice variants.
  • SEQ ID Nos: 7, 9, and 11 are the amino acid sequences of the mouse GRK6A, GRK6B, and GRK6c splice variants.
  • SEQ ID Nos: 12, 14, and 16 are the nucleic acid sequences of the human GRK6A, GRK6B, and GRK6c splice variants.
  • SEQ ID Nos: 13, 15, and 17 are the amino acid sequences of the human GRK6A, GRK6B, and GRK6c splice variants.
  • the present inventors have determined the role of GRK6 in desensitization of GPCRs and have constructed a transgenic mouse that has a functionally disrupted GRK6 gene.
  • the present inventors have determined that GRK6 is a target for modulating desensitization of GPCRs and have designed methods for the identification of compounds that target GRK6 and alter GPCR desensitization.
  • the present inventors describe methods of evaluating the compounds for the treatment of disease and describe the use of such compounds for the treatment of disease.
  • a "replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • linear DNA molecules e.g., restriction fragments
  • viruses e.g., plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • An "origin of replication” refers to those DNA sequences that participate in the initiation of DNA synthesis.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3" (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5* direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA” boxes and “CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgamo sequences in addition to the -10 and -35 consensus sequences.
  • An "expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a "signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
  • oligonucleotide as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand.
  • non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
  • the terms "restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are "substantially homologous" when at least about 65% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • ⁇ AR is a GPCR termed a ⁇ -adrenergic receptor.
  • Internalization of a GPCR is the translocation of a GPCR from the cell surface membrane to an intracellular vesicular membrane, where it may be inaccessible to substances remaining outside the cell.
  • Carboxyl-terminal tail means the carboxyl-terminal tail of a GPCR following membrane span 7. The carboxyl-terminal tail of many GPCRs begins shortly after the conserved NPXXY motif that marks the end of the seventh transmembrane domain (i.e. what follows the NPXXY motif is the carboxyl-terminal tail of the GPCR).
  • the carboxyl-terminal tail may be relatively long (approximately tens to hundreds of amino acids), relatively short (approximately tens of amino acids), or virtually non-existent (less than approximately ten amino acids).
  • carboxyl-terminal tail shall mean all three variants (whether relatively long, relatively short, or virtually non-existent), and may or may not contain palmitoylated cysteine residue(s).
  • "Class A receptors" preferably do not translocate together with arrestin proteins to endocytic vesicles or endosomes in association with arrestin-GFP in HEK- 293 cells.
  • DACs mean any desensitization active compounds. Desensitization active compounds are any compounds that influence the GPCR desensitization mechanism by either stimulating or inhibiting the process. DACs may influence the GPCR desensitization pathway by acting on any cellular component of the process, as well as any cellular structure implicated in the process, including but not limited to: arrestins, GRKs, GPCRs, phosphoinositide 3-kinase, AP-2 protein, clathrin, protein phosphatases, and the like.
  • DACs may include, but are not limited to, compounds that inhibit arrestin translocating to a GPCR, compounds that inhibit arrestin binding to a GPCR, compounds that stimulate arrestin translocating to a GPCR, compounds that stimulate arrestin binding to a GPCR, compounds that inhibit GRK phosphorylation of a GPCR, compounds that stimulate GRK phosphorylation of a GPCR, compounds that stimulate or inhibit GRK binding to a GPCR, compounds that inhibit protein phosphatase dephosphorylation of a GPCR, compounds that stimulate protein phosphatase dephosphorylation of a GPCR, compounds that prevent GPCR internalization or recycling to the cell surface, compounds that regulate the release of arrestin from a GPCR, antagonists of a GPCR, inverse agonists and the like.
  • DACs may inhibit or stimulate the GPCR desensitization process and may not bind to the same ligand binding site of the GPCR as traditional agonists and antagonists of the GPCR.
  • DACs may act independently of the GPCR, i.e., they do not have high specificity for one particular GPCR or one particular type of GPCRs.
  • DACs may bind the same site(s) as agonist or antagonist but do not desensitize the receptor (perhaps by not altering the receptor to be properly phosphorylated or bind to arrestin or any other protein).
  • DACs may bind to allosteric sites on the receptor and inhibit or enhance desensitization.
  • Detectable molecule means any molecule capable of detection by spectroscopic, photochemical, biochemical, immunochemical, electrical, radioactive, and optical means, including but not limited to, fluorescence, phosphorescence, and bioluminescence and radioactive decay.
  • Detectable molecules include, but are not limited to, GFP, luciferase, ⁇ -galactosidase, rhodamine-conjugated antibody, and the like.
  • Detectable molecules include radioisotopes, epitope tags, affinity labels, enzymes, fluorescent groups, chemiluminescent groups, and the like.
  • Detectable molecules include molecules which are directly or indirectly detected as a function of their interaction with other molecule(s).
  • GFP Green Fluorescent Protein which refers to various naturally occurring forms of GFP which may be isolated from natural sources or genetically engineered, as well as artificially modified GFPs. GFPs are well known in the art. See, for example, U.S. Patent Nos. 5,625,048; 5,777,079; and 6,066,476. It is well understood in the art that GFP is readily interchangeable with other fluorescent proteins, isolated from natural sources or genetically engineered, including but not limited to, yellow fluorescent proteins (YFP), red fluorescent proteins (RFP), cyan fluorescent proteins (CFP), blue fluorescent proteins, luciferin, UV excitable fluorescent proteins, or any wave-length in between. As used herein, “GFP” shall mean all fluorescent proteins known in the art.
  • "Unknown or Orphan Receptor” means a GPCR whose function and/or ligands are unknown.
  • Downstream means toward a carboxyl-terminus of an amino acid sequence, with respect to the amino-terminus.
  • Upstream means toward an amino-terminus of an amino acid sequence, with respect to the carboxyl-terminus.
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced a potential site in order to allow formation of disulfide bridges with another Cys.
  • a His may be introduced as a particularly "catalytic" residue (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis).
  • Pro may be introduced because of its particularly planar structure, which induces ⁇ -turns in the protein's structure.
  • Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • an "immunoglobulin” includes antibodies and antibody fragments with immunogenic activity. Preferred immunogenic activity is where the immunoglobulin binds to a modified GPCR. An even more preferable immunoglobulin is one that can distinguish between a modified GPCR and a wild-type GPCR.
  • the term “antibody” refers to immunoglobulins, including whole antibodies as well as fragments thereof that recognize or bind to specific epitopes. The term antibody encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.
  • immunoglobulins are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope.
  • Antibody fragments include those portions known in the art as Fab, Fab', F(ab') 2 , F(v), and scFv which portions are preferred for use in the therapeutic methods described herein.
  • Fab and F(ab") 2 portions of antibody fragments are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibodies by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to
  • Theofilopolous et al. Fab' antibody portions are also well-known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation with a reagent such as ⁇ iodoacetamide.
  • An antibody containing intact antibody portions is preferred herein.
  • An "antibody combining site” is that structural portion of an antibody comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular epitope on an antigen.
  • a monoclonal antibody may therefore contain a plurality of antibody combining sites, each immunospecific for a different antigen, e.g., a bispecific (chimeric) monoclonal antibody.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • therapeutically effective amount is used herein to mean an amount sufficient to prevent, and preferably reduce some feature of pathology such as for example, elevated blood pressure, respiratory output, etc.
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • Hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine (A) and thymine (T) are complementary nucleobases that pair through the formation of hydrogen bonds.
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65 °C for both hybridization and wash. However, one skilled in the art will appreciate that such “standard hybridization conditions” are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like.
  • Standard hybridization conditions are whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20 °C below the predicted or determined Tm with washes of higher stringency, if desired.
  • animal is meant any member of the animal kingdom including vertebrates (e.g., frogs, salamanders, chickens, or horses) and invertebrates (e.g., worms, etc.).
  • animals e.g., frogs, salamanders, chickens, or horses
  • invertebrates e.g., worms, etc.
  • animals include livestock animals (e.g., ungulates, such as cattle, buffalo, horses, sheep, pigs and goats), as well as rodents (e.g., mice, hamsters, rats and guinea pigs), canines, felines, primates, lupine, camelid, cervidae, rodent, avian and ichthyes.
  • Antagonist(s) include all agents that interfere with wild-type and/or modified GPCR binding to an agonist, wild-type and/or modified GPCR desensitization, wild-type and/or modified GPCR binding arrestin, wild-type and/or modified GPCR endosomal localization, internalization, and the like, including agents that affect the wild-type and/or modified GPCRs as well as agents that affect other proteins involved in wild-type and/or modified GPCR signaling, desensitization, endosomal localization, resensitization, and the like.
  • GPCR means G protein-coupled receptor and includes GPCRs naturally occurring in nature, as well as GPCRs which have been modified. Such modified GPCRs are described in U.S.S.N. 09/993,844 and U.S.S.N. 10/054,616.
  • Abnormal GPCR desensitization and "abnormal desensitization” mean that the GPCR desensitization pathway is disrupted such that the balance between active receptor and desensitized receptor is altered with respect to wild-type conditions. Either there is more active receptor than normal or there is more desensitized receptor than wild-type conditions.
  • Abnormal GPCR desensitization may be the result of a GPCR that is constitutively active or constitutively desensitized, leading to an increase above normal in the signaling of that receptor or a decrease below normal in the signaling of that receptor.
  • Bio sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject; wherein said sample can be blood, serum, a urine sample, a fecal sample, a tumor sample, a cellular wash, an oral sample, sputum, biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture.
  • Concurrent administration means that the compounds are administered at the same point in time or sufficiently close in time that the results observed are essentially the same as if the two or more compounds were administered at the same point in time.
  • Consed abnormality means an abnormality in the GPCR pathway, including but not limited to, abnormalities in GPCRs, GRKs, arrestins, AP-2 protein, clathrin, protein phosphatase and the like, that may cause abnormal GPCR signaling.
  • This abnormal GPCR signaling may contribute to a GPCR-related disease.
  • Desensitized GPCR means a GPCR that presently does not have ability to respond to agonist and activate conventional G protein signaling.
  • Desensitization pathway means any cellular component of the desensitization process, as well as any cellular structure implicated in the desensitization process and subsequent processes, including but not limited to, arrestins, GRKs, GPCRs, AP-2 protein, clathrin, protein phosphatases, and the like.
  • the polypeptides may be detected, for example, in the cytoplasm, at a cell membrane, in clathrin-coated pits, in endocytic vesicles, endosomes, any stages in between, and the like.
  • GPCR signaling means GPCR induced activation of G proteins. This may result in, for example, cAMP production.
  • G protein-coupled receptor kinase includes any kinase that has the ability to phosphorylate a GPCR.
  • Homo sapiens GPCR means a naturally occurring GPCR in a Homo sapiens.
  • Inverse agonist means a compound that, upon binding to the GPCR, inhibits the basal intrinsic activity of the GPCR.
  • An inverse agonist is a type of antagonist.
  • Modified GRK means a GRK modified such that it alters desensitization.
  • Naturally occurring GPCR means a GPCR that is present in nature.
  • Oxygen ligand means a ligand compound that, upon binding to a receptor, leads to the perception of an odor including a synthetic compound and/or recombinantly produced compound including agonist and antagonist molecules.
  • Optorant receptor means a receptor protein normally found on the surface of olfactory neurons which, when activated (normally by binding an odorant ligand) leads to the perception of an odor.
  • pharmaceutically acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.
  • Primed antibody means a recombinant antibody containing primate variable sequences or antigen binding portions, and human constant domain sequences.
  • Sensitized GPCR means a GPCR that presently has ability to respond to agonist and activate conventional G protein signaling.
  • GRK6 includes GRK6 splice variants, including GRK6a, GRK6b, GRK6c, and GRK6d and a GRK6, of a human, a primate, a feline, a canine, a porcine, a bovine, a caprine, an ovine, or other animals.
  • Modulation includes at least an up-regulation or down-regulation of the expression, or an increase or decrease in activity of a protein.
  • Modulation of a protein includes the up-regulation, down-regulation, increase or decrease in activity of a protein or compound that regulates a protein. Modulation also includes the regulation of the gene, the mRNA, or any other step in the synthesis of the protein of interest.
  • a "GRK6 related disease” refers to a disease affected by GRK6, particularly
  • a GRK6 related disease also includes diseases affected by GPCRs that may be phosphorylated and/or regulated by GRK6, such as the dopamine receptor. Such diseases include Parkinson's, schizophrenia, depression,
  • GPCR desensitization refers to GPCR desensitization in which
  • the GRK6 affects the desensitization.
  • the GRK6 may directly phosphorylate the GPCR, or otherwise affect the desensitization of the GPCR.
  • An "overexpressed" protein refers to a protein that is expressed at levels greater than wild-type expression levels.
  • GPCRs and desensitization [0089] The exposure of a GPCR to agonist produces rapid attenuation of its signaling ability that involves uncoupling of the receptor from its cognate heterotrimeric G-protein.
  • the cellular mechanism mediating agonist-specific or homologous desensitization is a two-step process in which agonist-occupied receptors are phosphorylated by a G protein-coupled receptor kinases (GRKs) and then bind an arrestin protein.
  • GRKs G protein-coupled receptor kinases
  • G-protein coupled receptor kinases phosphorylate intracellular domains of GPCRs. After phosphorylation, an arrestin protein associates with the GRK-phosphorylated receptor and uncouples the receptor from its cognate G protein. The interaction of the arrestin with the phosphorylated GPCR terminates GPCR signaling and produces a non-signaling, desensitized receptor.
  • the arrestin bound to the desensitized GPCR targets the GPCR to clathrin- coated pits or other cellular machinery for endocytosis (i.e., internalization) by functioning as an adaptor protein, which links the GPCR to components of the endocytic machinery, such as adaptor protein-2 (AP-2) and clathrin.
  • the internalized GPCRs are dephosphorylated and are recycled back to the cell surface desensitized, or are retained within the cell and degraded.
  • the stability of the interaction of arrestin with the GPCR is one factor that dictates the rate of GPCR dephosphorylation, recycling, and resensitization.
  • GRK1 Seven distinct GRK genes are known, named GRK1 through GRK7, that were classified into three distinct groups.
  • GRK6 is a member of the GRK4 subfamily of GRKs, which also contains GRK4 and GRK5.
  • Multiple GRK enzymes are found in brain regions, but the relative physiological importance of each GRK to any given neurotransmitter, prior to the present invention, was unclear.
  • Brain dopaminergic transmission is critically involved in numerous vital functions, such as movement control, emotion and affect, and its dysfunction is believed to be central in several pathological conditions, including addiction.
  • Physiological responses to dopamine are controlled by a family of G-protein coupled dopamine receptors (including D1-D5), that are expressed in specific brain areas.
  • Sensitivity of dopamine receptors to endogenous and exogenous ligands is known to be an important modulator of dopamine-related functions in physiology and pathology.
  • Supersensitivity of dopamine signaling has been described in several brain disorders, including addiction. Particularly, it is believed that sensitization, an early biochemical and behavioral manifestation of cellular plasticity leading to addiction, is associated with long-term changes in dopamine receptor responsiveness.
  • GPCR G protein-coupled receptor
  • GRKs G protein-coupled receptor kinases
  • mice are supersensitive to the locomotor-stimulating effect of psychostimulants, including cocaine and amphetamine, and displayed little further sensitization to chronic treatment with cocaine.
  • these mice demonstrated an enhanced coupling of striatal dopamine receptors to G proteins and augmented locomotor response to the dopamine agonist apomorphine in dopamine-depleted animals.
  • postsynaptic dopamine receptors are physiological targets for GRK6, and suggests that these regulatory mechanisms contribute to central dopaminergic supersensitivity observed in drug abuse and other pathological conditions, such as Parkinson's disease.
  • GRK6 Altering the expression and activity of GRK6 will be useful in treatment of diseases associated with dopamine receptor supersensitivity, such as schizophrenia, depression, Tourette Syndrome, and Parkinson's disease [0096] This supersensitivity is correlated with an increased coupling of dopamine receptors to G proteins, caused by diminished dopamine receptor desensitization. These results indicate that dopamine receptor regulation by GRK6 plays an important role in setting the basal tone of dopamine signaling in the striatum and that diminished GRK6 function may be a predisposing factor affecting drug sensitivity.
  • Knock-out mice and animals For use as disease models and to test compounds identified herein, modified GRK6 transgenic and knock-out mice and animals may be produced and utilized.
  • modified GRK6 transgenic and knock-out mice and animals For use as disease models, to test compounds identified herein, and to identify GPCRs phosphorylated by GRK6, transgenic and knock-out mice and animals comprising modified components of the desensitization pathway described herein may be produced and utilized.
  • the animal may, for example, be a mouse or an animal as listed herein. Examples related to knock-out animals are described herein. Certain non- limiting embodiments refer specifically to a knock-out mouse, but are intended to encompass animals as described herein.
  • the cells of the mouse containing at least one inactive endogenous GRK6 gene may be a complete knockout or homozygous for the inactive endogenous GRK6 gene, or the mouse may be a partial knockout or heterozygous for the inactive endogenous GRK6 gene.
  • the knockout mouse may be useful for verification that a compound is in fact a GRK6 modulator.
  • the knockout mouse of the present invention may be used as a model for comparison with wild-type mice that have been treated with a GRK6 modulator. This comparison may be used to verify that the compound administered to the wild-type mice is a GRK6 modulator.
  • the knockout mouse may also be useful for verification that a compound is in fact a GRK6 activator or inhibitor.
  • partial knockout mice that have been treated with a GRK6 activator or inhibitor may be used as a model for comparison with wild-type mice and complete knockout mice. This comparison may be used to verify that the compound administered is a GRK6 activator or inhibitor.
  • the production of GRK6 knockout mice can be carried out in view of the disclosure provided herein and in light of techniques known to those skilled in the art, such as described in U.S.S.N. 09/469,554, filed December 22, 1999, U.S. Patents Nos.
  • mice for carrying out the present invention are as disclosed below. Sequences described in Figure 10 include the disrupted GRK6 of the transgenic animal, as well as constructs used in making the transgenic animal.
  • the present invention is also related to methods of testing a compound for the ability to modulate GRK6.
  • the test compound may be administered to a wild-type non-human animal; and the locomotor response of the wild-type non-human animal exposed to the compound will be compared to the locomotor response of the non-human transgenic animal that has a disrupted GRK6 gene. Tests of locomotor response would be performed as described in the Examples. Other physical and cellular responses may be monitored, as described in the Examples.
  • the present invention relates to methods of screening for compounds which modulate GRK6-associated desensitization.
  • a cell is provided which includes GRK6 and a GPCR. The cell is contacted with a candidate modulator. The cell is monitored for GRK6-associated desensitization. Methods of monitoring desensitization are described herein, and in U.S.S.N. 09/993,844 filed on November 5, 2001 , U.S.S.N. 10/054,616 filed on January 22, 2002, and U.S.S.N. 10/101 ,235 filed on March 19, 2002, which are hereby incorporated by reference in their entirety.
  • the GRK6- associated desensitization may be monitored by determining the cellular distribution of the GRK6, GPCR, or arrestin in the presence of the compound as compared to the cellular distribution in the absence of the compound.
  • the difference between the cellular distribution of the GRK6, GPCR, or arrestin in the presence or absence of the compound(s) may be correlated to modulation of GRK6 activity.
  • the candidate modulator may be a pure compound, or may be a heterogeneous mixture of compounds. The mixture may contain certain compounds that modulate GRK6 and other compounds which do not modulate GRK6.
  • the cellular distribution of the GRK6, GPCR, or arrestin may be determined.
  • the present invention relates to methods of identifying compounds that modulate GRK6-associated desensitization.
  • a cell is provided which includes GRK6, a GPCR, and an arrestin, wherein one of the molecules is detectably labeled and the GRK6 is overexpressed.
  • the cell is contacted with a candidate modulator.
  • the cellular distribution of the GRK6, GPCR, or arrestin in the presence of the compound is compared to the cellular distribution in the absence of the compound.
  • the difference between the cellular distribution of the GRK6, GPCR, or arrestin in the presence or absence of the compound(s) is correlated to modulation of GRK6 activity.
  • Such methods are described herein, and in U.S.S.N.
  • the GRK6 is overexpressed.
  • the labeled molecule may be localized in the cytosol, plasma membrane, clathrin-coated pits, endocytic vesicles, or endosomes.
  • the detectable molecule may be a radioisotope, an epitope tag, an affinity label, an enzyme, a fluorescent group, or a chemiluminescent group.
  • the molecule may be detectably labeled due to its interaction with another molecule, which may be detectably labeled.
  • the present invention further relates to methods of inhibiting desensitization of the dopamine receptor in a cell. These methods may include contacting the cell with a compound.
  • the compound may be an antisense oligonucleotide, or another compound as described herein.
  • the antisense oligonucleotide may inhibit expression of a nucleic acid encoding GRK6, or another gene that affects GRK6 activity.
  • Methods of detecting the intracellular location of the detectably labeled arrestin, the intracellular location of a detectably labeled GPCR, the intracellular location of a detectably labeled GRK, or interaction of the detectably labeled molecule with a GPCR or any other cell structure including for example, the concentration of arrestin, GRK, or GPCR at a cell membrane, colocalization of arrestin with GPCR in endosomes, and concentration of arrestin or GPCR in clathrin-coated pits, and the like, will vary dependent upon the detectable molecule(s) used.
  • any optical method may be used where a change in the fluorescence, bioluminescence, or phosphorescence may be measured due to a redistribution or reorientation of emitted light.
  • Such methods include, for example, polarization microscopy, BRET, FRET, evanescent wave excitation microscopy, and standard or confocal microscopy.
  • arrestin may be conjugated to GFP and the arrestin-GFP conjugate may be detected by confocal microscopy.
  • arrestin may conjugated to a GFP and the GPCR or GRK may be conjugated to an immunofluorescent molecule, and the conjugates may be detected by confocal microscopy.
  • arrestin may conjugated to a GFP and the carboxy-terminus of the GPCR may be conjugated to a luciferase and the conjugates may be detected by bioluminescence resonance emission technology.
  • arrestin may be conjugated to a luciferase and GPCR may be conjugated to a GFP, and the conjugates may be detected by bioluminescence resonance emission technology.
  • the methods of the present invention are directed to detecting GPCR activity. The methods of the present invention allow enhanced monitoring of the GPCR pathway in real time.
  • the localization pattern of the detectable molecule is determined.
  • alterations of the localization pattern of the detectable molecule may be determined.
  • the localization pattern may indicate cellular localization of the detectable molecule.
  • Molecules may also be detected by their interaction with another detectably labeled molecule, such as an antibody.
  • the cells used in the methods of assaying of the present invention may comprise a conjugate of a GRK protein and a detectable molecule, and the like.
  • the detectable molecule allows detection of molecules interacting with the detectable molecule, as well as the molecule itself.
  • GRKs naturally occurring and engineered variants, may be used in the present invention.
  • GRKs may interact to a detectable level with all forms of
  • Detectable molecules that may be used include, but are not limited to, molecules that are detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, radioactive, and optical means, including but not limited to bioluminescence, phosphorescence, and fluorescence. These detectable molecules should be a biologically compatible molecule and should not compromise the biological function of the molecule and must not compromise the ability of the detectable molecule to be detected. Preferred detectable molecules are optically detectable molecules, including optically detectable proteins, such that they may be excited chemically, mechanically, electrically, or radioactively to emit fluorescence, phosphorescence, or bioluminescence.
  • More preferred detectable molecules are inherently fluorescent molecules, such as fluorescent proteins, including, for example, Green Fluorescent Protein (GFP).
  • the detectable molecule may be conjugated to the GRK protein by methods as described in Barak et al. (U.S. Patent Nos. 5,891 ,646 and 6,110,693).
  • the detectable molecule may be conjugated at the front-end, at the back-end, or in the middle.
  • the GPCRs may also be conjugated with a detectable molecule.
  • the carboxyl-terminus of the GPCR is conjugated with a detectable molecule. If the GPCR is conjugated with a detectable molecule, proximity of the GPCR with the GRK may be readily detected. In addition, if the GPCR is conjugated with a detectable molecule, compartmentalization of the GPCR with the GRK may be readily confirmed.
  • the detectable molecule used to conjugate with the GPCRs may include those as described above, including, for example, optically detectable molecules, such that they may be excited chemically, mechanically, electrically, or radioactively to emit fluorescence, phosphorescence, or bioluminescence. Preferred optically detectable molecules may be detected by immunofluorescence, luminescence, fluorescence, and phosphorescence.
  • the GPCRs may be antibody labeled with an antibody conjugated to an immunofluorescence molecule or the GPCRs may be conjugated with a luminescent donor.
  • the GPCRs may be conjugated with, for example, luciferase, for example, Renilla luciferase, or a rhodamine-conjugated antibody, for example, rhodamine-conjugated anti-HA mouse monoclonal antibody.
  • the carboxyl-terminal tail of the GPCR may be conjugated with a luminescent donor, for example, luciferase.
  • the GPCR, preferably the carboxyl-terminal tail also may a be conjugated with GFP as described in L. S. Barak et al. Internal Trafficking and Surface Mobility of a Functionally Intact ⁇ 2-Adrenergic Receptor-Green Fluorescent Protein Conjugate, Mol. Pharm. (1997) 51 , 177 - 184.
  • the cells of the present invention may express at least one GRK, and GPCR, wherein at least one of the molecules is detectably labeled.
  • Cells useful in the present invention include eukaryotic and prokaryotic cells, including, but not limited to, bacterial cells, yeast cells, fungal cells, insect cells, nematode cells, plant cells, and animal cells. Suitable animal cells include, but are not limited to, HEK cells, HeLa cells, COS cells, and various primary mammalian cells.
  • An animal model expressing a conjugate of a GRK6 and a detectable molecule throughout its tissues or within a particular organ or tissue type, may also be used in the present invention.
  • a substrate may have deposited thereon a plurality of cells of the present invention.
  • the substrate may be any suitable biologically substrate, including but not limited to, glass, plastic, ceramic, semiconductor, silica, fiber optic, diamond, biocompatible monomer, or biocompatible polymer materials.
  • DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.
  • Such operative linking of a DNA sequence of this invention to an expression control sequence includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCR1 , pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage ⁇ , e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • phage DNAS e.g., the numerous derivatives of phage ⁇ , e.g.,
  • any of a wide variety of expression control sequences ⁇ sequences that control the expression of a DNA sequence operatively linked to it - may be used in these vectors to express the DNA sequences of this invention.
  • useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast -mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • a wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.
  • These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, plant cells, nematode cells, and animal cells, such as HEK-293, CHO, RU, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1 , COS 7, BSC1 , BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.
  • eukaryotic and prokaryotic hosts such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, plant cells, nematode cells, and animal cells, such as HEK-293, CHO, RU, B-W
  • Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.
  • modified GRK6 analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention.
  • Analogs, such as fragments may be produced, for example, by pepsin digestion of GRK6 material.
  • Other analogs, such as muteins can be produced by standard site-directed mutagenesis of GRK6 coding sequences.
  • Analogs exhibiting "GRK6 activity" such as small molecules, whether functioning as promoters or inhibitors, may be identified by known in vivo and/or in vitro assays.
  • a DNA sequence encoding a modified GRK6 can be prepared synthetically rather than cloned.
  • the DNA sequence can be designed with the appropriate codons for the GRK6 amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981 ); Nambair e. a/., Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).
  • Synthetic DNA sequences allow convenient construction of genes which will express GRK6 analogs or "muteins".
  • DNA encoding muteins can be made by site-directed mutagenesis of native or modified GRK6 genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.
  • a general method for site-specific incorporation of unnatural amino acids into proteins is described in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science, 244:182-188 (April 1989). This method may be used to create analogs with unnatural amino acids.
  • Method of evaluating treatments of GRK6-associated disease Provided in the present invention are methods of evaluating treatments of GRK6-associated disease.
  • the compound that modulates GRK6 will be administered to a wild-type non-human animal.
  • the locomotor responses of this animal will be compared to the locomotor responses of the transgenic animal with a functionally disrupted GRK6 gene. Means of examining the locomotor responses are described in the examples.
  • the present invention is related to methods of treating or diagnosing a disease.
  • the disease treatment may involve administering a compound that modulates GRK6.
  • the compound may directly or indirectly modulate GRK6.
  • the compound may be an antisense molecule or an immunoglobulin.
  • the disease may by Parkinson's disease, schizophrenia, depression, Tourette Syndrome, or drug-addiction.
  • the methods of disease diagnosis relate to the detection of the GRK6 protein, nucleic acid, or activity in a sample. Such methods include detection using immunoglobulins, nucleic acids, and antisense molecules.
  • the methods of disease treatment of the present invention include the concurrent administration of the compound that modulates GRK6 with an additional compound.
  • the additional compound may directly or indirectly affect dopamine levels.
  • Such compounds include L-DOPA, cocaine, and morphine.
  • the compound that modulates GRK6 may increase the effectiveness of the additional compound.
  • the concurrent administration of the compound that modulates GRK6 may decrease the amount of the additional compound required by the patient.
  • the present invention relates to methods of treating a human or non-human subject suffering from a GPCR-related disease, such as cardiovascular disease, heart failure, asthma, nephrogenic diabetes insipidus, hypertension, Parkinson's disease, schizophrenia, depression, Tourette Syndrome, or drug-addiction.
  • a GPCR-related disease such as cardiovascular disease, heart failure, asthma, nephrogenic diabetes insipidus, hypertension, Parkinson's disease, schizophrenia, depression, Tourette Syndrome, or drug-addiction.
  • Such treatment can be performed either by administering to a subject in need of such treatment, an amount of the compound identified by the present method sufficient to treat the GPCR-related disease, or at least to lessen the symptoms thereof.
  • Treatment may also be effected by administering to the subject the naked modified nucleic acid sequences of the invention, such as by direct injection, microprojectile bombardment, delivery via liposomes or other vesicles, or by means of a vector which can be administered by one of the foregoing methods. Gene delivery in this manner may be considered gene therapy.
  • the naked modified nucleic acid sequences comprise modified GRK6 proteins of the present invention.
  • an appropriate inhibitor of the GRK could be introduced to block the phosphorylation of the GPCR by the GRK.
  • instances in which insufficient activation of a G protein or second messenger is taking place could be remedied by introduction of additional quantities of the GRK or its chemical or pharmaceutical cognates, analogs, fragments and the like.
  • instances in which excess activation of a G protein or second messenger is taking place could be remedied by introduction of decreased quantities of the GRK or its chemical or pharmaceutical cognates, analogs, fragments and the like.
  • a subject therapeutic composition includes, in a mixture, a pharmaceutically acceptable excipient (carrier) and a compound that modulates a GRK, as described herein as an active ingredient.
  • the composition comprises a drug capable of modulating the phosphorylation of the GPCR by a GRK.
  • compositions which contain polypeptides, analogs or active fragments as active ingredients are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents that enhance the effectiveness of the active ingredient.
  • a GRK6 modulating compound obtained by the methods disclosed herein can be formulated into the therapeutic composition as neutralized pharmaceutically ' acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the therapeutic compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent (i.e., carrier, or vehicle).
  • diluent i.e., carrier, or vehicle.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compositions lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range which includes the IC50 (i.e., the concentration of the test composition which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test composition which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of modulation of GPCR activity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.001 to 30, preferably about 0.01 to about 25, and more preferably about 0.1 to 20 milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.
  • treatment of a subject with a therapeutically effective amount of the composition(s) can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with the composition in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of the composition used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the therapeutic compositions may further include an effective amount of the GRK6 modulating compound and one or more of the following active ingredients: an antibiotic, a steroid, and the like.
  • prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
  • active form i.e., drug
  • prodrug versions of the oligonucleotides of the invention can be prepared as SATE ((S-acetyl-2-thioethyl) phosphate) derivatives according to the methods disclosed for example in WO 93/24510 and in WO 94/26764.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • the compounds for modulating any of the disclosed genes, gene transcripts or proteins encoded thereby include antisense compounds as well as other modulatory compounds.
  • Pharmaceutically acceptable base addition salts for use with antisense as well as other modulatory compounds are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.
  • metals used as cations are sodium, potassium, magnesium, calcium, and the like.
  • suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, e.g., Berge et al., "Pharmaceutical Salts," J. Pharma. Sci., 1977, 66: 1-19).
  • the base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
  • the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.
  • the free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.
  • a "pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines.
  • Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates.
  • Other suitable pharmaceutically acceptable salts are known in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid); with organic carboxylic, sulfonic, sulfo- or phospho- acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid
  • Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation.
  • Suitable pharmaceutically acceptable cations are well known in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.
  • salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.
  • acid addition salts formed with inorganic acids for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like
  • salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygal
  • antisense compounds and other modulatory compounds described herein can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound or other modulatory compound to a suitable pharmaceutically acceptable diluent or carrier.
  • Use of the compounds and methods of the invention may also be useful prophylactically.
  • the antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding a gene identified using the systematic discovery technique or a mRNA transcript thereof. Such hybridization allows the use of sandwich and other assays to easily be constructed to exploit this fact.
  • Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding a gene or gene transcript identified by a systematic discovery method can be detected by means known in the art. Such means may include, for example, conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means.
  • Kits using such detection means for detecting the level of a transcript of a gene in a sample may also be prepared.
  • the present invention also includes pharmaceutical antisense compositions and formulations which include the antisense compounds and other modulatory compounds and compositions of the invention.
  • the pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. [00156] In certain embodiments, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment.
  • compositions may be combined with a carrier so that an effective dosage is delivered, based on the desired activity.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer, salts, flavoring, coloring and sweetening agents as appropriate.
  • compositions for oral administration may be suitably formulated to give controlled release of the active composition.
  • the compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray, presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the composition and a suitable powder base such as lactose or starch.
  • compositions may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compositions may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials for example as an emulsion in an acceptable oil
  • ion exchange resins for example as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • compositions e.g., gene, gene transcript or protein product modulatory agents as described herein
  • Pharmaceutical compositions include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • the pharmaceutical formulations of the present invention which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature, these formulations vary in the components and the consistency of the final product.
  • the preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.
  • the compositions of the present invention may be prepared and formulated as emulsions. See, e.g., Idson, in Pharmaceutical Dosage Forms v. 1 , p.
  • Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other.
  • emulsions may be either water-in-oil (w/o) or of the oil-in-water (o/w) variety.
  • Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed.
  • compositions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.
  • Such complex formulations often provide certain advantages that simple binary emulsions do not.
  • Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion.
  • a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion.
  • Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms v. 1 , p. 199 (Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York).
  • Synthetic surfactants also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage FormsN. 1 , p. 285; Idson, in Pharmaceutical Dosage Forms, v. 1 , p. 199).
  • Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion.
  • the ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations.
  • HLB hydrophile/lipophile balance
  • Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms).
  • Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia.
  • Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers, especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, non-swelling clays (e.g., bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate), pigments and nonpolar solids (e.g., carbon or glyceryl tristearate).
  • polar inorganic solids such as heavy metal hydroxides, non-swelling clays (e.g., bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate), pigments and nonpolar solid
  • non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, v.1 p.385 (Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York)).
  • Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers, such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
  • polysaccharides e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth
  • cellulose derivatives e.g., carboxymethylcellulose and carboxypropylcellulose
  • synthetic polymers
  • emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives.
  • preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.
  • Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.
  • Antioxidants used may be free radical scavengers (e.g., tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene) or reducing agents (e.g., ascorbic acid and sodium metabisulfite), and antioxidant synergists (e.g., citric acid, tartaric acid, and lecithin).
  • free radical scavengers e.g., tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene
  • reducing agents e.g., ascorbic acid and sodium metabisulfite
  • antioxidant synergists e.g., citric acid, tartaric acid, and lecithin
  • Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint.
  • Rosoff in Pharmaceutical Dosage Forms, v. 1 , p. 245 (Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York); Idson, in Pharmaceutical Dosage Forms).
  • Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
  • the compositions of oligonucleotides and nucleic acids are formulated as microemulsions.
  • a microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamicaliy stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, v. 1 , p. 245).
  • microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system.
  • microemulsions have also been described as thermodynamicaliy stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in Controlled Release of Drugs: Polymers and Aggregate Systems, 185-215 (Rosoff, M., Ed., 1989, VCH Publishers, New York).
  • Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte.
  • microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, 271 (Mack Publishing Co., Easton, Pa., 1985).
  • Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with co-surfactants.
  • ionic surfactants etraglycerol monolaurate
  • MO310 tetraglycerol monooleate
  • PO310 hexaglycerol monooleate
  • PO500 hexa
  • the co-surfactant usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
  • aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol.
  • the oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs.
  • Lipid based microemulsions both o/w and w/o have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides er a/., Pharm. Res., 1994, 11 :1385-90; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13: 205).
  • Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., 1994; Ho et al., J. Pharm. Sci., 1996, 85: 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications.
  • microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids and other active agents from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids and other active agents within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.
  • Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention.
  • Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories — surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Crit. Rev. Therap. Drug Carrier Systems, 1991 , p. 92). Each of these classes has been discussed above. [00181] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, are useful because of their specificity and the duration of action. As used in the present invention, the term "liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
  • Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo. Selection of the appropriate liposome depending on the agent to be encapsulated would be evident given what is known in the art.
  • liposomes obtained from natural phospholipids are biocompatible and biodegradable; (b) liposomes can incorporate a wide range of water and lipid soluble drugs; (c) liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
  • Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
  • Another embodiment also contemplates the use of liposomes for topical administration.
  • advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
  • Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin.
  • Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.
  • Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes that interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Comm., 1987, 147:980-985). [00188] Liposomes that are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs.
  • Another contemplated liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine.
  • Neutral liposome compositions for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).
  • Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.
  • PC phosphatidylcholine
  • Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • sterically stabilized liposomes which refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids are also contemplated.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1 , or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. See, e.g., Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53: 2778) described liposomes comprising a nonionic detergent, 2C12 15G, that contains a PEG moiety. Ilium et al. (FEBS Lett, 1984, 167: 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives.
  • Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.).
  • Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).
  • U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
  • Methods of encapsulating nucleic acids in liposomes is also known in the art. See, WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes.
  • HLB hydrophile/lipophile balance
  • Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure.
  • Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters.
  • Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.
  • the polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
  • Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.
  • the most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
  • the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic.
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
  • Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids and other agents, particularly oligonucleotides, to the skin of animals.
  • Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991 , p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
  • Another embodiment of the invention contemplates pharmaceutical compositions comprising surfactants.
  • Surfactants are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced.
  • these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Crit. Rev. Therap. Drug Carrier Systems, 1991 , 92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al., J. Pharm.
  • Another embodiment contemplates the use of various fatty acids and their derivatives to act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcamitines, acylcholines, C1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate
  • compositions comprising the active agents of the invention may further comprise bile salts.
  • the physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, N.Y., 1996, pp. 934-935).
  • bile salts includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.
  • the bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al.,
  • compositions comprising chelating agents.
  • Chelating agents can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced.
  • chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618: 315-39).
  • Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Lee et al., 1991 ; Muranishi, 1990; Buur et al., J. Control Rel., 1990, 14: 43-51).
  • EDTA disodium ethylenediaminetetraacetate
  • citric acid e.g., sodium salicylate, 5-methoxysalicylate and homovanilate
  • N-acyl derivatives of collagen laureth-9
  • N-amino acyl derivatives of beta-diketones enamines
  • Non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants, but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, 1990).
  • This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1 -alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., 1991 ); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39: 621-6).
  • agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention.
  • cationic lipids such as lipofectin (Junichi et al., U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731 ), are also known to enhance the cellular uptake of oligonucleotides.
  • compositions of the present invention also incorporate carrier compounds in the formulation.
  • carrier compound or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.
  • carrier compound typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.
  • the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5: 115-121 ; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6: 177-183).
  • the pharmaceutical compositions disclosed herein may also comprise a excipients.
  • these excipients include a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids or other active agents to an animal.
  • the excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid or other active agent and the other components of a given pharmaceutical composition.
  • Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxyprop
  • compositions of the present invention can also be used to formulate the compositions of the present invention.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Formulations for topical administration of nucleic acids and other contemplated active agents may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.
  • the solutions may also contain buffers, diluents and other suitable additives.
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids or other contemplated active agents can be used.
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • compositions of the invention may contain one or more antisense compound or other active agents. Two or more combined compounds may be used together or sequentially.
  • the present invention further includes the preparation of antisense oligonucleotides and ribozymes that may be used to interfere with the expression of GRK6, and the like at the translational level.
  • the antisense and ribozymes may be used to interfere with the expression of a GRK6, and the like having discrete point mutations that increases its affinity for arrestin in suspect target cells.
  • This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule. (See Weintraub, Sci Am. 1990 Jan;262(1 ):40-6; Marcus-Sekura, Anal Biochem. 1988 Aug 1 ;172(2):289-95). In the cell, they hybridize to that mRNA, forming a double stranded molecule. The ceil does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein.
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns.
  • RNA molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, Gene. 1988 Dec 20;73(2):259-71). Because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • the DNA sequences described herein may thus be used to prepare antisense molecules against, and ribozymes that cleave mRNAs for GRK6s and their ligands.
  • the antisense molecules and ribozymes may be particularly useful for GRK ⁇ s having mutations that alter their affinity for GPCRs.
  • the invention further provides antibodies, preferably monoclonal antibodies, which specifically bind to the polypeptides of the invention.
  • Methods are also provided for producing antibodies in a host animal.
  • the methods of the invention comprise immunizing an animal with at least one GRK6 -derived immunogenic component, wherein the immunogenic component comprises one or more of the polypeptides encoded by any one of SEQ ID NO: 1 - SEQ ID NO: 3 or sequence-conservative or function-conservative variants thereof; or polypeptides that are contained within any ORFs, including complete protein-coding sequences, of which any of SEQ ID NO: 1 - SEQ ID NO: 3 forms a part; or polypeptide sequences contained within any of SEQ ID NO: 1 - SEQ ID NO: 3; or polypeptides of which any of SEQ ID NO: 1 - SEQ ID NO: 3 forms a part.
  • Host animals include any warm blooded animal, including without limitation mammals and birds. Such antibodies have utility as reagents for immunoassays to evaluate the abundance and distribution of GRK6 antigens.
  • Antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of GRK6 and/or their biologically active fragments or subunits may possess certain diagnostic or therapeutic applications.
  • the GRK6a, GRK6b, GRK6c, GRK6d or fragments or subunits thereof may be used to produce both polyclonal and monoclonal antibodies, to GRK6 or subunits thereof, in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.
  • small molecules that mimic or antagonize the activity(ies) of the GRK6 of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.
  • Fragments of the GRK6 sequence may be prepared synthetically as peptides and covalently conjugated to a carrier protein (such as Keyhole limpet hemocyanin) or fused directly to the coding region of a carrier protein (such as glutathione S-transferase) and expressed as a unit.
  • a carrier protein such as Keyhole limpet hemocyanin
  • a carrier protein such as glutathione S-transferase
  • Such carrier conjugated are injected into the host species to allow antibodies to be produced. This method was employed to make an antibody against the last 30 unique residues of the GRK6B splice variant.
  • the present invention likewise extends to the development of antibodies against GRK6, including naturally raised and recombinantly prepared antibodies.
  • the antibodies could be used to screen expression libraries to obtain the gene or genes that encode subunits of the GRK6.
  • Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for additional diagnostic use conjunctive with their capability of modulating GRK6 activity.
  • the anti-GRK6 antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb).
  • the anti-modified-GPCR antibody fragments used herein be in the form of Fab, Fab', F(ab') 2 , F(v), or scFv.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Methods for producing monoclonal anti-GRK6 antibodies are also well- known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA, 80:4949-4953 (1983).
  • the GRK6 or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen in the before described procedure for producing anti-GRK6 monoclonal antibodies.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibodies into the medium.
  • the hybridomas are screened for the ability to produce an antibody that immunoreacts with the GRK6 or peptide analog.
  • the antibody-containing medium is then collected.
  • the antibody can then be further isolated by well-known techniques.
  • Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., Using Antibodies: A Laboratory Manual, Harlow, Ed and Lane, David (Cold Spring Harbor Press, 1999).
  • Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media, inbred mice and the like.
  • An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/l glucose, 20 mM glutamine, and 20% fetal calf serum.
  • DMEM Dulbecco's minimal essential medium
  • a preferred inbred mouse strain is the Balb/c.
  • Methods for producing polyclonal anti-polypeptide antibodies are well-known in the art. See U.S. Patent No. 4,493,795 to Nestor et al.
  • a monoclonal antibody, and immunologically active fragments thereof can be prepared using the hybridoma technology described in Using Antibodies: A Laboratory Manual, Harlow, Ed and Lane, David (Cold Spring Harbor Press, 1999), which is incorporated herein by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with a GRK6. Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000 MW.
  • PEG polyethylene glycol
  • Fused hybrids are selected by their sensitivity to HAT (hypoxanthine, aminopterin, thymidine) supplemented media.
  • Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present GRK6 and their ability to inhibit specified GRK6 activity in target cells.
  • the three Triple-Lox vectors described for the GRK5 knockout were modified to have new multiple cloning sites, and phage e carrying fragments of the mouse GRK6 gene from the 129/SvJ strain were obtained and sequenced as described.
  • the targeting vector ( Figure 2A) contained the 7-kb Nhe ⁇ -Not ⁇ fragment (exons 10-15), loxP site, 2.75-kb Xba ⁇ -Nhe ⁇ fragment (exons 3 through 9), loxP site, TK-NEO marker gene cassette, loxP site, and 1.3-kb Xba ⁇ gene fragment (exon 2).
  • Growth and selection of targeted ES cells and creation of chimeric mice was performed as described in B.
  • F1 heterozygote animals were bred with transgenic mice bearing CMV-Cre (backcrossed to a C57BI/6J genetic background to induce deletion of the floxed cassettes. From offspring of these crosses, GRK6 knockout (GRK6-KO) animals were obtained, in which both the exon 3-9 cassette and the TK-NEO marker gene cassettes were deleted. Deletion of exons 3 through 9 leads to a GRK6 that lacks most of the amino terminal RGS-like domain as well as half of the conserved catalytic domain elements (i.e., the gene is inactive) (Fig 2A). Genotyping was routinely performed on tail tip DNA using a PCR method utilizing three primers to simultaneously detect the wild type and mutant loci ( Figure 2B).
  • HEK293 cells ATCC, Rockville, MD
  • MEM fetal calf serum
  • penicillin/streptomycin at 37 °C under 5% C0 2 /95% air.
  • Cells are transfected with appropriate expression plasmid DNAs using calcium phosphate co-precipitation.
  • Free floating coronal or saggital sections of 25 ⁇ m were incubated 32 h at 4 °C with a mixture of antibodies against DARPP-32 (mouse, 1 :5000, D97520, BD Transduction laboratories, K) and GRK6 (rabbit, 1:50, sc-566, Santa Cruz Biotechnologies, Inc.).
  • the second immunoreaction step was performed by incubation (1 h at room temperature) each of the following antibodies: fluorescein-isothiocyanate-labelled goat antirabbit IgG and Texas-Red-labelled goat anti-mouse IgG (Vector laboratories).
  • mice were placed in activity monitor, 30-60 min later they were injected with drugs or vehicle i.p., and locomotor activity was monitored for the following 90 min.
  • cocaine sensitization experiments mice were treated chronically with cocaine (20 mg/kg, i.p.) for 5 days and their responses to challenging dose of cocaine were analyzed at day 7.
  • mice were pre-treated with a combination of reserpine (5 mg/kg, i.p. 20 h before the experiment) and ⁇ -methyl-p-tyrosine (250 mg/kg, i.p., 1h before the experiment). This treatment resulted in depletion of striatal dopamine to less than 0.75% in both wild type and GRK6-KO mice. Mice were completely immobilized by this treatment. Dopamine-depleted mice were treated with vehicle or D1/D2 dopamine receptor agonist apomorphine (0.2-1 mg/kg, s.c.) and locomotor activity was immediately analyzed as described above.
  • mice were anesthetized and dialysis probes were implanted into the right striatum.
  • the dialysis probe was connected to a syringe pump and perfused with artificial CSF.
  • Quantitative "low perfusion" rate (70 nl/min) microdialysis experiments were conducted in freely moving mice for determination of basal extracellular dopamine levels in striatum.
  • "conventional" microdialysis method perfusion flow rate 1 ⁇ l/min in freely moving animals was employed.
  • striatal tissue from wild type and GRK6-KO mice were homogenized in a sucrose buffer (0.32M sucrose, 4.2mM Hepes, pH 7.4). Homogenates were centrifuged at 1 ,500 x g for 15 min and supematants were collected and re-centrifuged at 10,000 x g for 15 min.
  • the resulting pellets were washed and resuspended in buffer (0.02 % ascorbic acid, 50 ⁇ M pargyline, 50 mM Tris-HCl, 125 mM NaCl, 5mM KCI, 1 mM CaCI 2 , 1 mM MgCI 2 , 10 mM glucose; pH 7.4).
  • buffer 0.2 % ascorbic acid, 50 ⁇ M pargyline, 50 mM Tris-HCl, 125 mM NaCl, 5mM KCI, 1 mM CaCI 2 , 1 mM MgCI 2 , 10 mM glucose; pH 7.4
  • Synaptosomal samples were incubated at 37 °C for 2 min with 20 nM of [ 3 H]-dopamine (31.6 Ci/mmol). Non-specific uptake was carried out in the presence of 5 ⁇ M mazindol.
  • Incubations were then stopped by adding 3 ml cold wash buffer (50 mM Tris-HCl, 5 mM KCI, 1 mM CaCI 2 , 1 mM MgCI 2 , 10 mM glucose, pH 7.4) and vacuum-filtered through glass microfiber filters. The filters were then washed 2 times with cold wash buffer and placed in vials containing scintillation cocktail.
  • cold wash buffer 50 mM Tris-HCl, 5 mM KCI, 1 mM CaCI 2 , 1 mM MgCI 2 , 10 mM glucose, pH 7.4
  • D2R 20 ⁇ g of cell membrane proteins were incubated in a buffer containing 20 mM HEPES, pH 7.4, 10 mM MgCI 2 , 150 mM NaCl, 3 ⁇ M GDP, and 0.1 nM [ 35 S]GTP ⁇ S for 1 hr at room temperature.
  • D3R 20 ⁇ g of cell membrane proteins were incubated in a buffer containing 25 mM HEPES, pH 7.4, 120 mM NaCl, 1.8 mM KCI, 20 mM MgCI 2 , 20 ⁇ M GDP, 0.2 nM [ 35 S]GTP ⁇ S, and 1 mM sodium deoxycholate for 2 hr at 30 °C. Incubation mixtures were filtered with GF/B filter and washed with 10 mM sodium phosphate buffer.
  • GRK6 expressed in neuronal populations containing a key dopaminergic signaling molecule
  • GRK6 GRK6 mRNA in the striatum was found to be higher than that of other GRKs (GRK2, GRK3 and GRK5), suggesting that GRK6 might be a predominant receptor kinase in this brain area.
  • GRK6 protein was found in the same neuronal population that expresses DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, apparent molecular weight of 32,000 Da), a key molecule involved in dopaminergic signaling mediated both by D1-like and by D2-like dopamine receptors, and a phenotypic marker of the medium-size spiny GABA neurons of the mammalian striatum (Fig. 1 ). These neurons represent a major striatal cell group receiving dopaminergic input and are believed to be critically involved in cellular mechanisms of addiction.
  • FIG. 1 illustrates that GRK6 is present in striatal neurons expressing DARPP-32.
  • Upper-left Immunofluorescence analysis reveals GRK6 immunoreactivity in the striatal neurons of WT mouse (+0.74 from bregma).
  • Upper-right Lack of GRK6 immunoreactivity in the striatal neurons of GRK6-KO mouse.
  • Lower-left DARPP-32 immunoreactivity in the striatal neurons of WT mouse.
  • GRK6 and DARPP-32 are co-localized in the same neuronal population in the striatum of WT mouse.
  • GRK6 immunoreactivity was detected using a commercially available anti-GRK6 antibody (rabbit, 1 :50, sc-566, Santa Cruz Biotechnologies, Inc.). Similar observations were made using another anti-GRK6 antiserum.
  • GRK6 is also expressed in large cholinergic striatal cells, which do not express DARPP-32, but can be labeled with anti - choline acetyltransferase antibody. Scale bar is equal 50 ⁇ m.
  • GRK6 transgenic mouse was targeted by homologous recombination in embryonic stem cells (Fig. 2A-C).
  • the heterozygote and homozygote GRK6-KO mice are viable and present no gross anatomical or behavioral abnormalities, although GRK6-KO mice demonstrate reduced lymphocyte chemotaxis.
  • locomotor activity tests unchallenged knockout mice were not different from wild type littermates either in horizontal (Fig.3A,B) or vertical activities.
  • acute cocaine (20 mg/kg, i.p.) administration resulted in a markedly enhanced locomotor response in GRK6 mutant mice (Fig.
  • mice In this paradigm, the GRK6-KO mice exhibited a more pronounced and longer lasting locomotor activation, as measured by horizontal (Fig. 3A,B) and vertical activities in response to cocaine (10-30 mg/kg, i.p.) than did wild type littermate mice. Interestingly, the mice heterozygous for GRK6 deletion were as responsive to cocaine as GRK6 "null" mice (Fig. 3A,B), suggesting that even minor changes in GRK6 levels or activity may result in significant behavioral alterations.
  • FIG. 1 A. Schematic diagram of the wild type GRK6 locus, the GRK6/lox targeting vector, the integrated targeting construct, and the Cre recombinase-deleted GRK6 locus (GRK6-KO).
  • GRK6 exons are shown as open boxes, and numbered from the first coding exon. LoxP sites are shown as filled triangles, and the location of the Southern blot probe as a hatched box. Relevant Nhel restriction sites are indicated.
  • C GRK6 protein expression by Western blotting.
  • Membrane proteins from brainstem and striatum of wild type and GRK6-KO animals were subjected to immunoblotting using an anti-GRK6 antiserum.
  • GRK6-KO homozygote animals exhibit a loss of the 68-kDa immunoreactive band compared to wild type animals (Arrow).
  • the 69-kDa band is a non-specific interaction, since it is present in GRK5 and GRK4 homozygote animals and is not recognized by other GRK6 antiserum.
  • FIG. 3 Cocaine supersensitivity in GRK6 mutant mice.
  • GRK6 heterozygous and GRK6-KO mice are significantly different from WT controls in responses to cocaine (p ⁇ 0.001 , two-way analysis of variance (ANOVA).
  • GRK6 heterozygous and GRK6-KO mice are significantly different from WT controls in responses to cocaine (p ⁇ 0.001 , two-way ANOVA).
  • GRK6-KO animals were as responsive to cocaine on the 1st day as were wild type mice following the sensitization protocol (Fig. 3C). As might be expected, GRK6-KO mice were substantially less affected by this sensitization regimen. In fact, analysis of total distance traveled for 90 min did not reveal significant differences between day 1 and day 7 (Fig. 3C). Nonetheless, in the first 15 min after cocaine administration, sensitized GRK6-KO mice did exhibit a slightly enhanced response (Fig. 3C, legend). Taken together, these data imply that in the absence of pharmacological treatment, the GRK6-KO mice may already be essentially "pre-sensitized" to cocaine.
  • the locomotor responses of GRK6 transgenic mice were enhanced in the presence of increased dopamine, induced by amphetamine and ⁇ -phenylethylamine. It is well established that the locomotor stimulating action of cocaine is mediated by the blockade of the dopamine transporter (DAT) and the resultant elevation of extracellular dopamine in the striatum and related brain areas. Another psychostimulant known to markedly enhance central dopaminergic transmission via complex interaction with the DAT is amphetamine. Similarly to cocaine, amphetamine-induced locomotor activation was significantly enhanced in both GRK6 heterozygous and "null" mice, (Fig. 4A).
  • DAT dopamine transporter
  • GRK6 mutant mice Enhanced locomotor effects of c/-amphetamine and ⁇ - phenylethylamine in GRK6 mutant mice.
  • GRK6 heterozygous and GRK6-KO mice are significantly different from WT controls in responses to d-amphetamine. p ⁇ 0.001 , two-way ANOVA.
  • B Two-way ANOVA.
  • Example 6 GRK6-transgenic mice did not differ from wild-type mice in measured neurochemical parameters [00243] Behavioral supersensitivity to psychostimulants could be explained either by alterations in presynaptic dopaminergic function in these mice leading to augmented extracellular dopamine levels or by altered postsynaptic receptor responsiveness. To test the status of striatal presynaptic dopaminergic transmission in mutant mice, a set of neurochemical approaches was used (Fig. 5). GRK6-KO mice were not different from wild type controls in any of the neurochemical parameters examined.
  • Example 7 GRK6 modulation results in dopamine receptors that are more efficiently coupled to their G proteins [00245]
  • GRK6 was co-expressed with either D2 or D3 dopamine receptors (D2R or D3R) in HEK293 cells and analysis of dopamine-stimulated [ 35 S]GTP ⁇ S binding was performed.
  • D2R or D3R dopamine receptors
  • substantially impaired G protein coupling was observed when these receptors were co-expressed with GRK6 (Fig. 6B,C).
  • co-expression of GRK6 enhanced the basal (unstimulated) translocation of ⁇ -arrestin2 to D2R or D3R.
  • GRK6 appears to induce a basal level of desensitization of D2/D3 dopamine receptors that is associated with increased ⁇ -arrestin2 binding. This is in agreement with many previous studies that have shown in other receptor systems that membrane-associated GRK6 induces basal (activation-independent) receptor phosphorylation, and suggests that this basal receptor phosphorylation tone is physiologically important, at least for the dopamine receptors.
  • G-proteins A. [ 35 S]GTP ⁇ S binding to striatal membranes from mutant and wild type mice. Total [ 35 S]GTP ⁇ S binding is portrayed after subtracting unstimulated [ 35 S]GTP ⁇ S binding from each point. [ 35 S]GTP ⁇ S binding to striatal membranes was determined after stimulation with quinpirole. Percent stimulated [ 35 S]GTP ⁇ S binding was calculated by dividing unstimulated [ 35 S]GTP ⁇ S binding into each agonist-stimulated point. Nonlinear regressions were used to calculate the EC 50 parameters (WT: 2.0 ⁇ 0.5 ⁇ M; GRK6-KO: 1.9 ⁇ 0.6 ⁇ M).
  • Wild type and GRK6-KO mice were treated with reserpine (5 mg/kg, i.p.) to deplete intraneuronal storage of monoamines including dopamine, and with ⁇ -methyl-p-tyrosine (250 mg/kg, i.p.) to inhibit dopamine synthesis (20h and 1 h before the experiment, respectively). Both mutant and control mice were completely immobilized by this treatment. Locomotion in dopamine-depleted wild type and mutant mice was restored by administration of mixed D1/D2 dopamine receptor agonist apomorphine (0.2-1 mg/kg, s.c.) (Fig.7A-C).
  • GRK6-KO mice showed a markedly enhanced locomotor response to apomorphine in comparison to wild type litteramates, directly demonstrating that postsynaptic dopamine receptor responsiveness is enhanced in GRK6 mutant mice. Furthermore, these data suggest that a decrease in GRK6 levels or activity could enhance the behavioral effects of dopamine agonists in this animal model. [00249] Figure 7. Dopamine agonist effect is enhanced in dopamine-depleted GRK6-KO mice. To deplete brain dopamine, animals were treated with a combination of reserpine (5 mg/kg, i.p.) and ⁇ -methyl-p-tyrosine (250 mg/kg, i.p.) as described in Materials and Methods. A.
  • Example 9 GRK6 modulation increased effectiveness of compounds to treat Parkinson's disease
  • Chem. 274, 32248-32257 Zhang, J. , Barak, L. S. , Anborgh, P. H. , Laporte, S. A. , Caron, M. G. & Ferguson, S.

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Abstract

La présente invention concerne des procédés permettant de traiter une maladie en modifiant la protéine kinase GR 6 (GRK6). On a la possibilité, pour cela, de modifier l'expression ou l'activité de la protéine, par exemple. La présente invention peut être utilisée pour diagnostiquer une maladie, en détectant l'expression ou l'actvité de la GRK6. L'invention concerne également une souris déficiente en GRK6, des variantes de l'épissure de la GRK6, et des procédés d'utilisation. L'invention concerne aussi des procédés d'identification de composés modifiant l'activité de la GRK6. L'invention concerne enfin le traitement de maladies par modification de l'expression ou de l'actvité de la GRK6.
EP03763111A 2002-07-03 2003-07-03 Procedes de recherche systematique de composes destines a l'activite de modulation des grk6 Withdrawn EP1540332A1 (fr)

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WO2005113788A2 (fr) * 2004-05-21 2005-12-01 Bayer Healthcare Ag Diagnostics et therapeutiques pour des maladies associees a la kinase 6 associee au recepteur couple a une proteine g (grk6)
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US9784726B2 (en) 2013-01-08 2017-10-10 Atrogi Ab Screening method, a kit, a method of treatment and a compound for use in a method of treatment
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