WO2002031125A2 - Regulation de l'enzyme semblable a phospholipase a2 humaine - Google Patents
Regulation de l'enzyme semblable a phospholipase a2 humaine Download PDFInfo
- Publication number
- WO2002031125A2 WO2002031125A2 PCT/EP2001/011703 EP0111703W WO0231125A2 WO 2002031125 A2 WO2002031125 A2 WO 2002031125A2 EP 0111703 W EP0111703 W EP 0111703W WO 0231125 A2 WO0231125 A2 WO 0231125A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- phospholipase
- enzyme
- polypeptide
- seq
- polynucleotide
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the invention relates to the area of enzyme regulation. More particularly, the invention relates to human phospholipase A2-like enzyme and its regulation.
- the secretory phospholipase A2 (PLA2) superfamily comprises a number of heterogeneous enzymes whose common feature is to hydrolyze the Serial No. -2 fatty acid acyl ester bond of phosphoglycerides.
- PLA2 activity generates precursors for the biosynthesis of biologically active lipids, hydroxy fatty acids, and platelet-activating factor. Kramer et al., FEBS Lett. 1997 Jun 23;410(l):49-53, 1997.
- PLA2 hydrolysis of the sn-2 ester bond in phospliolipids generates free fatty acids, such as arachidonic acid and lysophospholipids.
- Arachidonic acid is processed into bioactive lipid mediators of inflammation, such as lyso-platelet-activating factor and eicosanoids.
- the synthesis of arachidonic acid from membrane phospholipids is the rate-limiting step in the biosynthesis of the four major classes of eicosanoids (prostaglandins, prostacyclins, thromboxanes and leukotrienes) involved in pain, fever, and inflammation.
- leukotriene-B4 is known to function in a feedback loop which further increases PLA2 activity.
- PLA2s first described as components of snake venoms and pancreatic juice and later defined in mammals, have traditionally been classified into several major groups and subgroups based on their amino acid sequence, disulfide bond location, and divalent cation requirements.
- the PLA2s of groups I, II, and III consist of low molecular weight, secreted, Ca 2+ -dependent proteins, Group IV PLA2s are primarily 85-kDa,
- Ca 2+ -dependent cytosolic phospholipases and an additional group comprises the Ca 2+ -independent cytosolic phospholipases.
- the first PLA2s to be extensively characterized were the Group I, II, and III PLA2s found in snake and bee venoms. These venom PLA2s share many features with mammalian PLA2s, including a common catalytic mechanism, the same Ca+ requirement, and conserved primary and tertiary structures. In addition to their role in the digestion of prey, the venom PLA2s display neurotoxic, myotoxic, anti- coagulant, and proinflammatory effects in mammalian tissues. This diversity of pathophysiological effects is due to the presence of specific, high affinity receptors for these enzymes on various cells and tissues. Lambeau et al., J. Biol. Chem. 270, 5534-40, 1995.
- At least four different groups of PLA2s have been characterized in mammalian cells, including Group I (pancreatic), Groups IIA, and IIC (inflammatory), and Group V (expressed in the heart).
- the pancreatic PLA2s function in the digestion of dietary lipids and have been proposed to play a role in cell proliferation, smooth muscle contraction, and acute lung injury.
- the inflammatory PLA2s are potent mediators of inflammatory processes and are highly expressed in serum and synovial fluids of patients with inflammatory disorders.
- These Group II PLA2s are found in most human cell types assayed and are expressed in diverse pathological processes such as septic shock, intestinal cancers, rheumatoid arthritis, and epidermal hyperplasia.
- a Group V PLA2 has been cloned from brain tissue and is strongly expressed in heart tissue. Other PLA2s have been cloned from various human tissues and cell lines, suggesting a large diversity of PLA2s.
- a human PLA2 was recently cloned from fetal lung, and based on its structural properties, appears to be the first member of a new group of mammalian PL As, referred to as Group X. Chen et ah, J. Biol. Chem. 269, 2365-68, 1994; Kennedy et al, J. Biol. Chem. 270, 22378-85, 1995; Komada et ah, Biochem. Biophys. Res. Commun. 168, 1059-65, 1990; and Cupillard et ah, J. Biol. Chem. 272, 15745-52, 1997).
- One embodiment of the invention is a phospholipase A2-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:
- amino acid sequences which are at least about 41% identical to the amino acid sequence shown in SEQ ID NO: 2;
- Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
- a test compound is contacted with a phospholipase A2-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 41% identical to the amino acid sequence shown in SEQ ID NO: 2; and
- Binding between the test compound and the phospholipase A2-like enzyme polypeptide is detected.
- a test compound which binds to the phospholipase A2-like enzyme polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation.
- the agent can work by decreasing the activity of the phospholipase A2-like enzyme.
- Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
- a test compound is contacted with a polynucleotide encoding a phospholipase A2-like enzyme polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:
- nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;
- a test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation.
- the agent can work by decreasing the amount of the phospholipase A2-like enzyme through interacting with the phospholipase A2-like enzyme mRNA.
- Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation.
- a test compound is contacted with a phospholipase A2-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:
- amino acid sequences which are at least about 41% identical to the amino acid sequence shown in SEQ ID NO: 2;
- a phospholipase A2-like enzyme activity of the polypeptide is detected.
- a test compound which increases phospholipase A2-like enzyme activity of the polypeptide relative to phospholipase A2-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation.
- a test compound which decreases phospholipase A2-like enzyme activity of the polypeptide relative to phospholipase A2-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.
- Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
- a test compound is contacted with a phospholipase A2-like enzyme product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of:
- nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;
- Binding of the test compound to the phospholipase A2-like enzyme product is detected.
- a test compound which binds to the phospholipase A2-like enzyme product is thereby identified as a potential agent for decreasing extracellular matrix degradation.
- Still another embodiment of the invention is a method of reducing extracellular matrix degradation.
- a cell is contacted with a reagent which specifically binds to a polynucleotide encoding a phospholipase A2-like enzyme polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:
- nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1 ;
- Phospholipase A2-like enzyme activity in the cell is thereby decreased.
- the invention thus provides a human phospholipase A2-like enzyme which can be used to identify test compounds which may act, for example, as activators or inhibitors at the enzyme's active site.
- Human phospholipase A2-like enzyme and fragments thereof also are useful in raising specific antibodies which can block the enzyme and effectively reduce its activity.
- Fig. 1 shows the DNA-sequence encoding a phospholipase A2-like enzyme Polypeptide (SEQ ID NO: 1).
- Fig. 2 shows the amino acid sequence deduced from the DNA-sequence of Fig.1 (SEQ ID NO: 2).
- Fig. 3 shows the amino acid sequence of the protein identified by embl Accession No. AF065215 (SEQ ID NO: 3).
- Fig. 4 shows the DNA sequence of EST EMBLJAA365197IHSZZ0541 (SEQ ED NO: 4).
- Fig. 5 shows the amino acid sequence of the protein identified by SwissProt Accession No. 077793
- Fig. 6 shows the BLASTP alignment of human phospholipase A2-like enzyme (SEQ ID NO: 2) with the protein identified with embl Accession No. AF065215 (SEQ ID NO: 3).
- Fig. 7 shows the BLASTP alignment of 366_protein (SEQ ID NO: 2) against swiss
- Fig. 8 shows the BLASTP-alignment of 366_protein against swiss
- Fig. 9 shows the BLASTP-alignment of 366jprotein against pdbj IC JY
- Fig. 10 shows the BLASTP-alignment of 366_protein against ⁇ db
- Fig. 11 shows the Prosite and BLOCKS search results.
- Fig. 12 shows the HJVJ PFAM-alignment of 366_ rotein against pfamjhmm
- Fig. 13 shows the H_V--MPFAM-alignment of 366_protein against pfam]hmm
- Fig. 14 shows the BLOCKS search results.
- Fig. 15 shows the results of the gene expression profiling of phospholipase A2- like enzyme mRNA in a whole-body screen.
- Fig. 16 shows the results of the gene expression profiling of phospholipase A2- like enzyme mRNA in a blood-lung screen.
- the invention relates to an isolated polynucleotide encoding a phospholipase A2-like enzyme polypeptide and being selected from the group consisting of:
- amino acid sequences which are at least about 41% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2;
- Human phospholipase A2-like enzyme comprises the amino acid sequence shown in SEQ ID NO: 2. Alignments of SEQ ID NO: 2 with known phospholipase A2 enzymes are shown in Figs. 6-10. A coding sequence for SEQ ID NO: 2 is shown in SEQ ID NO: 1. A related EST (SEQ ID NO: 4) is expressed in the pineal gland.
- Human phospholipase A2-like enzyme of the invention is expected to be useful for the same purposes as previously identified phospholipase A2 enzymes. Human phospholipase A2-like enzyme is believed to be useful in therapeutic methods to treat disorders such as asthma, cancer, inflammation, CNS disorders, cardiovascular disorders, chronic obstructive pulmonary disease, diabetes, and obesity. Human phospholipase A2-like enzyme also can be used to screen for human phospholipase A2-like enzyme activators and inhibitors.
- Human phospholipase A2-like enzyme polypeptides comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 795 contiguous amino acids selected from the amino acid sequence shown in SEQ ED NO: 2 or a biologically active variant thereof, as defined below.
- a phospholipase A2 ike enzyme polypeptide of the invention therefore can be a portion of a phospholipase A2-like enzyme protein, a full-length phospholipase A2- like enzyme protein, or a fusion protein comprising all or a portion of a phospholipase A2-like enzyme protein.
- phospholipase A2-like enzyme polypeptides Preferably, naturally or non-naturally occurring phospholipase
- A2-like enzyme polypeptide variants have amino acid sequences which are at least about 41, 45, 50, 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 96, 96, or 98% identical to the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof. Percent identity between a putative phospholipase A2-like enzyme polypeptide variant and an amino acid sequence of SEQ ID NO: 2 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).
- Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions.
- Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
- Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a phospholipase A2-like enzyme polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active phos- pholipase A2-like enzyme polypeptide can readily be determined by assaying for phospholipase A2 activity, as described for example, in the specific examples, below.
- Fusion proteins are useful for generating antibodies against phospholipase A2-like enzyme polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of a phospholipase A2-like enzyme polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
- a phospholipase A2-like enzyme polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
- the first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 795 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO: 2 or biologically active variants of SEQ ID NO: 2, as defined below, those described above.
- the first polypeptide segment also can comprise full-length phospholipase A2-like enzyme protein.
- the second polypeptide segment can be a full-length protein or a protein fragment.
- Proteins commonly used in fusion protein construction include ⁇ -galactosidase, ⁇ - glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horse- radish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
- epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
- Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
- a fusion protein also can be engineered to contain a cleavage site located between the phospholipase A2-like enzyme polypeptide-encoding sequence and the heterologous protein sequence, so that the phospholipase A2-like enzyme polypeptide can be cleaved and purified away from the heterologous moiety.
- a fusion protein can be synthesized chemically, as is known in the art.
- a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology.
- Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from the complement of SEQ ED NO: 1 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
- kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal,
- Species homologs of human phospholipase A2-like enzyme polypeptide can be obtained using phospholipase A2-like enzyme polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of phospholipase A2-like enzyme polypeptide, and expressing the cDNAs as is known in the art.
- a phospholipase A2-like enzyme polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a phospholipase A2-like enzyme polypeptide.
- a coding sequence for human phospholipase A2-like enzyme is shown in SEQ ED NO: 1.
- nucleotide sequences encoding human phospholipase A2-like enzyme polypeptides, as well as homologous nucleotide sequences which are at least about
- nucleotide sequences shown in SEQ ED NO: 1 or its complement also are phospholipase A2-like enzyme polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affme gap search with a gap open penalty of
- cDNA Complementary DNA
- species homologs, and variants of phospholipase A2-like enzyme polynucleotides which encode biologically active phospholipase A2-like enzyme polypeptides also are phospholipase A2-like enzyme polynucleotides.
- Polynucleotides comprising at least 6, 7, 8, 9, 10, 12, 15, 18, 20, or 25 contiguous nucleotides of SEQ ID NO: 1 or its complement also are phospholipase A2-like enzyme polynucleotides.
- Such polynucleotides can be used, for example, as hybridization probes or antisense olig- nucleotides.
- Variants and homologs of the phospholipase A2-like enzyme polynucleotides described above also are phospholipase A2-like enzyme polynucleotides.
- homologous phospholipase A2-like enzyme polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known phospholipase A2- like enzyme polynucleotides under stringent conditions, as is known in the art.
- homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.
- Species homologs of the phospholipase A2-like enzyme polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast.
- Human variants of phospholipase A2-like enzyme polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T m of a double-stranded DNA decreases by 1-1.5°C with every 1% decrease in homology (Bonner et ah, J. Mol. Biol. 81, 123 (1973).
- Variants of human phospholipase A2-like enzyme polynucleotides or phospholipase A2-like enzyme poly- nucleotides of other species can therefore be identified by hybridizing a putative homologous phospholipase A2-like enzyme polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 or the complement thereof to form a test hybrid.
- the melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
- Nucleotide sequences which hybridize to phospholipase A2-like enzyme poly- nucleotides or their complements following stringent hybridization and/or wash conditions also are phospholipase A2-like enzyme polynucleotides.
- Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et ah, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
- T m a combination of temperature and salt concentration should be chosen that is approximately 12-20°C below the calculated T m of the hybrid under study.
- the T m of a hybrid between a phospholipase A2-like enzyme polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):
- Stringent wash conditions include, for example, 4X SSC at 65°C, or 50% formamide, 4X SSC at 42°C, or 0.5X SSC, 0.1% SDS at 65°C.
- Highly stringent wash conditions include, for example, 0.2X SSC at 65°C.
- a phospholipase A2-like enzyme polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
- Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated phospholipase A2-like enzyme polynucleotides.
- restriction enzymes and probes can be used to isolate polynucleotide fragments which comprises phospholipase A2-like nucleotide sequences.
- Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.
- Human phospholipase A2-like enzyme cDNA molecules can be made with standard molecular biology techniques, using phospholipase A2-like enzyme mRNA as a template. Human phospholipase A2-like enzyme cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
- the partial sequence disclosed herein can be used to identify the corresponding full length gene from which it was derived.
- the partial sequence can be nick-translated or end-labeled with 32 P using polynucleotide kinase using labeling methods known to those with skill in the art (BASIC METHODS IN MOLECULAR BIOLOGY, Davis et ah, eds., Elsevier Press, N.Y., 1986).
- a lambda library prepared from human tissue can be directly screened with the labeled sequences of interest or the library can be converted en masse to pBluescript (Stratagene Cloning Systems, La Jolla, Calif. 92037) to facilitate bacterial colony screening (see Sambrook et ah, MOLECULAR
- filters with bacterial colonies containing the library in pBluescript or bacterial lawns containing lambda plaques are denatured, and the DNA is fixed to the filters.
- the filters are hybridized with the labeled probe using hybridization conditions described by Davis et ah, 1986.
- the partial sequences, cloned into lambda or pBluescript, can be used as positive controls to assess background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification.
- the resulting autoradio- grams are compared to duplicate plates of colonies or plaques; each exposed spot corresponds to a positive colony or plaque.
- the colonies or plaques are selected, expanded and the DNA is isolated from the colonies for further analysis and sequencing.
- Positive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial sequence and the other primer from the vector.
- Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot Analysis.
- the complete sequence of the clones can be determined, for example after exonuclease m digestion (Mc- Combie et ah, Methods 3, 33-40, 1991).
- a series of deletion clones are generated, each of which is sequenced. The resulting overlapping sequences are assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a highly accurate final sequence.
- PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements.
- restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
- Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988).
- Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C.
- the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
- capture PCR involves PCR amplifi- cation of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et ah, PCR Methods Applic. 1, 111-119, 1991).
- multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
- Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
- capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
- capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
- Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
- Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
- Human phospholipase A2-like enzyme polypeptides can be obtained, for example, by purification from human cells, by expression of phospholipase A2-like enzyme polynucleotides, or by direct chemical synthesis.
- Human phospholipase A2-like enzyme polypeptides can be purified from any cell which expresses the enzyme, including host cells which have been transfected with phospholipase A2-like enzyme expression constructs.
- a purified phospholipase A2- like enzyme polypeptide is separated from other compounds which normally associate with the phospholipase A2-like enzyme polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
- a preparation of purified phospholipase A2-like enzyme polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99%> pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.
- the polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
- Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding phospholipase A2-like enzyme polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et ah, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.
- a variety of expression vector/host systems can be utilized to contain and express sequences encoding a phospholipase A2-like enzyme polypeptide.
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,
- TMV TMV
- bacterial expression vectors e.g., Ti or pBR322 plasmids
- animal cell systems e.g., TMV, TMV, TMV, TMV, TMV, or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
- control elements or regulatory sequences are those non-translated regions of the vector ⁇ enhancers, promoters, 5' and 3' untranslated regions ⁇ which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the
- BLUESCREPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
- the baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable.
- vectors based on SV40 or EB V can be used with an appropriate selectable marker.
- Bacterial and Yeast Expression Systems Bacterial and Yeast Expression Systems.
- a number of expression vectors can be selected depending upon the use intended for the phospholipase A2-like enzyme polypeptide. For example, when a large quantity of a phospholipase A2-like enzyme polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUE- SCRIPT (Stratagene).
- a sequence encoding the phospholipase A2-like enzyme polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
- pIN vectors Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989
- pGEX vectors Promega, Madison, Wis.
- GST glutathione S-transferase
- fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
- Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
- yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
- constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
- sequences encoding phospholipase A2-like enzyme polypeptides can be driven by any of a number of promoters.
- viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987).
- plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al, EMBOJ. 3, 1671-1680, 1984; Brogue et al, Science 224, 838-843, 1984; Winter et ah, Results Prohl. Cell Differ. 17, 85-105, 1991).
- constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.
- pathogen-mediated transfection Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).
- An insect system also can be used to express a phospholipase A2-like enzyme polypeptide.
- Autographa californica nuclear poly- hedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
- Sequences encoding phospholipase A2- like enzyme polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
- Successful insertion of phospholipase A2-like enzyme polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
- the recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which phospholipase A2-like enzyme polypeptides can be expressed
- a number of viral-based expression systems can be used to express phospholipase
- A2-like enzyme polypeptides in mammalian host cells For example, if an adeno- virus is used as an expression vector, sequences encoding phospholipase A2-like enzyme polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing a phospholipase A2-like enzyme polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984).
- transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
- RSV Rous sarcoma virus
- HACs Human artificial chromosomes
- 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
- Specific initiation signals also can be used to achieve more efficient translation of sequences encoding phospholipase A2-like enzyme polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a phospholipase A2-like enzyme polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al, Results Probl. Cell Differ. 20, 125-162, 1994).
- a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed phospholipase A2-like enzyme polypeptide in the desired fashion.
- modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
- Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
- Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.
- ATCC American Type Culture Collection
- Stable expression is preferred for long-term, high-yield production of recombinant proteins.
- cell lines which stably express phospholipase A2-like enzyme polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium.
- the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced phospholipase A2-like enzyme sequences.
- Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986.
- herpes simplex virus thymidine kinase (Wigler et al, Cell 11, 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al, Cell 22, 817-23, 1980) genes which can be employed in tk ⁇ or aprf cells, respectively.
- antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
- dhfr confers resistance to methotrexate (Wigler et al, Proc. Natl. Acad. Sci.
- npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al, J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988).
- Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Biol 55, 121-131, 1995).
- marker gene expression suggests that the phospholipase A2-like enzyme polynucleotide is also present, its presence and expression may need to be confirmed.
- a sequence encoding a phospholipase A2-like enzyme polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode a phospholipase A2-like enzyme polypeptide can be identified by the absence of marker gene function.
- a marker gene can be placed in tandem with a sequence encoding a phospholipase A2-like enzyme polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the phospholipase A2-like enzyme polynucleotide.
- host cells which contain a phospholipase A2-like enzyme polynucleotide and which express a phospholipase A2-like enzyme polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
- the presence of a polynucleotide sequence encoding a phospholipase A2-like enzyme polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a phospholipase A2-like enzyme polypeptide.
- Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a phospholipase A2-like enzyme polypeptide to detect transformants which contain a phospholipase A2-like enzyme polynucleotide.
- A2-like enzyme polypeptide using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- FACS fluorescence activated cell sorting
- a two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a phospholipase A2-like enzyme polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al, SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al, J. Exp. Med. 158, 1211-1216, 1983).
- Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding phospholipase A2-like enzyme polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
- sequences encoding a phospholipase A2-like enzyme polypeptide can be cloned into a vector for the production of an mRNA probe.
- RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like. Expression and Purification of Polypeptides
- Host cells transformed with nucleotide sequences encoding a phospholipase A2-like enzyme polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
- the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides which encode phospholipase A2-like enzyme polypeptides can be designed to contain signal sequences which direct secretion of soluble phospholipase A2-like enzyme polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane- bound phospholipase A2-like enzyme polypeptide.
- purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system
- cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the phospholipase A2-like enzyme polypeptide also can be used to facilitate purification.
- One such expression vector provides for expression of a fusion protein containing a phospholipase A2-like enzyme polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et al, Prot. Exp.
- enterokinase cleavage site provides a means for purifying the phospholipase A2-like enzyme polypeptide from the fusion protein.
- Vectors which contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993.
- Sequences encoding a phospholipase A2-like enzyme polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al Nucl. Acids Res. Symp. Ser. 225-232, 1980).
- a phospholipase A2-like enzyme polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995).
- Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of phospholipase A2-like enzyme polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
- the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND
- composition of a synthetic phospholipase A2-like enzyme polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the phospholipase A2-like enzyme polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein. Production of Altered Polypeptides
- codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
- nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter phospholipase A2-like enzyme polypeptide- encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
- DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
- site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
- Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab,
- F(ab') 2 , and Fv which are capable of binding an epitope of a phospholipase A2-like enzyme polypeptide.
- a phospholipase A2-like enzyme polypeptide typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope.
- epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.
- An antibody which specifically binds to an epitope of a phospholipase A2-like enzyme polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
- immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.
- an antibody which specifically binds to a phospholipase A2-like enzyme polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
- antibodies which specifically bind to phospholipase A2-like polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a phospholipase A2-like enzyme polypeptide from solution.
- Human phospholipase A2-like enzyme polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies.
- a phospholipase A2-like enzyme polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
- a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
- various adjuvants can be used to increase the immunological response.
- adjuvants include, but are not limited to, Freund' s adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g.
- BCG Bacilli Calmette-Guerin
- Corynebacterium parvum are especially useful.
- Monoclonal antibodies which specifically bind to a phospholipase A2-like enzyme polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl. Acad. Sci. 80, 2026-2030, 1983; Cole et al, Mol. Cell Biol. 62, 109-120, 1984).
- Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
- Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
- humanized antibodies can be produced using recombinant methods, as described in GB2188638B.
- Antibodies which specifically bind to a phospholipase A2-like enzyme polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
- single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to phospholipase A2-like enzyme polypeptides.
- Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).
- Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Eur. J, Cancer Prev. 5, 507-11).
- Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15,
- a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
- single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al, 1995, Int. J. Cancer 61, 497-501; Nicholls et al, 1993, J. Immunol. Meth. 165, 81- 91).
- Antibodies which specifically bind to phospholipase A2-like enzyme polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837,
- chimeric antibodies can be constructed as disclosed in WO 93/03151.
- Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.
- Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a phospholipase A2-like enzyme polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
- Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of phospholipase A2-like enzyme gene products in the cell.
- Antisense oligonucleotides can be deoxyribonucleotides, ribomicleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.
- Modifications of phospholipase A2-like enzyme gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to he COn.t-.oL, 5', or regulatory regions of the phospholipase A2-Iike enzyme gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
- An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
- Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a phospholipase A2-like enzyme polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent phospholipase A2-like enzyme nucleotides, can provide sufficient targeting specificity for phospholipase A2-like enzyme mRNA.
- each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
- Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
- One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular phospholipase A2-like enzyme polynucleotide sequence.
- Antisense oligonucleotides can be modified without affecting their ability to hybridize to a phospholipase A2-like enzyme polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
- internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
- Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
- modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 215, 3539-3542, 1987.
- Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673).
- ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
- Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
- the coding sequence of a phospholipase A2-like enzyme polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the phospholipase A2-like enzyme polynucleotide.
- Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988).
- the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
- the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).
- RNA target Specific ribozyme cleavage sites within a phospholipase A2-like enzyme RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate phospholipase A2-like enzyme RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
- hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
- Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease phospholipase A2-like enzyme expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
- a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
- ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells. Differentially Expressed Genes
- genes whose products interact with human phospholipase A2-like enzyme may represent genes which are differentially expressed in disorders including, but not limited to, asthma, cancer, inflammation, CNS disorders, cardiovascular disorders, chronic obstructive pulmonary disease, diabetes, and obesity. Further, such genes may represent genes which are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human phospholipase A2-like enzyme gene or gene product may itself be tested for differential expression.
- the degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques.
- standard characterization techniques such as differential display techniques.
- Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.
- RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique which does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al, ed. dislike CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.
- Transcripts within the collected RNA samples which represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subtractive hybridization (Hedrick et al, Nature 308, 149-53; Lee et al, Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), differential display (Liang & Pardee, Science 257, 967-71, 1992; U.S. Patent 5,262,311), and microarrays.
- the differential expression information may itself suggest relevant methods for the treatment of disorders involving the human phospholipase A2-like enzyme.
- treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human phospholipase A2-like enzyme.
- the differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human phospholipase A2-like enzyme gene or gene product are up-regulated or down-regulated.
- the invention provides assays for screening test compounds which bind to or modulate the activity of a phospholipase A2-like enzyme polypeptide or a phospholipase A2-like enzyme polynucleotide.
- a test compound preferably binds to a phospholipase A2-like enzyme polypeptide or polynucleotide. More preferably, a test compound decreases or increases phospholipase A2-like by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
- Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
- the com- pounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced re- combinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
- the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule Hbraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.
- Test compounds can be screened for the ability to bind to phospholipase A2-like enzyme polypeptides or polynucleotides or to affect phospholipase A2-like enzyme activity or phospholipase A2-like enzyme gene expression using high throughput screening.
- high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
- the most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l.
- many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
- free format assays or assays that have no physical barrier between samples, can be used.
- an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by
- Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change. Yet another example is described by Salmon et al, Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.
- test samples are placed in a porous matrix.
- One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
- a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
- the test compound is preferably a small molecule which binds to and occupies, for example, the active site of the phospholipase A2-like enzyme polypeptide, such that normal biological activity is prevented.
- small molecules include, but are not limited to, small peptides or peptide-like molecules.
- either the test compound or the phospholipase A2-like enzyme polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
- a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
- Detection of a test compound which is bound to the phospholipase A2-like enzyme polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
- binding of a test compound to a phospholipase A2-like enzyme poly- peptide can be determined without labeling either of the interactants.
- a microphysiometer can be used to detect binding of a test compound with a phos- pholipase A2-like enzyme polypeptide.
- a microphysiometer e.g., CytosensorTM
- a microphysiometer is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a phospholipase A2-like enzyme polypeptide (McConnell et al,
- Determining the ability of a test compound to bind to a phospholipase A2-like enzyme polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63,
- BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcoreTM). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
- SPR surface plasmon resonance
- a phospholipase A2-like enzyme polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268, 12046-12054, 1993; Bartel et al, BioTechniques 14, 920-924, 1993;
- the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
- the assay utilizes two different DNA constructs.
- polynucleotide encoding a phospholipase A2-like enzyme polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
- a DNA sequence that encodes an unidentified protein (“prey" or "sample” can be fused to a polynucleotide that codes for the activation domain of the known transcription factor.
- the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the phospholipase A2-like enzyme polypeptide.
- a reporter gene e.g., LacZ
- either the phospholipase A2-like enzyme polypeptide (or polynucleotide) or the test compound can be bound to a solid support.
- Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
- Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
- Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a phospholipase A2- like enzyme polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
- the phospholipase A2-like enzyme polypeptide is a fusion protein comprising a domain that allows the phospholipase A2-like enzyme polypeptide to be bound to a solid support.
- glutathione-S-transferase fiision proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed phospholipase A2-like enzyme polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
- Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
- phospholipase A2-like enzyme polypeptide or polynucleotide
- test compound can be immobilized utilizing conjugation of biotin and streptavidin.
- Biotinylated phospholipase A2-like enzyme polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
- antibodies which specifically bind to a phospholipase A2-like enzyme polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the phospholipase A2-like enzyme polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
- GST-immobilized complexes include immunodetection of complexes using antibodies which specifically bind to the phospholipase A2-like enzyme polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the phospholipase A2-like enzyme polypeptide, and SDS gel electrophoresis under non- reducing conditions. Screening for test compounds which bind to a phospholipase A2-like enzyme polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a phospholipase A2-like enzyme polypeptide or polynucleotide can be used in a cell-based assay system.
- a phospholipase A2-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a phospholipase A2-like enzyme polypeptide or polynucleotide is determined as described above.
- Test compounds can be tested for the ability to increase or decrease the phospholipase A2 activity of a human phospholipase A2-like enzyme polypeptide.
- phospholipase A2 activity can be measured, for example, as described in the specific examples, below.
- Enzyme assays can be carried out after contacting either a purified phospholipase A2-like enzyme polypeptide, a cell membrane preparation, or an intact cell with a test compound.
- a test compound which decreases a phospholipase A2 activity of a phospholipase A2-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing phospholipase A2-like enzyme activity.
- a test compound which increases a phospholipase A2 activity of a human phospholipase A2-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human phospholipase A2-like enzyme activity.
- test compounds which increase or decrease phospholipase A2-like enzyme gene expression are identified.
- a phospholipase A2-like enzyme polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the phospholipase A2-like enzyme polynucleotide is determined.
- the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
- the test compound can then be identified as a modulator of expression based on this comparison.
- test compound when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
- test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
- the level of phospholipase A2-like enzyme mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
- the presence of polypeptide products of a phospholipase A2-like enzyme polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
- polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a phospholipase A2-like enzyme polypeptide.
- Such screening can be carried out either in a cell-free assay system or in an intact cell.
- Any cell which expresses a phospholipase A2-like enzyme polynucleotide can be used in a cell-based assay system.
- the phospholipase A2-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above.
- Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.
- compositions of the invention can comprise, for example, a phospholipase A2-like enzyme polypeptide, phospholipase A2-like enzyme polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a phospholipase A2-like enzyme polypeptide, or mimetics, activators, or inhibitors of a phospholipase A2-like enzyme polypeptide activity.
- compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
- agent such as stabilizing compound
- the compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.
- compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
- Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
- compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
- Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
- disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
- Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- suitable coatings such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
- Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
- Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
- the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
- compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as
- Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
- suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
- Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
- Non-lipid polycationic amino polymers also can be used for delivery.
- the suspension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
- penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
- compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
- the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
- the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
- compositions can be placed in an appropriate container and labeled for treatment of an indicated condition.
- labeling would include amount, frequency, and method of administration.
- Human phospholipase A2-like enzyme can be regulated to treat cancer.
- Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recruit blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression. Various combinations of these aberrant physiologies, along with the acquisition of drug-resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue.
- Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins.
- proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities.
- Activators and/or inhibitors of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity.
- Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxicological analyses form the basis for drug development and subsequent testing in humans.
- the present invention provides methods for treating neoplastic diseases. Specifically, tumor necrosis, the killing of neoplastic cells and a reduction in the size of a neoplastic mass, can be induced in a mammal suffering from a neoplastic disease by administering an effective amount of an agent which stimulates human phospholipase A2-like enzyme activity or promotes arachidonic acid release, an indication of phospholipase A2-like enzyme activity.
- the stimulation of phospholipase A2-like enzyme activity results in the generation of eicosanoids, leading to the recruitment/activation of cell which promote an inflammatory response.
- the inflammatory response triggers the mammal's immune system to destroy the neoplastic cells.
- the response initiated by the treatment of the present invention results in a reduction in the severity of the neoplastic disease, a reduction of the tumor burden in the mammal, and a significant reduction in the number of neoplastic cells and neoplasm mass.
- Agents ⁇ vhich selectively block PLA2 activity in neurons and/or glial cells and/or astrocytes or monocytes can be used to inhibit A ⁇ -mediated neuronal degeneration, such as that which results from exposure of such cells to pathogenic forms of Ap as occurs in Alzheimer's Disease and related A ⁇ -induced neurodegenerative conditions. It is likely, therefore, that inhibitors of human phospholipase A2-like enzyme also can be used to inhibit neuronal degeneration resulting from A ⁇ toxicity.
- CNS disorders which may be treated include brain injuries, cerebrovascular diseases and their consequences, Parkinson's disease, corticobasal degeneration, motor neuron disease, dementia, including ALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke, post-traumatic brain injury, and small-vessel cerebrovascular disease.
- Dementias such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia and Parkinsonism linked to chromosome 17, frontotemporal dementias, including Pick's disease, progressive nuclear palsy, corticobasal degeneration, Huntington's disease, thalamic degeneration, Creutzfeld- Jakob dementia, HIV dementia, schizophrenia with dementia, and Korsakoff s psychosis also can be treated.
- cognitive-related disorders such as mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, vascular cognitive impairment, attention deficit disorders, attention deficit hyperactivity disorders, and memory disturbances in children with learning disabilities, by regulating the activity of human phospholipase
- Pain that is associated with CNS disorders also can be treated by regulating the activity of human phospholipase A2-like enzyme. Pain which can be treated includes that associated with central nervous system disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation).
- central nervous system disorders such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation).
- Non-central neuropathic pain includes that associated with post mastectomy pain, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HTV/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic poly- neuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with cancer and cancer treatment also can be treated, as can headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania.
- headache pain for example, migraine with aura, migraine without aura, and other migraine disorders
- episodic and chronic tension-type headache tension-type like headache, cluster headache, and chronic
- allergens typically elicit a specific IgE response and, although in most cases the allergens themselves have little or no intrinsic toxicity, they induce pathology when the IgE response in turn elicits an IgE-dependent or T cell-dependent hypersensitivity reaction.
- Hypersensitivity reactions can be local or systemic and typically occur within minutes of allergen exposure in individuals who have previously been sensitized to an allergen.
- the hypersensitivity reaction of allergy develops when the allergen is recognized by IgE antibodies bound to specific receptors on the surface of effector cells, such as mast cells, basophils, or eosinophils, which causes the activation of the effector cells and the release of mediators that produce the acute signs and symptoms of the reactions.
- Allergic diseases include asthma, allergic rhinitis (hay fever), atopic dermatitis, and anaphylaxis.
- Asthma is though to arise as a result of interactions between multiple genetic and environmental factors and is characterized by three major features: 1) intermittent and reversible airway obstruction caused by bronchoconstriction, increased mucus production, and thickening of the walls of the airways that leads to a narrowing of the airways, 2) airway hyperresponsiveness caused by a decreased control of airway caliber, and 3) airway inflammation.
- Certain cells are critical to the inflammatory reaction of asthma and they include T cells and antigen presenting cells, B cells that produce IgE, and mast cells, basophils, eosinophils, and other cells that bind IgE.
- effector cells accumulate at the site of allergic reaction in the airways and release toxic products that contribute to the acute pathology and eventually to the tissue destruction related to the disorder.
- Other resident cells such as smooth muscle cells, lung epithelial cells, mucus-producing cells, and nerve cells may also be abnormal in individuals with asthma and may contribute to the pathology. While the airway obstruction of asthma, presenting clinically as an intermittent wheeze and shortness of breath, is generally the most pressing symptom of the disease requiring immediate treatment, the inflammation and tissue destruction associated with the disease can lead to irreversible changes that eventually make asthma a chronic disabling disorder requiring long-tenn management.
- arachidonic acid is metabolized in mammals by two distinct pathways.
- U.S. Patent No. 5,070,207 The metabolism of arachidonic acid by cyclooxygenase enzymes results in the production of prostaglandins and thromboxanes.
- the physiological activity of the prostaglandins has akeady been amply elucidated in recent years.
- prostaglandins arise from the endoperoxides PGG 2 and PGH 2 by the cyclooxygenase pathway of arachidonic acid metabolism. These endoperoxides are also the precursors of the thromboxanes (Tx) A 2 and B 2 .
- TxA 2 is a vasoconstrictor which stimulates platelet aggregation.
- the vasoconstrictive and platelet aggregating properties of the thromboxanes are balanced by another product arising from the endoperoxides in the cyclooxygenase pathway, prostacyclin (PGI 2 ), which is a vasodilator with platelet aggregation inhibitory activity.
- prostacyclin synthesis is impaired and/or platelet activation is enhanced, then thrombosis and vasoconstriction is favored.
- the role of prostanoids in haemostasis and thrombosis are reviewed by Gryglewski, CRC Crit. Rev. Biochem.
- Cyclooxygenase metabolites are known to participate directly in the inflammatory response. See Higgs et al, Annals of Clinical Research 16, 287-99, 1984. This is through their vasodepressor activities, participation in pain and fever, augmentation of peptide mediator vascular permeability and edema forming properties. Finally, various aspects of cell mediated immunity are influenced by cyclooxygenase products.
- the other pathway of AA metabolism involves lipoxygenase enzymes and results in the production of a number of oxidative products called leukotrienes.
- the latter are designated by the LT nomenclature system, and the most significant products of the lipoxygenase metabolic pathway are the leukotrienes B 4 , C 4 , and D .
- the substance denominated slow-reacting substance of anaphylaxis (SRS-A) has been shown to consist of a mixture of leukotrienes, with LTC 4 and LTD 4 as the primary products and having varying amounts of other leukotriene metabolites . See Bach et al, J. Immun. 215, 115-18, 1980; Biochem. Biophys. Res. Commun. 93, 1121-26, 1980.
- LTC 4 and LTD 4 are potent bronchoconstrictors of the human bronchi (Dahlen et al, Nature 288, 484-486 (1980) and Piper, Int. Arch. Appl. Immunol. 76, suppl. 1, 43 (1985)0, which stimulate the release of mucus from airways in vitro [Marom et al, Am. Rev. Resp. Dis.
- Phospholipase A2 is the critical rate limiting enzyme in the arachidonic acid (AA) cascade, because it is responsible for the hydrolysis of esterified AA from the C-2 position of membrane phospholipids. This reaction generates two products: (1) free AA which is then available for subsequent metabolism by either the cyclooxygenase or lipoxygenase enzymes, and (2) lysophospholipid.
- AA arachidonic acid
- PAF platelet activating factor
- the anti-inflammatory steroids are thought to inhibit eicosanoid synthesis by inducing the synthesis of a PLA2 inhibitory protein denominated macrocortin, lipomodulin or lipocortin [see Flower et al, Nature, London, 278, 456 (1979) and
- PLA2 As the initial step leading to subsequent conversion of AA to the various eicosanoids by the cyclooxygenase and lipoxygenase pathways, the PLA2 -mediated release of AA from membrane phospholipids is a critical event in attempting to deal with the various physiological manifestations which are based on the activity of the eicosanoids and/or PAF.
- PLA2 has been shown to be required for platelet aggregation [Pickett et al, Biochem. J., 160, 405 (1976)], cardiac contraction and excitation [Geisler et a , Pharm. Res. Commun., 9, 117 (1977)], as well as prostaglandin synthesis [Vogt, Adv. Prostagl.
- PLA2 Thromb. Res., 3, 89 (1978)] the inhibition of PLA2 is indicated in the therapeutic treatment of both PAF induced or cyclooxygenase and/or lipoxygenase pathway product-mediated physiological conditions.
- human phospholipase A2-like enzyme inhibitors are a rational approach to the prevention, removal or amelioration of such conditions as allergy, anaphylaxis, asthma and inflammation.
- Cytosolic phospholipase A2 (PLA2; EC 3.1.1.4 ) catalyzes the release of arachidonic acid from membrane phospholipids.
- Arachidonic acid in turn serves as the precursor for a wide spectrum of biologic effectors, collectively known as lipid mediators, such as lysophospholipids, prostaglandins, and leukotrienes, that are involved in hemodynamic regulation, inflammatory responses, and other cellular processes. Lipid mediators also play crucial roles in the pathogenesis of inflammatory diseases including asthma. Therefore, inhibition of PLA2 activity is a good therapeutic approach for inflammatory diseases. Attempts have been made in the past to develop known PLA2 inhibitors into pharmaceuticals; however, many of them have failed mainly due to general toxicity.
- PLA2G4A homologs In a database search for PLA2G4A homologs, Pickard et al. (Pickard, R. T.et al: J. Biol. Chem. 274: 8823-8831, 1999) identified PLA2G4B, also called cPLA2- ⁇ , and assembled its full-length cDNA using 5 -prime RACE, cDNA library screening, and RT-PCR.
- the PLA2G4B protein contains a C2 calcium- and lipid-binding domain and shares approximately 30% sequence identity with PLA2G4A and PLA2G4C, including total conservation of 4 catalytically essential residues. Using a baculovirus system, they demonstrated calcium-dependent enzymatic activity.
- the novel phospholipase A2-like enzyme disclosed herein shows 40% identity to human cytosolic phospholipase A2- ⁇ .
- the gene appears to be highly expressed in kidney, lung, and skeletal muscle (see Fig. 15).
- it was found to be expressed relatively highly in both tonsil and normal bronchial tracheal epithelial cells (see Fig. 16). Due to its high expression in kidney, lung, and skeletal muscle, regulation of phospholipase A2-like enzyme is expected to be useful in the treatment of inflammatory diseases that involve these tissues and are mediated in part by metabolites of arachidonic acid.
- Regulation of phospholipase A2-like enzyme is potentially particularly effective in the treatment of inflamed lung tissues since the accessibility of the lungs to aerosolized compounds allows the topical delivery of a therapeutic, thereby potentially avoiding the systemic toxicity previously experienced with other PLA2 inhibitors.
- Lysophospholipids have also been implicated as potential mediators of sudden cardiac death, Corr et al, "Lethal Arrhythmias Resulting from Myocardial Ischemia and Infarction", Rosen & Patti, eds., Kluwer Academic Publishers, Boston, 91-014 (1989).
- the addition of lysophospholipids to normoxic myocardial tissue in vitro induces electrophysiological alterations that are similar to those observed in the ischemic heart in vivo. Corr et al, Circ. Res, 55, 135-54 (1984).
- lysophospholipid accumulation in the ischemic dog heart in vivo has been correlated with the frequency of cardiac arrhythmias, Kinnaird et al, Lipids,
- ATP levels decrease.
- Treatment of rat neonatal myocytes with the glycolytic inhibitor iodoacetate lowers the levels of ATP which results in the release of arachidonic acid and morphological alterations of the myocytes, Chien et al, J. Clin. Invest., 75, 1770-80 (1985).
- One PLA2 inhibitor U26,384 prevented the release of arachidonic acid, phospholipid degradation, sarcolemmal membrane defects and the release of creatine kinase that was induced by the treatment of rat neonatal myocytes with iodoacetate, Sen et al, J. Clin. Invest., 82, 1333-38 (1988).
- Chronic obstructive pulmonary disease Inhibitors of human phospholipase A2-like enzyme also can be used to treat other conditions mediated by products of the oxidation of arachidonic acid, such as chronic obstructive pulmonary disease, emphysema, and acute respiratory distress syndrome.
- Chronic obstructive pulmonary (or airways) disease COPD is a condition defined physiologically as airflow obstruction that generally results from a mixture of emphysema and peripheral airway obstruction due to chronic bronchitis (Senior & Shapiro, Pulmonary Diseases and Disorders, 3d ed., New York, McGraw-Hill, 1998, pp.
- Emphysema is characterized by destruction of alveolar walls leading to abnormal enlargement of the air spaces of the lung.
- Chronic bronchitis is defined clinically as the presence of chronic productive cough for three months in each of two successive years.
- airflow obstruction is usually progressive and is only partially reversible.
- Chronic inflammation of the airways is a key pathological feature of COPD (Senior & Shapiro, 1998).
- the inflammatory cell population comprises increased numbers of macrophages, neutrophils, and CD8 + lymphocytes.
- Inhaled irritants such as cigarette smoke, activate macrophages which are resident in the respiratory tract, as well as epithelial cells leading to release of chemokines (e.g., interleukin-8) and other chemotactic factors.
- chemokines e.g., interleukin-8
- chemotactic factors act to increase the neutrophil/monocyte trafficking from the blood into the lung tissue and airways.
- Neutrophils and monocytes recruited into the airways can release a variety of potentially damaging mediators such as proteolytic enzymes and reactive oxygen species.
- Obesity may be treated by regulating human phospholipase A2-like enzyme.
- Obesity and overweight are defined as an excess of body fat relative to lean body mass. An increase in caloric intake or a decrease in energy expenditure or both can bring about this imbalance leading to surplus energy being stored as fat.
- Obesity is associated with important medical morbidities and an increase in mortality. The causes of obesity are poorly understood and may be due to genetic factors, environmental factors or a combination of the two to cause a positive energy balance.
- anorexia and cachexia are characterized by an imbalance in energy intake versus energy expenditure leading to a negative energy balance and weight loss.
- Agents that either increase energy expenditure and/or decrease energy intake, absorption or storage would be useful for treating obesity, overweight, and associated comorbidities.
- Agents that either increase energy intake and/or decrease energy expenditure or increase the amount of lean tissue would be useful for treating cachexia, anorexia and wasting disorders.
- This gene, translated proteins and agents which modulate this gene or portions of the gene or its products are useful for treating obesity, overweight, anorexia, cachexia, wasting disorders, appetite suppression, appetite enhancement, increases or decreases in satiety, modulation of body weight, and/or other eating disorders such as bulimia.
- this gene translated proteins and agents which modulate this gene or portions of the gene or its products are useful for treating obesity/overweight-associated comorbidities including hypertension, type 2 diabetes, coronary artery disease, hyperlipidemia, stroke, gallbladder disease, gout, osteoarthritis, sleep apnea and respiratory problems, some types of cancer including endometrial, breast, prostate, and colon cancer, thrombolic disease, polycystic ovarian syndrome, reduced fertility, complications of pregnancy, menstrual irregularities, hirsutism, stress incontinence, and depression.
- obesity/overweight-associated comorbidities including hypertension, type 2 diabetes, coronary artery disease, hyperlipidemia, stroke, gallbladder disease, gout, osteoarthritis, sleep apnea and respiratory problems, some types of cancer including endometrial, breast, prostate, and colon cancer, thrombolic disease, polycystic ovarian syndrome, reduced fertility, complications of pregnancy, menstrual irregularities, hirsu
- Diabetes mellitus is a common metabolic disorder characterized by an abnormal elevation in blood glucose, alterations in lipids and abnormalities (complications) in the cardiovascular system, eye, kidney and nervous system. Diabetes is divided into two separate diseases: type 1 diabetes (juvenile onset), which results from a loss of cells which make and secrete insulin, and type 2 diabetes
- Type 1 diabetes is initiated by an autoimuune reaction that attacks the insulin secreting cells (beta cells) in the pancreatic islets.
- Agents that prevent this reaction from occurring or that stop the reaction before destruction of the beta cells has been accomplished are potential therapies for this disease.
- Other agents that induce beta cell proliferation and regeneration also are potential therapies.
- Type II diabetes is the most common of the two diabetic conditions (6% of the population).
- the defect in insulin secretion is an important cause of the diabetic condition and results from an inability of the beta cell to properly detect and respond to rises in blood glucose levels with insulin release.
- Therapies that increase the response by the beta cell to glucose would offer an important new treatment for this disease.
- the defect in insulin action in Type II diabetic subjects is another target for therapeutic intervention. Agents that increase the activity of the insulin receptor in muscle, liver, and fat will cause a decrease in blood glucose and a normalization of plasma lipids.
- the receptor activity can be increased by agents that directly stimulate the receptor or that increase the intracellular signals from the receptor.
- Other therapies can directly activate the cellular end process, i.e. glucose transport or various enzyme systems, to generate an insulin-like effect and therefore a produce beneficial outcome. Because overweight subjects have a greater susceptibility to Type II diabetes, any agent that reduces body weight is a possible therapy.
- Type I and Type diabetes can be treated with agents that mimic insulin action or that treat diabetic complications by reducing blood glucose levels.
- agents that reduces new blood vessel growth can be used to treat the eye complications that develop in both diseases.
- test compound identified as described herein in an appropriate animal model.
- an agent identified as described herein e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a phospholipase
- A2-like enzyme polypeptide binding molecule can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
- an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
- this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
- a reagent which affects phospholipase A2-like enzyme activity can be administered to a human cell, either in vitro or in vivo, to reduce phospholipase A2-like enzyme activity.
- the reagent preferably binds to an expression product of a human phospholipase A2-like enzyme gene. If the expression product is a protein, the reagent is preferably an antibody.
- an antibody can be added to a preparation of stem cells which have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
- the reagent is delivered using a liposome.
- the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours.
- a liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human.
- the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.
- a liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell.
- the transfection efficiency of a liposome is about 0.5 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, more preferably about 1.0 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, and even more preferably about 2.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 10 6 cells.
- a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 mn, and even more preferably between about 200 and 400 nm in diameter.
- Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.
- a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome. Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Patent 5,705,151).
- polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 ⁇ g to about 5 ⁇ g of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 ⁇ g of polynucleotides is combined with about 8 nmol liposomes.
- antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery.
- Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al Trends in Biotechnol. 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci.
- a therapeutically effective dose refers to that amount of active ingredient which increases or decreases phospholipase A2-like enzyme activity relative to the phospholipase A2-like enzyme activity which occurs in the absence of the therapeutically effective dose.
- the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
- the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
- Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in 50%) of the population) and LD 5 o (the dose lethal to 50%) of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
- the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 5 o/ED 5 o.
- compositions which exhibit large therapeutic indices are preferred.
- the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
- the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
- the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
- Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect.
- Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
- Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
- Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
- Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
- polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun,” and DEAE- or calcium phosphate-mediated transfection.
- Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about 50 ⁇ g/kg, about 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg of patient body weight.
- effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA.
- the reagent is preferably an antisense oligonucleotide or a ribozyme.
- Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
- a reagent reduces expression of a phospholipase A2-like enzyme gene or the activity of a phospholipase A2-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent.
- any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
- the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
- any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
- Human phospholipase A2-like enzyme also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences which encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding phospholipase A2-like enzyme in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.
- Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method.
- cloned DNA segments can be employed as probes to detect specific DNA segments.
- the sensitivity of this method is greatly enhanced when combined with PCR.
- a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
- the sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.
- DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g. , Myers et al, Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl.
- the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. h addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
- Altered levels of a phospholipase A2-like enzyme also can be detected in various tissues.
- Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.
- the polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-phospholipase A2-like enzyme polypeptide obtained is transfected into human embryonic kidney 293 cells. From these cells extracts are obtained.
- Human phospholipase A2-like enzyme activity is determined by an in vitro hydrolysis assay with vesicles containing l-palmitoyl-2-[l- 14 C]oleoyl phosphatidylcholine (Sigma Chemical Co.).
- Human phospholipase A2-like enzyme phospholipase A2 activity is demonstrates by analysis of the cleavage products isolated from the hydrolysis reaction mixture.
- Vesicles containing l-palmitoyl-2-[l- 14 C]oleoyl phosphatidylcholine are prepared by mixing 2.0 ⁇ Ci of the radiolabeled phospholipid with 12.5 mg of unlabeled l-pahnitoyl-2-oleoyl phosphatidylcholine and drying the mixture under N 2 . Two and a half ml of 150 mM Tris-HCl, pH 7.5, is added, and the mixture is sonicated and centrifuged. The supernatant may be stored at 4°C. The final reaction mixture contains 0.25 ml of Hanks buffered salt solution supplemented with 2.0 mM taurochenodeoxycholate, 1.0% bovine serum albumin, 1.0 mM CaC12, pH 7.4,
- the lipids are separated by thin layer chromatography, using a two solvent system of chloroform:methanol:acetic acid:water (65:35:8:4) until the solvent front is halfway up the plate, the process is then continued with hexane:ether:acetic acid (86:16:1) until the solvent front is at the top of the plate.
- the lipid-containing areas are visualized with 12 vapor; the spots are scraped, and their radioactivity is determined by scintillation counting. The amount of radioactivity released as fatty acids will increase.
- the cell extracts as mentioned above are added to the assay mixture while the amount of radioactivity released as lyso-phosphatidylcholine will remain low.
- the Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human phospholipase A2-like polypeptides in yeast.
- the phospholipase A2-like enzyme-encoding DNA sequence is derived from SEQ ID NO: 1.
- the DNA sequence is modified by well known methods in such a way that it contains at its 5'-end an initiation codon and at its 3'-end an enterokinase cleavage site, a His6 reporter tag and a termination codon.
- at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZ B with the corresponding restriction enzymes the modified
- DNA sequence is ligated into pPICZB.
- This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter.
- the resulting pPICZ/md-His6 vector is used to transform the yeast.
- the yeast is cultivated under usual conditions in 5 liter shake flasks and the re- combinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea.
- the bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, CA) according to manufacturer's instructions.
- Purified human phospholipase Purified human phospholipase
- Purified phospholipase A2-like enzyme polypeptides comprising a glutathione-S- transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
- Human phospholipase A2-like enzyme polypeptides comprise the amino acid sequence shown in SEQ ED NO: 2.
- the test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
- the buffer solution containing the test compounds is washed from the wells.
- Binding of a test compound to a phospholipase A2-like enzyme polypeptide is detected by fluorescence measurements of the contents of the wells.
- a test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a phospholipase A2-like enzyme polypeptide.
- a test compound is administered to a culture of human cells transfected with a phospholipase A2-like enzyme expression construct and incubated at 37°C for 10 to 45 minutes.
- a culture of the same type of cells which have not been transfected is incubated for the same time without the test compound to provide a negative control.
- RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979).
- Northern blots are prepared using 20 to 30 ⁇ g total RNA and hybridized with a 32 P-labeled phospholipase A2-like enzyme-specific probe at 65°C in Express-hyb (CLONTECH).
- the probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO: 1.
- a test compound which decreases the phospholipase A2-like enzyme-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of phospholipase A2-like enzyme gene expression.
- Human phospholipase A2-like enzyme activity can be demonstrated by an in vitro hydrolysis assay with vesicles containing l-palmitoyl-2-[l- 14 C]oleoyl phosphatidylcholine (Sigma Chemical Co.). Human phospholipase A2-like enzyme phospholipase A2 activity is demonstrates by analysis of the cleavage products isolated from the hydrolysis reaction mixture.
- the final reaction mixture contains 0.25 ml of Hanks buffered salt solution supplemented with 2.0 mM taurochenodeoxycholate, 1.0% bovine serum albumin, 1.0 mM CaC12, pH 7.4, 150 ⁇ g of l-palmitoyl-2-[l- I4 C]oleoyl phosphatidylcholine vesicles, and various amount of human phospholipase A2-like enzyme diluted in PBS. After incubation for 30 min at 37°C, 20 ⁇ g each of lyso-phosphatidylcholine and oleic acid are added as carriers and each sample is extracted for total lipids.
- the lipids are separated by thin layer chromatography, using a two solvent system of chloroform:methanol: acetic acid:water (65:35:8:4) until the solvent front is halfway up the plate, the process is then continued with hexane:ether:acetic acid (86:16:1) until the solvent front is at the top of the plate.
- the lipid-containing areas are visualized with 12 vapor; the spots are scraped, and their radioactivity is determined by scintillation counting.
- the amount of radioactivity released as fatty acids will increase as a greater amount of Human phospholipase A2-like enzyme is added to the assay mixture while the amount of radioactivity released as lyso-phosphatidylcholine will remain low. This demonstrates that human phospholipase A2-like enzyme cleaves at the Serial No.-2 and not the Serial No.-l position, exhibiting phospholipase A2 activity.
- test compound is administered to a culture of human cells transfected with a phospholipase A2-like enzyme expression construct and incubated at 37°C for 10 to 45 minutes.
- a culture of the same type of cells which have not been transfected is incubated for the same time without the test compound to provide a negative control.
- a test compound which decreases the phospholipase A2 activity of the phospholipase A2-like enzyme relative to the phospholipase A2 activity in the absence of the test compound is identified as an inhibitor of phospholipase A2-like enzyme activity.
- Human phospholipase A2-like enzyme or biologically active fragments thereof are labeled with 125 1 Bolton-Hunter reagent (Bolton et al, Biochem. J. 133, 529, 1973).
- Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled human phospholipase A2-like enzyme, washed, and any wells with labeled human phospholipase A2-like enzyme complex are assayed.
- Data obtained using different concentrations of human phospholipase A2-like enzyme are used to calculate values for the number, affinity, and association of human phospholipase A2-like enzyme with the candidate molecules.
- RT-PCR human phospholipase-like enzyme is involved in cancer
- expression is determined in the following tissues: adrenal gland, bone marrow, brain, cerebellum, colon, fetal brain, fetal liver, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, uterus, and peripheral blood lymphocytes.
- Expression in the following cancer cell lines also is determined: DU-145 (prostate), NCI-H125 (lung), HT-29 (colon), COLO-205
- colon A-549 (lung), NCI-H460 (lung), HT-116 (colon), DLD-1 (colon), MDA-
- MD-231 (breast), LS174T (colon), ZF-75 (breast), MDA-MN-435 (breast), HT-1080, MCF-7 (breast), and U87. Matched pairs of malignant and normal tissue from the same patient also are tested.
- fetal and adult brain muscle, heart, lung, kidney, liver, thymus, testis, colon, placenta, trachea, pancreas, kidney, gastric mucosa, colon, liver, cerebellum, skin, cortex (Alzheimer's and normal), hypothalamus, cortex, amygdala, cerebellum, hippocampus, choroid, plexus, thalamus, and spinal cord.
- the initial expression panel consists of RNA samples from respiratory tissues and inflammatory cells relevant to COPD: lung (adult and fetal), trachea, freshly isolated alveolar type II cells, cultured human bronchial epithelial cells, cultured small airway epithelial cells, cultured bronchial sooth muscle cells, cultured H441 cells (Clara-like), freshly isolated neutrophils and monocytes, and cultured monocytes (macrophage-like).
- Body map profiling also is carried out, using total RNA panels purchased from Clontech.
- the tissues are adrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung, mammary gland, pancreas, prostate, salivary gland, skeletal muscle, small intestine, spleen, stomach, testis, thymus, trachea, thyroid, and uterus.
- the expression of human phospholipase- like enzyme in cells derived from normal individuals with the expression of cells derived from COPD-affected individuals is compared.
- U87MG, BE(2)C, T986, KANTS, MO59K, CHP234, C6 (rat), SK-N-F1, SK-PU- DW, PFSK-1, BE(2)M17, and MCIXC also are tested for human phospholipase-like enzyme expression.
- the expression of human phospholipase-like enzyme in cells derived from normal individuals with the expression of cells derived from obese individuals is compared.
- lung or immune tissues brain, heart, kidney, liver, lung, trachea, bone marrow, colon, small intestine, spleen, stomach, thymus, mammary gland, skeletal muscle, prostate, testis, uterus, cerebellum, fetal brain, fetal liver, spinal cord, placenta, adrenal gland, pancreas, salivary gland, thyroid, peripheral blood leukocytes, lymph node, and tonsil.
- lung and immune system cells are screened to localize expression to particular cell subsets: lung microvascular endothelial cells, bronchial/tracheal epithelial cells, bronchial/tracheal smooth muscle cells, lung fibroblasts, T cells (T helper 1 subset, T helper 2 subset, NKT cell subset, and cytotoxic T lymphocytes), B cells, mononuclear cells (monocytes and macrophages), mast cells, eosinophils, neutrophils, and dendritic cells.
- T cells T helper 1 subset, T helper 2 subset, NKT cell subset, and cytotoxic T lymphocytes
- B cells mononuclear cells (monocytes and macrophages)
- mast cells eosinophils, neutrophils, and dendritic cells.
- human phospholipase A2-like enzyme is involved in the disease process of diabetes
- the following whole body panel is screened to show predominant or relatively high expression: subcutaneous and mesenteric adipose tissue, adrenal gland, bone marrow, brain, colon, fetal brain, heart, hypothalamus, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spleen, stomach, testis, thymus, thyroid, trachea, and uterus.
- Human islet cells and an islet cell library also are tested.
- the expression of human phospholipase A2- ⁇ ke enzyme in cells derived from normal individuals with the expression of cells derived from diabetic individuals is compared.
- Quantitative expression profiling is performed by the form of quantitative PCR analysis called "kinetic analysis" firstly described in
- the probe is cleaved by the 5 '-3' endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al, Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al, Genome Res. 6, 986-94, 1996, and Gibson et al, Genome Res. 6, 995-1001, 1996).
- the amplification of an endogenous control can be performed to standardize the amount of sample RNA added to a reaction.
- the control of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous control can be independently quantified in the same tube if probes labeled with different dyes are used.
- RNA extraction and cDNA preparation Total RNA from the tissues listed above are used for expression quantification. RNAs labeled “from autopsy” are extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer's protocol.
- RNA Fifty ⁇ g of each RNA are treated with DNase I for 1 hour at 37°C in the following reaction mix: 0.2 U/ ⁇ l RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/ ⁇ l RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; lO mM MgCl 2 ; 50 mM NaCl; and 1 mM DTT.
- RNA is extracted once with 1 volume of phenokchloroform:- isoamyl alcohol (24:24:1) and once with chloroform, and precipitated with 1/10 volume of 3 MNaAcetate, pH5.2, and 2 volumes of ethanol.
- RNA from the autoptic tissues Fifty ⁇ g of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and spectro- photometric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is 200 ng/ ⁇ L. Reverse transcription is carried out with 2.5 ⁇ M of random hexamer primers.
- Probes can be labeled at the 5' end with FAM (6-carboxyfluorescein) and at the 3' end with TAMRA (6-carboxytetramethylrhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.
- FAM 6-carboxyfluorescein
- TAMRA 6-carboxytetramethylrhodamine
- the assay reaction mix is as follows: IX final TaqMan Universal PCR Master Mix (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from
- Overnight fasted normal rats or mice have elevated rates of gluconeogenesis as do streptozotocin-induced diabetic rats or mice fed ad libitum.
- Rats are made diabetic with a single intravenous injection of 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kg i.p. for 5 consecutive days.
- Blood glucose is measured from tail-tip blood and then compounds are administered via different routes (p.o., i.p., i.v., s.c). Blood is collected at various times thereafter and glucose measured. Alternatively, compounds are administered for several days, then the animals are fasted overnight, blood is collected and plasma glucose measured. Compounds that inhibit glucose production will decrease plasma glucose levels compared to the vehicle- treated control group.
- Both ob/ob and db/db mice as well as diabetic Zucker rats are hyperglycemic, hyperinsulinemic and insulin resistant.
- the animals are pre-bled, their glucose levels measured, and then they are grouped so that the mean glucose level is the same for each group.
- Compounds are administered daily either q.d. or b.i.d. by different routes (p.o., i.p., s.c.) for 7-28 days. Blood is collected at various times and plasma glucose and insulin levels determined. Compounds that improve insulin sensitivity in these models will decrease both plasma glucose and insulin levels when compared to the vehicle-treated control group.
- Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the administration of a glucose load.
- compounds are administered by different routes (p.o., i.p., s.c. or i.v.) to overnight fasted normal rats or mice.
- an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later.
- Plasma insulin levels are determined.
- Compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose.
- animals are bled at the appropriate time after compound administration, then given either an oral or intraperitoneal glucose load (lg/kg), bled again after 15, 30, 60 and 90 minutes and plasma glucose levels determined.
- Compounds that increase insulin levels will decrease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.
- test compounds which regulate pristanoyl-CoA oxidase-like enzyme are administered by different routes (p.o., i.p., s.c, or i.v.) to overnight fasted normal rats or mice.
- an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later. Plasma insulin levels are determined.
- Test compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose.
- mice When measuring glucose disappearance, animals are bled at the appropriate time after compound administration, then given either an oral or intraperitoneal glucose load (lg/kg), bled again after 15, 30, 60, and 90 minutes and plasma glucose levels determined. Test compounds that increase insulin levels will decrease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.
- Overnight fasted normal rats or mice have elevated rates of gluconeogenesis as do streptozotocin-induced diabetic rats or mice fed ad libitum.
- Rats are made diabetic with a single intravenous injection of 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kg i.p. for 5 consecutive days.
- Blood glucose is measured from tail-tip blood and then compounds are administered via different routes (p.o., i.p., i.v., s.c). Blood is collected at various times thereafter and glucose measured. Alternatively, compounds are administered for several days, then the animals are fasted overnight, blood is collected and plasma glucose measured. Compounds that inhibit glucose production will decrease plasma glucose levels compared to the vehicle- treated control group.
- Both ob/ob and db/db mice as well as diabetic Zucker rats are hyperglycemic, hyperinsulinemic and insulin resistant.
- the animals are pre-bled, their glucose levels measured, and then they are grouped so that the mean glucose level is the same for each group.
- Compounds are administered daily either q.d. or b.i.d. by different routes (p.o., i.p., s.c.) for 7-28 days. Blood is collected at various times and plasma glucose and insulin levels determined. Compounds that improve insulin sensitivity in these models will decrease both plasma glucose and insulin levels when compared to the vehicle-treated control group.
- Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the administration of a glucose load.
- compounds are administered by different routes (p.o., i.p., s.c. or i.v.) to overnight fasted normal rats or mice.
- an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later.
- Plasma insulin levels are determined.
- Compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose.
- animals are bled at the appropriate time after compound administration, then given either an oral or intraperitoneal glucose load (lg/kg), bled again after 15, 30, 60 and 90 minutes and plasma glucose levels determined.
- Compounds that increase insulin levels will decrease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.
- Acute pain is measured on a hot plate mainly in rats.
- Two variants of hot plate testing are used: In the classical variant animals are put on a hot surface (52 to 56 ⁇ C) and the latency time is measured until the animals show nocifensive behavior, such as stepping or foot licking.
- the other variant is an increasing temperature hot plate where the experimental animals are put on a surface of neutral temperature. Subsequently this surface is slowly but constantly heated until the animals begin to lick a hind paw. The temperature which is reached when hind paw licking begins is a measure for pain threshold.
- Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to pain testing.
- application routes i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal
- Persistent pain is measured with the formalin or capsaicin test, mainly in rats.
- a solution of 1 to 5% formalin or 10 to 100 ⁇ g capsaicin is injected into one hind paw of the experimental animal.
- the animals show nocifensive reactions like flinching, licking and biting of the affected paw.
- the number of nocifensive reactions within a time frame of up to 90 minutes is a measure for intensity of pain.
- Compounds are tested against a vehicle treated control group.
- Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t, i.c.v., s.c, intradermal, transdermal) prior to formalin or capsaicin administration.
- application routes i.v., i.p., p.o., i.t, i.c.v., s.c, intradermal, transdermal
- Neuropathic pain is induced by different variants of unilateral sciatic nerve injury mainly in rats.
- the operation is performed under anesthesia.
- the first variant of sciatic nerve injury is produced by placing loosely constrictive ligatures around the common sciatic nerve.
- the second variant is the tight ligation of about the half of the diameter of the common sciatic nerve.
- a group of models is used in which tight ligations or transections are made of either the L5 and L6 spinal nerves, or the L%> spinal nerve only.
- the fourth variant involves an axotomy of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact whereas the last variant comprises the axotomy of only the tibial branch leaving the sural and common nerves uninjured. Control animals are treated with a sham operation.
- the nerve injured animals develop a chronic mechanical allodynia, cold allodynioa, as well as a thermal hyperalgesia.
- Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc-Life Science Instruments, Woodland Hills, SA,
- Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy), or by means of a cold plate of 5 to 10 ⁇ C where the nocifensive reactions of the affected hind paw are counted as a measure of pain intensity.
- a further test for cold induced pain is the counting of nocifensive reactions, or duration of nocifensive responses after plantar administration of acetone to the affected hind limb.
- Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to pain testing.
- application routes i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal
- Inflammatory pain is induced mainly in rats by injection of 0.75 mg car- rageenan or complete Freund's adjuvant into one hind paw.
- the animals develop an edema with mechanical allodynia as well as thermal hyperalgesia.
- Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc-Life Science Instruments, Woodland Hills, SA, USA).
- Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy, Paw thermal stimulator, G. Ozaki, University of California, USA).
- Plant Test Ugo Basile, Comerio, Italy
- Paw thermal stimulator G. Ozaki, University of California, USA
- Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc-Life Science Instruments, Woodland Hills, SA, USA).
- Compounds are tested against diabetic and non-diabetic vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to pain testing.
- application routes i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal
- Degeneration of the dopaminergic nigrostriatal and striatopallidal pathways is the central pathological event in Parkinson's disease. This disorder has been mimicked experimentally in rats using single/sequential unilateral stereotaxic injections of 6-OH-DA into the medium forebrain bundle (MFB).
- MFB medium forebrain bundle
- mice Male Wistar rats (Harlan Winkelmann, Germany), weighing 200 ⁇ 250 g at the beginning of the experiment, are used. The rats are maintained in a temperature- and humidity-controlled environment under a 12 h light/dark cycle with free access to food and water when not in experimental sessions. The following in vivo protocols are approved by the governmental authorities. All efforts are made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques. Animals are administered pargyline on the day of surgery (Sigma, St.
- DA nigrostriatal pathway 4 ⁇ l of 0.01% ascorbic acid-saline containing 8 ⁇ g of 6-OHDA HBr (Sigma) are injected into the left medial fore-brain bundle at a rate of 1 ⁇ l/min (2.4 mm anterior, 1.49 mm lateral, -2.7 mm ventral to Bregma and the skull surface). The needle is left in place an additional 5 min to allow diffusion to occur.
- Forelimb akinesia is assessed three weeks following lesion placement using a modified stepping test protocol.
- the animals are held by the experimenter with one hand fixing the hindlimbs and slightly raising the hind part above the surface.
- One paw is touching the table, and is then moved slowly sideways (5 s for 1 m), first in the forehand and then in the backhand direction.
- the number of adjusting steps is counted for both paws in the backhand and forehand direction of movement.
- the sequence of testing is right paw forehand and backhand adjusting stepping, followed by left paw forehand and backhand directions.
- the test is repeated three times on three consecutive days, after an initial training period of three days prior to the first testing.
- Forehand adjusted stepping reveals no consistent differences between lesioned and healthy control animals. Analysis is therefore restricted to backhand adjusted stepping.
- Balance Test
- Balance adjustments following postural challenge are also measured during the stepping test sessions.
- the rats are held in the same position as described in the stepping test and, instead of being moved sideways, tilted by the experimenter towards the side of the paw touching the table. This maneuver results in loss of balance and the ability of the rats to regain balance by forelimb movements is scored on a scale ranging from 0 to 3. Score 0 is given for a normal forelimb placement. When the forelimb movement is delayed but recovery of postural balance detected, score 1 is given. Score 2 represents a clear, yet insufficient, forelimb reaction, as evidenced by muscle contraction, but lack of success in recovering balance, and score 3 is given for no reaction of movement. The test is repeated three times a day on each side for three consecutive days after an initial training period of three days prior to the first testing.
- a modified version of the staircase test is used for evaluation of paw reaching behavior three weeks following primary and secondary lesion placement.
- Plexiglass test boxes with a central platform and a removable staircase on each side are used.
- the apparatus is designed such that only the paw on the same side at each staircase can be used, thus providing a measure of independent forelimb use.
- the double staircase is filled with 7 x 3 chow pellets (Precision food pellets, formula: P, purified rodent diet, size 45 mg; Sandown Scientific) on each side.
- pellets Precision food pellets, formula: P, purified rodent diet, size 45 mg; Sandown Scientific
- MPTP neurotoxin l-methyl-4-phenyl-l,2,3,6-tetrahydro-pyridine
- DAergic mesencephalic dopaminergic
- MPTP leads to a marked decrease in the levels of dopamine and its metabolites, and in the number of dopaminergic terminals in the striatum as well as severe loss of the tyrosme hydroxylase (TH)-immunoreactive cell bodies in the substantia nigra, pars compacta.
- TH tyrosme hydroxylase
- mice are perfused transcardially with 0.01 M PBS (pH 7.4) for 2 min, followed by 4% paraformaldehyde (Merck) in PBS for 15 min.
- the brains are removed and placed in 4% paraformaldehyde for 24 h at 4°C. For dehydration they are then transfe ⁇ ed to a 20% sucrose (Merck) solution in 0.1 M PBS at 4°C until they sink.
- the brains are frozen in methylbutan at -20°C for 2 min and stored at -70°C.
- sledge microtome (mod. 3800-Frigocut, Leica) 25 ⁇ m sections are taken from the genu of the corpus callosum (AP 1.7 mm) to the hippocampus (AP 21.8 mm) and from AP 24.16 to AP 26.72. Forty-six sections are cut and stored in assorters in 0.25 M Tris buffer (pH 7.4) for immunohistochemistry.
- TH free-floating tyrosine hydroxylase
- the system logs the fall as the end of the experiment for that mouse, and the total time on the rotarod, as well as the time of the fall and all the set-up parameters, are recorded.
- the system also allows a weak current to be passed through the base grid, to aid training.
- the object recognition task has been designed to assess the effects of experimental manipulations on the cognitive performance of rodents.
- a rat is placed in an open field, in which two identical objects are present.
- the rats inspects both objects during the first trial of the object recognition task.
- a second trial after a retention interval of for example 24 hours, one of the two objects used in the first trial, the 'familiar' object, and a novel object are placed in the open field.
- the inspection time at each of the objects is registered.
- the basic measures in the OR task is the time spent by a rat exploring the two object the second trial. Good retention is reflected by higher exploration times towards the novel than the 'familiar' object.
- Administration of the putative cognition enhancer prior to the first trial predominantly allows assessment of the effects on acquisition, and eventually on consolidation processes.
- Administration of the testing compound after the first trial allows to assess the effects on consolidation processes, whereas administration before the second trial allows to measure effects on retrieval processes.
- the passive avoidance task assesses memory performance in rats and mice.
- the inhibitory avoidance apparatus consists of a two-compartment box with a light compartment and a dark compartment.
- the two compartments are separated by a guillotine door that can be operated by the experimenter.
- a threshold of 2 cm separates the two compartments when the guillotine door is raised.
- the illumination in the dark compartment is about 2 lux.
- the light intensity is about 500 lux at the center of the floor of the light compartment.
- Two habituation sessions, one shock session, and a retention session are given, separated by inter-session intervals of 24 hours.
- the rat is allowed to explore the apparatus for 300 sec.
- the rat is placed in the light compartment, facing the wall opposite to the guillotine door. After an accommodation period of 15 sec. the guillotine door is opened so that all parts of the apparatus can be visited freely. Rats normally avoid brightly lit areas and will enter the dark compartment within a few seconds.
- the shock session the guillotine door between the compartments is lowered as soon as the rat has entered the dark compartment with its four paws, and a scrambled 1 mA footshock is administered for 2 sec.
- the rat is removed from the apparatus and put back into its home cage. The procedure during the retention session is identical to that of the habituation sessions.
- the step-through latency that is the first latency of entering the dark compartment (in sec.) during the retention session is an index of the memory performance of the animal; the longer the latency to enter the dark compartment, the better the retention is.
- the Morris water escape task measures spatial orientation learning in rodents.
- Abundant extra-maze cues are provided by the furniture in the room, including desks, computer equipment, a second water tank, the presence of the experimenter, and by a radio on a shelf that is playing softly.
- the animals receive four trials during five daily acquisition sessions.
- a trial is started by placing an animal into the pool, facing the wall of the tank. Each of four starting positions in the quadrants north, east, south, and west is used once in a series of four trials; their order is randomized.
- the escape platform is always in the same position.
- a trial is terminated as soon as the animal had climbs onto the escape platform or when 90 seconds have elapsed, whichever event occurs first. The animal is allowed to stay on the platform for 30 seconds. Then it is taken from the platform and the next trial is started. If an animal did not find the platfo ⁇ n within 90 seconds it is put on the platform by the experimenter and is allowed to stay there for 30 seconds.
- an additional trial is given as a probe trial: the platform is removed, and the time the animal spends in the four quadrants is measured for 30 or 60 seconds.
- the probe trial all animals start from the same start position, opposite to the quadrant where the escape platform had been positioned during acquisition.
- rats or mice with specific brain lesions which impair cognitive functions, or animals treated with compounds such as scopolamine or MK-801, which interfere with normal learning, or aged animals which suffer from cognitive deficits, are used.
- the T-maze spontaneous alternation task assesses the spatial memory performance in mice.
- the start arm and the two goal arms of the T-maze are provided with guillotine doors which can be operated manually by the experimenter.
- a mouse is put into the start arm at the beginning of training.
- the guillotine door is closed.
- the 'forced trial' either the left or right goal arm is blocked by lowering the guillotine door.
- the mouse After the mouse has been released from the start arm, it will negotiate the maze, eventually enter the open goal arm, and return to the start position, where it will be confined for 5 seconds, by lowering the guillotine door.
- the animal can choose freely between the left and right goal arm (all guillotine-doors opened) during 14 'free choice' trials. As soon a the mouse has entered one goal arm, the other one is closed. The mouse eventually returns to the start arm and is free to visit whichever go alarm it wants after having been confined to the start arm for 5 seconds. After completion of 14 free choice trials in one session, the animal is removed from the maze. During training, the animal is never handled.
- the percent alternations out of 14 trials is calculated. This percentage and the total time needed to complete the first forced trial and the subsequent 14 free choice trials (in s) is analyzed.
- Cognitive deficits are usually induced by an injection of scopolamine, 30 min before the start of the training session. Scopolamine reduced the per-cent alternations to chance level, or below.
- a cognition enhancer which is always administered before the training session, will at least partially, antagonize the scopolamine-induced reduction in the spontaneous alternation rate.
- the cell line used for testing is the human colon cancer cell line HCT116.
- Cells are cultured in RPMI-1640 with 10-15% fetal calf serum at a concentration of 10,000 cells per milliliter in a volume of 0.5 ml and kept at 37°C in a 95% air/5%CO atmosphere.
- Phosphorothioate oligoribonucleotides are synthesized on an Applied Biosystems Model 380B DNA synthesizer using phosphoroamidite chemistry. A sequence of 24 bases complementary to the nucleotides at position 1 to 24 of SEQ ID NO: 1 is used as the test oligonucleotide. As a control, another (random) sequence is used: 5'-TCA
- oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate buffered saline at the desired concentration. Purity of the oligonucleotides is tested by capillary gel electrophoresis and ion exchange HPLC. The purified oligonucleotides are added to the culture medium at a concentration of 10 ⁇ M once per day for seven days.
- test oligonucleotide for seven days results in significantly reduced expression of human phospholipase A2-like enzyme as determined by Western blotting. This effect is not observed with the control oligonucleotide.
- the number of cells in the cultures is counted using an automatic cell counter. The number of cells in cultures treated with the test oligonucleotide (expressed as 100%) is compared with the number of cells in cultures treated with the control oligonucleotide. The number of cells in cultures treated with the test oligonucleotide is not more than 30% of control, indicating that the inhibition of human phospholipase A2-like enzyme has an anti-proliferative effect on cancer cells.
- This non-tumor assay measures the ability of a compound to reduce either the endogenous level of a circulating hormone or the level of hormone produced in response to a biologic stimulus.
- Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c).
- test compound p.o., i.p., i.v., i.m., or s.c
- Plasma is assayed for levels of the hormone of interest. If the normal circulating levels of the hormone are too low and/or variable to provide consistent results, the level of the hormone may be elevated by a pre-treatment with a biologic stimulus (i.e., LHRH may be injected i.m.
- a biologic stimulus i.e., LHRH may be injected i.m.
- mice were fed at a dosage of 30 ng/mouse to induce a burst of testosterone synthesis).
- the timing of plasma collection would be adjusted to coincide with the peak of the induced hormone response.
- Compound effects are compared to a vehicle- treated control group.
- An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value ⁇ 0.05 compared to the vehicle control group.
- Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol, these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇ 0.05 as compared to the vehicle control group.
- specific readout assay protocol these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇
- Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.) according to a predetermined schedule and for a predetermined duration (i.e., 1 week).
- animals are weighed, the target organ is excised, any fluid is expressed, and the weight of the organ is recorded.
- Blood plasma may also be collected. Plasma may be assayed for levels of a hormone of interest or for levels of test agent.
- Organ weights may be directly compared or they may be normalized for the body weight of the animal. Compound effects are compared to a vehicle-treated control group. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value ⁇ 0.05 compared to the vehicle control group.
- Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol.
- Cell proliferation is determined by measuring a marker of cell number (i.e., MTT or LDH). The cell number and change in cell number from the starting inoculum are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇ 0.05 as compared to the vehicle control group.
- Hydron pellets with or without growth factors or cells are implanted into a micropocket surgically created in the rodent cornea.
- Compound administration may be systemic or local (compound mixed with growth factors in the hydron pellet).
- Corneas are harvested at 7 days post implantation immediately following intracardiac infusion of colloidal carbon and are fixed in 10% formalin. Readout is qualitative scoring and/or image analysis. Qualitative scores are compared by Rank Sum test. Image analysis data is evaluated by measuring the area of neovascularization (in pixels) and group averages are compared by Student's t-test (2 tail). Significance is p ⁇ 0.05 as compared to the growth factor or cells only group.
- Matrigel containing cells or growth factors, is injected subcutaneously. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Matrigel plugs are harvested at predetermined time point(s) and prepared for readout. Readout is an ELISA-based assay for hemoglobin concentration and/or histological examination (i.e. vessel count, special staining for endothelial surface markers: CD31, factor-8). Readouts are analyzed by Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p ⁇ 0.05 as compared to the vehicle control group.
- Tumor cells or fragments are implanted subcutaneously on
- Vehicle and/or compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting at a time, usually on Day 1, prior to the ability to measure the tumor burden.
- Body weights and tumor measurements are recorded 2-3 times weekly. Mean net body and tumor weights are calculated for each data collection day.
- Anti-tumor efficacy may be initially determined by comparing the size of treated (T) and control (C) tumors on a given day by a
- Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p ⁇ 0.05.
- Tumor cells are injected intraperitoneally or intracranially on
- Tumor cells or fragments are implanted subcutaneously and grown to the desired size for treatment to begin. Once at the predetermined size range, mice are randomized into treatment groups. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
- Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay.
- Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value 0.05 compared to the vehicle control group.
- Tumor cells or fragments, of mammary adenocarcinoma origin are implanted directly into a surgically exposed and reflected mammary fat pad in rodents. The fat pad is placed back in its original position and the surgical site is closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
- Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay.
- Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size.
- Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value ⁇ 0.05 compared to the vehicle control group.
- this model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ, or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
- Tumor cells or fragments, of prostatic adenocarcinoma origin are implanted directly into a surgically exposed dorsal lobe of the prostate in rodents.
- the prostate is externalized through an abdominal incision so that the tumor can be implanted specifically in the dorsal lobe while verifying that the implant does not enter the seminal vesicles.
- the successfully inoculated prostate is replaced in the abdomen and the incisions through the abdomen and skin are closed.
- Hormones may also be administered to the rodents to support the growth of the tumors.
- Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected.
- the size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope.
- An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
- This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the lungs), or measuring the target organ weight (i.e., the regional lymph nodes). The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
- Tumor cells of pulmonary origin may be implanted intra- bronchially by making an incision through the skin and exposing the trachea.
- the trachea is pierced with the beveled end of a 25 gauge needle and the tumor cells are inoculated into the main bronchus using a flat-ended 27 gauge needle with a 90° bend.
- Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected.
- the size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope.
- An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
- This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the contralateral lung), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
- Tumor cells of gastrointestinal origin may be implanted intracecally by making an abdominal incision through the skin and externalizing the intestine. Tumor cells are inoculated into the cecal wall without penetrating the lumen of the intestine using a 27 or 30 gauge needle. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope.
- An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the liver), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
- Tumor cells are inoculated s.c and the tumors allowed to grow to a predetermined range for spontaneous metastasis studies to the lung or liver. These primary tumors are then excised. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule which may include the period leading up to the excision of the primary tumor to evaluate therapies directed at inhibiting the early stages of tumor metastasis. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined.
- Survival data is used to generate Kaplan-Meier curves. Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment for both of these endpoints.
- Tumor cells are injected into the tail vein, portal vein, or the left ventricle of the heart in experimental (forced) lung, liver, and bone metastasis studies, respectively.
- Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment.
- Expression profiling is based on a quantitative polymerase chain reaction (PCR) analysis, also called kinetic analysis, first described in Higuchi et al., 1992 and Higuchi et al., 1993.
- PCR polymerase chain reaction
- the principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies.
- mRNA messenger RNA
- cDNA DNA copy
- quantitative RT-PCR quantitative reverse transcription-polymerase chain reaction
- RNA from different human tissues was performed to investigate the tissue distribution of phospholipase A2-like enzyme, Cytosolic phospholipase A2 beta homolog mRNA.
- 25 .mu.g of total RNA from various tissues was used as a template to synthsize first-strand cDNA using the
- First-strand cDNA synthesis was carried out according to the manufacturer's protocol using oligo (dT) to hybridize to the 3' poly A tails of mRNA and prime the synthesis reaction. Approximately 10 ng of the first-strand cDNA was then used as template in a polymerase chain reaction. In other cases, 10 ng of commercially available cDNAs (Human Immune System MTC Panel and Human Blood Fractions MTC Panel, Clontech Laboratories, Palo Alto, CA, USA) were used as template in a polymerase chain reaction.
- the polymerase chain reaction was performed in a LightCycler (Roche Molecular Biochemicals, Indianapolis, IN, USA), in the presence of the DNA-binding fluorescent dye SYBR Green I which binds to the minor groove of the DNA double helix, produced only when double-stranded DNA is successfully synthesized in the reaction (Morrison et al., 1998). Upon binding to double-stranded DNA, SYBR Green I emits light that can be quantitatively measured by the LightCycler machine.
- the polymerase chain reaction was carried out using oligonucleotide primers 366_DNA-L4 (GAAGTACTGCCTGGACCGAGGA) and 366_DNA-R3
- G3PDH glyceraldehyde-3-phosphatase
- HPRT hypoxanthine guanine phophoribosyl transferase
- beta-actin beta-actin
- PBGD porphobilinogen deaminase
- the level of housekeeping gene expression is considered to be relatively constant for all tissues (Adams et al., 1993, Adams et al., 1995, Liew et al., 1994) and therefore can be used as a gauge to approximate relative numbers of cells per .mu.g of total RNA used in the cDNA synthesis step. Except for the use of a slightly different set of housekeeping genes and the use of the LightCycler system to measure expression levels, the normalization procedure was similar to that described in the RNA Master Blot User Manual, Apendix C (1997, Clontech Laboratories, Palo Alto, CA, USA). In brief, expression levels of the five housekeeping genes in all tissue samples were measured in three independent reactions per gene using the
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AU2002224774A AU2002224774A1 (en) | 2000-10-10 | 2001-10-10 | Human phospholipase a2-like enzyme |
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US23843300P | 2000-10-10 | 2000-10-10 | |
US60/238,433 | 2000-10-10 | ||
US25805200P | 2000-12-27 | 2000-12-27 | |
US60/258,052 | 2000-12-27 |
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PCT/EP2001/011703 WO2002031125A2 (fr) | 2000-10-10 | 2001-10-10 | Regulation de l'enzyme semblable a phospholipase a2 humaine |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005056837A2 (fr) * | 2003-11-26 | 2005-06-23 | Applera Corporation | Polymorphismes genetiques associes a des troubles cardiovasculaires et a une reponse au medicament, leurs procedes de detection et d'utilisation |
US7258855B2 (en) | 2000-09-19 | 2007-08-21 | Kyowa Hakko Kogyo Co., Ltd. | Polypeptides having phospholipase A2 activity |
US20130045947A1 (en) * | 2010-02-11 | 2013-02-21 | Indiana University Research And Technology Corporation | Pla2activity as a marker for ovarian and other gynecologic cancers |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US6025178A (en) * | 1997-03-28 | 2000-02-15 | Eli Lilly And Company | Human phospholipase A2 and related nucleic acid compounds |
WO2001053326A1 (fr) * | 1999-12-23 | 2001-07-26 | Hyseq, Inc. | Procedes et elements ayant trait a des polypeptides et des polynucleotides de type phospholipase |
WO2002031162A2 (fr) * | 2000-10-10 | 2002-04-18 | Bayer Aktiengesellschaft | Regulation de l'enzyme du type phospholipase a2 humaine |
WO2002031160A2 (fr) * | 2000-10-10 | 2002-04-18 | Bayer Aktiengesellschaft | Regulation de l'enzyme de type a2 phospholipase humaine |
-
2001
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Non-Patent Citations (10)
Title |
---|
DATABASE EMBL [Online] cDNA, 403 bp, 24 September 2001 (2001-09-24) "Human cytosolic PLA2 beta cDNA" retrieved from EBI Database accession no. AAD11385 XP002212984 -& WO 01 53326 A1 (HYSEQ, INC.) 26 July 2001 * |
DATABASE EMBL [Online] DNA, 2409 bp, 18 June 2002 (2002-06-18) retrieved from EBI Database accession no. AX417785 XP002212987 -& WO 02 31162 A2 (BAYER AKTIENGESELLSCHAFT) 18 April 2002 * |
DATABASE EMBL [Online] DNA, 306 pb, 6 July 2002 (2002-07-06) retrieved from EBI Database accession no. AX453671 XP002213000 -& WO 02 31160 A2 (BAYER AKTIENGESELLSCHAFT) 18 April 2002 * |
DATABASE EMBL [Online] DNA, 3085 bp, 18 June 2001 (2001-06-18) retrieved from EBI Database accession no. AR137924 XP002212985 -& US 6 197 569 B1 (ELI LILLY AND COMPANY) 6 March 2001 (2001-03-06) * |
DATABASE EMBL [Online] DNA, 3085 bp, 9 August 2001 (2001-08-09) retrieved from EBI Database accession no. AR156371 XP002212986 -& US 6 242 206 B1 (ELI LILLY AND COMPANY) 5 June 2001 (2001-06-05) * |
DATABASE EMBL [Online] mRNA, 3085 pb, 18 May 2000 (2000-05-18) "Human PLA2 mRNA" retrieved from EBI Database accession no. AAZ88758 XP002212980 * |
DATABASE EMBL [Online] RNA, 3339 pb, 25 May 1999 (1999-05-25) "Homo sapiens cPLA2 beta mRNA" retrieved from EBI Database accession no. AF121908 XP002212983 * |
DATABASE EMBL [Online] RNA, 3352 pb, 2 November 1998 (1998-11-02) "Homo sapiens cPLA2 beta mRNA" retrieved from EBI Database accession no. AF065215 XP002212982 * |
DATABASE EMBL [Online] RNA, 7252 pb, 2 November 1998 (1998-11-02) "Homo sapiens cPLA2 beta precursor RNA" retrieved from EBI Database accession no. AF065216 XP002212981 * |
DENNIS E A: "The growing phospholipase A2 superfamily of signal transduction enzymes" TIBS TRENDS IN BIOCHEMICAL SCIENCES, ELSEVIER PUBLICATION, CAMBRIDGE, EN, vol. 22, no. 1, 1997, pages 1-2, XP004016851 ISSN: 0968-0004 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7258855B2 (en) | 2000-09-19 | 2007-08-21 | Kyowa Hakko Kogyo Co., Ltd. | Polypeptides having phospholipase A2 activity |
WO2005056837A2 (fr) * | 2003-11-26 | 2005-06-23 | Applera Corporation | Polymorphismes genetiques associes a des troubles cardiovasculaires et a une reponse au medicament, leurs procedes de detection et d'utilisation |
WO2005056837A3 (fr) * | 2003-11-26 | 2006-10-05 | Applera Corp | Polymorphismes genetiques associes a des troubles cardiovasculaires et a une reponse au medicament, leurs procedes de detection et d'utilisation |
US20130045947A1 (en) * | 2010-02-11 | 2013-02-21 | Indiana University Research And Technology Corporation | Pla2activity as a marker for ovarian and other gynecologic cancers |
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