Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • 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
  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

• Noxious chemical, thermal and mechanical stimuli excite peripheral nerve endings of small diameter sensory neurons (nociceptors) in sensory ganglia (eg., dorsal root, nodose and trigeminal ganglia) and initiate signals that are perceived as pain.
• sensory ganglia eg., dorsal root, nodose and trigeminal ganglia
• These neurons are crucial for the detection of harmful or potentially harmful stimuli (heat) and tissue damage (H + (local tissue acidosis), and/or stretch) which arise from changes in the extracellular space during inflammatory or ischaemic conditions (Wall and Melzack, 1994).
• Capsaicin (8-methyl-N-vanillyl-6-nonenamide), the main pungent ingredient in "hot” capsicum peppers, and its analogs interact at specific membrane recognition sites (vanilloid receptors), expressed almost exclusively by primary sensory neurons involved in nociception and neurogenic inflammation (Bevan and Szolcsanyi, 1990).
• Capsaicin is a very selective activator of thinly or unmyelinated nociceptive afferents (Szolcsanyi, 1993; Szolcsanyi, 1996).
• Capsaicin derivatives show structure-function relationships and their effects can be blocked by a selective antagonist capsazepine.
• RTX The ultra potent tricyclic diterpene resiniferatoxin (RTX; (Szolcsanyi et al., 1991)) binds with nanomolar affinity at the capsaicin binding site and has revealed a very localized distribution of capsaicin receptors to rat somatic and visceral primary sensory neurons (Szallasi et al., 1995). Interestingly, the density of RTX receptor sites in nodose and dorsal root ganglia increased after ligation of the vagal and sciatic nerves (Szallasi et al., 1995).
• VRl capsaicin
• the vanilloid (“capsaicin”) receptor VRl is activated by capsaicin and RTX, and activation of VRl is blocked by the antagonists capsazepine (CPZ; (Bevan et al., 1992)) and ruthenium red (RR; (Wood et al., 1988)) (Caterina et al., 1997).
• VRl is a ligand-gated non-selective cation channel that shows pronounced outward rectification (Caterina et al., 1997).
• rat VRl and VR2 and a partial cDNA sequence of human sequences were disclosed in the WIPO publication WO 99/09140.
• vanilloids such as capsaicin (Zostrix 0.025% and Zostrix HP 0.075%) have been used to mitigate neuropathic pain and to treat the intractable pain associated with postherpetic neuralgia, diabetic neuropathy, postmastectomy pain, complex regional pain syndromes and rheumatoid arthritis (Robbins et al., 1998; Rowbotham, 1994; Szallasi and Blumberg, 1996). With prolonged exposure to capsaicin, nociceptor cells become not only insensitive to this agonist but to other noxious stimuli as well (Szolcsanyi, 1993).
• capsaicin produces analgesia
• the mechanism by which capsaicin produces analgesia includes desensitization of nociceptive sensory neurons, and depletion of peptides from peripheral terminals, as well as damage to sensory nerves (Jancso et al., 1977; Rowbotham, 1994).
• the irritancy of capsaicin severely limits its use, and the discovery of novel compounds that block the acidic and/or thermal activation of capsaicin sensitive receptors is sought.
• capsaicin-sensitive fibers are involved in the repair mechanisms of the gastric mucosa. In most studies, capsaicin given into the stomach of rats or cats inhibited gastric acid secretion (Ome et al., 1997).”
• a DNA molecule encoding the human vanilloid receptor (hVRl) has been cloned and characterized.
• the biological and structural properties of these proteins are disclosed, as is the amino acid and nucleotide sequence.
• the recombinant protein is useful to identify modulators of the receptor VRl.
• Modulators identified in the assay disclosed herein are useful as therapeutic agents, which are candidates for the treatment of inflammatory conditions associated with capsaicin receptor activity and for use as analgesics for intractable pain associated with postherpetic neuralgia, diabetic neuropathy, postmastectomy pain, complex regional pain syndromes, arthritis (e.g., rheumatoid and osteoarthritis), as well as ulcers, neurodegenerative diseases, asthma, chronic obstructive pulmonary disease, irritable bowel syndrome, and psoriasis.
• the recombinant DNA molecules, and portions thereof are useful for isolating homologues of the DNA molecules, identifying and isolating genomic equivalents of the DNA molecules, and identifying, detecting or isolating mutant forms of the DNA molecules.
• FIGURE 2 The nucleotide sequence of hVRl is shown including 921 bp 5' UT and 1383 bp 3'UT.
• FIGURE 3 The amino acid sequence of hVRl is shown (839 amino acids).
• FIGURE 4- Functional expression of hVRl in Xenopus oocytes is shown: activation by capsaicin and resiniferatoxin and block of capsaicin response by capsazepine and ruthenium red.
• Capsaicin C; 1 ⁇ M
• Capsaicin is applied at the time indicated by the bar (left panel).
• Preincubation with 0.6 ⁇ M capsazepine (CPZ) for 2 min blocked residual current still present 6 min after the original response (small outward current in beginning of current trace (middle panel)) and completely blocked subsequent application of C (C + CPZ, middle panel).
• FIGURE 5- Dose response for capsaicin applied to Xenopus oocytes expressing hVRl.
• the responses to the indicated concentrations of capsaicin were bath applied to oocytes expressing 2.5 ng hVRl cRNA.
• Oocytes were continuously perfused for 6 min between agonist tests.
• n 4,3,4,2 oocytes for 0.1, 0.3, 1 and 3 ⁇ M agonist, respectively.
• FIGURE 7- Functional expression of hVRl in Xenopus oocytes is shown: activation by low pH (pH 5.5).
• a voltage ramp protocol was applied to a VRl expressing oocyte (-120 to +80 mV over 200 ms) and the whole cell currents elicited are shown before (lower current trace in each a-c) and after (current trace indicated with a solid circle) pH5.5 application.
• Initially oocytes were bathed in ND96 with 100 ⁇ M Ca2+, pH 8. Experiment was performed at room temperature (20 deg C). Arrow indicates 0 mV.
• Low pH activates an outwardly rectifying current (a) that is blocked by CPZ) (b). The effect of CPZ is reversible ( c ). In this example inward currents are very small, presumably due to the low levels of extracellular Ca 2+ .
• Whole cell currents recorded in the presence of low pH are indicated by the solid circles.
• FIGURE 8- Functional expression of hVRl in a mammalian cell line is shown: HEK293 cells were transiently transfected with hVRl (a) or vector alone (pcDNA3.1-zeo) (b) and 4 days later were tested for their response to vanilloid agonists and antagonists. Ca 2+ influx was measured using the Ca 2+ sensitive dye Fluo-4 on a FLIPR system. 1 : Cells were challenged with 1 ⁇ M capsaicin during the time indicated by the open bar (duration: about 1.5 min). 2: Cells were preincubated in 100 nM CPZ (solid bar) for about 1 min and then challenged with 1 ⁇ M Capsaicin (open bar) in the presence of 100 nM CPZ.
• FIG 9 A and B HEK293 cells stably expressing hVRl were tested for responsiveness to capsaicin and sensitivity to ruthenium red and capsazepine.
• the increase in intracellular Ca2+ evoked by 15 nM Capsaicin was blocked in a dose dependent manner by ruthenium red (A, B) and capsazepine (C).
• the parent cell line did not respond to capsaicin (top 2 rows).
• FIG. 10 HEK293 cells stably expressing hVRl show increased conductance in response to 100 nM capsaisin.
• the whole cell configuration of the patch clamp technique was used to record whole cell currents elicted by a voltage ramp protocol (bottom trace).
• the response to capsaicin was blocked by capsazepine and the effect was partially reversed after wash out of antagonist.
• FIG. 11 HEK293 cells stably expressing hVRl show increased conductance in response to RTX (160 nM).
• FIG. 12 A, B, C (A) HEK293 cells stably expressing hVRl show increased conductance in response to low (pH 4.5). (B) Inhibition of the pH response by hVRl antagonist CPZ at 1 micromolar. (C) pH response after CPZ washout. DETAILED DESCRIPTION
• the present invention relates to DNA encoding human VRl receptor that was isolated from a human thalamus cDNA library.
• Human VRl receptor refers to protein which can specifically function as a receptor.
• Other cells and cell lines may also be suitable for use to isolate human VRl receptor cDNA. Selection of suitable cells may be done by screening for human VRl receptor activity in cell extracts. Human VRl receptor activity can be monitored by performing an ⁇ H-[resiniferatoxin] binding assay (Acs et al., 1994; Szallasi and Blumberg, 1990; Szallasi et al., 1994; Szallasi et al., 1993; Szallasi et al., 1991) or by direct measurement of a capsaicin-, RTX- and/or low pH-induced Ca 2+ influx or non- selective cation currents through the hVRl receptor (Caterina et al., 1997). Cells that possess human VRl receptor activity in this assay may be suitable for the isolation of human VRl receptor DNA or mRNA.
• any of a variety of procedures known in the art may be used to molecularly clone human VRl receptor DNA. These methods include, but are not limited to, direct functional expression of the human VRl receptor genes following the construction of a human VRl receptor-containing cDNA library in an appropriate expression vector system. Another method is to screen human VRl receptor- containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the amino acid sequence of the human VRl receptor subunits. An additional method consists of screening a human VRl receptor-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the human VRl receptor protein. This partial cDNA is obtained by the specific PCR amplification of human VRl receptor DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence of the purified human VRl receptor protein.
• Another method is to isolate RNA from human VRl receptor-producing cells and translate the RNA into protein via an in vitro or an in vivo translation system.
• the translation of the RNA into a peptide a protein will result in the production of at least a portion of the human VRl receptor protein which an be identified by, for example, immunological reactivity with an anti-human VRl receptor antibody or by biological activity of human VRl receptor protein.
• pools of RNA isolated from human VRl receptor-producing cells can be analyzed for the presence of an RNA that encodes at least a portion of the human VRl receptor protein. Further fractionation of the RNA pool can be done to purify the human VRl receptor RNA from non-human VRl receptor RNA.
• the peptide or protein produced by this method may be analyzed to provide amino acid sequences, which in turn are used to provide primers for production of human VRl receptor cDNA, or the RNA used for translation can be analyzed to provide nucleotide sequences encoding human VRl receptor and produce probes for this production of human VRl receptor cDNA.
• This method is known in the art and can be found in, for example, Maniatis, T., Fritsch, E.F., Sambrook, J. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989.
• libraries as well as libraries constructed from other cells or cell types, may be useful for isolating human VRl receptor-encoding DNA.
• Other types of libraries include, but are not limited to, cDNA libraries derived from other cells and genomic DNA libraries that include YAC (yeast artificial chromosome) and cosmid libraries.
• cDNA libraries may be prepared from cells or cell lines which have human VRl receptor activity.
• the selection of cells or cell lines for use in preparing a cDN A library to isolate human VRl receptor cDNA may be done by first measuring cell associated human VRl receptor activity using the measurement of Capsaicin-associated biological activity or a capsaicin ligand binding assay.
• cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, in Maniatis, T., Fritsch, E.F., Sambrook, J., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).
• DNA encoding human VRl receptor may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in Maniatis, T., Fritsch, E.F., Sambrook, J. in Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).
• human VRl receptor protein may be purified and partial amino acid sequence determined by automated sequencers. It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 amino acids from the protein is determined for the production of primers for PCR amplification of a partial human VRl receptor DNA fragment.
• the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the human VRl receptor sequence but will be capable of hybridizing to human VRl receptor DNA even in the presence of DNA oligonucleotides with mismatches. The mismatched DNA oligonucleotides may still sufficiently hybridize to the human VRl receptor DNA to permit identification and isolation of human VRl receptor encoding DNA. DNA isolated by these methods can be used to screen DNA libraries from a variety of cell types, from invertebrate and vertebrate sources, and to isolate homologous genes.
• Human VRl receptor may exist as a full-length nascent or unprocessed polypeptide, or as partially processed polypeptides or combinations of processed polypeptides.
• the full-length nascent human VRl receptor polypeptide may be posttranslationally modified by specific proteolytic cleavage events, which result in the formation of fragments of the full-length nascent polypeptide.
• a fragment, or physical association of fragments may have the full biological activity associated with human VRl receptor, however, the degree of human VRl receptor activity may vary between individual human VRl receptor fragments and physically associated human VRl receptor polypeptide fragments.
• the cloned human VRl receptor DNA obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant human VRl receptor protein.
• Techniques for such man ripulations are fully described in Maniatis, T, et al., supra, and are well known in the art.
• Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria including E_. coli. blue-green algae, plant cells, insect cells, fungal cells including yeast cells, and animal cells.
• Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells or bacteria- fungal cells or bacteria- invertebrate cells.
• An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
• a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
• a strong promoter is one that causes mRNAs to be initiated at high frequency.
• Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
• mammalian expression vectors may be used to express recombinant human VRl receptor in mammalian cells.
• Commercially available mammalian expression vectors which may be suitable for recombinant human VRl receptor expression, include but are not limited to, pMAMneo (Clontech), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO- pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).
• bacterial expression vectors may be used to express recombinant human VRl receptor in bacterial cells.
• Commercially available bacterial expression vectors which may be suitable for recombinant human VRl receptor expression include, but are not limited to pET vectors (Novagen) and pQE vectors (Qiagen).
• a variety of fungal cell expression vectors may be used to express recombinant human VRl receptor in fungal cells such as yeast.
• Commercially available fungal cell expression vectors which may be suitable for recombinant human VRl receptor expression include but are not limited to pYES2 (InVitrogen) and Pichia expression vector (InVitrogen).
• insect cell expression vectors may be used to express recombinant human VRl receptor in insect cells.
• Commercially available insect cell expression vectors that may be suitable for recombinant expression of human VRl receptor include but are not limited to pBlueBacII (InVitrogen).
• DNA encoding human VRl receptor may be cloned into an expression vector for expression in a recombinant host cell.
• Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as coli.
• fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to drosophila and silkworm derived cell lines.
• Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH 3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), L-cells, and HEK-293 (ATCC CRL1573).
• CV-1 ATCC CCL 70
• COS-1 ATCC CRL 1650
• COS-7 ATCC CRL 1651
• CHO-Kl ATCC CCL 61
• 3T3 ATCC CCL 92
• NIH 3T3 ATCC CRL 1658
• HeLa ATCC CCL 2
• C127I ATCC CRL 1616
• the expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, lipofection, and electroporation.
• the expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce human VRl receptor protein. Identification of human VRl receptor expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-human VRl receptor antibodies, and the presence of host cell-associated human VRl receptor activity.
• Human VRl receptor DNA may also be performed using in vitro produced synthetic mRNA.
• Synthetic mRNA or mRNA isolated from human VRl receptor producing cells can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being generally preferred.
• human VRl receptor DNA molecules including, but not limited to, the following can be constructed: the full-length open reading frame of the human VRl receptor cDNA encoding the approximately 95,048 kDa protein from approximately base 1 to approximately base 2517 (these numbers correspond to first nucleotide of first methionine and last nucleotide before the first stop codon) and several constructs containing portions of the cDNA encoding human VRl receptor protein. All constructs can be designed to contain none, all or portions of the 5' or the 3' untranslated region of human VRl receptor cDNA.
• Human VRl receptor activity and levels of protein expression can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the human VRl receptor DNA cassette yielding optimal expression in transient assays, this human VRl receptor DNA construct is transferred to a variety of expression vectors, for expression in host cells including, but not limited to, mammalian cells, baculovirus-infected insect cells, coli. and the yeast £. cerevisiae.
• Host cell transfectants and microinjected oocytes may be used to assay both the levels of human VRl receptor channel activity and levels of human VRl receptor protein by the following methods. In the case of recombinant host cells, this involves the co-transfection of one or possibly two or more plasmids, containing the human VRl receptor DNA encoding one or more fragments or subunits. In the case of oocytes, this involves the co-injection of synthetic RNAs for human VRl receptor protein.
• cellular protein is metabolically labelled with, for example -"S-methionine for 24 hours, after which cell ly sates and cell culture supematants are harvested and subjected to immunoprecipitation with polyclonal antibodies directed against the human VRl receptor protein
• VRl receptor cDNA or oocytes injected with human VRl receptor mRNA Human VRl receptor activity is measured by specific ligand binding and biological characteristics of the host cells expressing human VRl receptor DNA.
• patch voltage clamp techniques can be used to measure receptor activity and quantitate human VRl receptor protein.
• oocytes patch clamp as well as two-electrode voltage clamp techniques can be used to measure VRl receptor activity and quantitate human VRl receptor protein by determining single channel and whole cell conductances.
• Levels of human VRl receptor protein in host cells are quantitated by immunoaffinity and/or ligand affinity techniques.
• Cells expressing human VRl receptor can be assayed for the number of human VRl receptor molecules expressed by measuring the amount of radioactive ligand binding to cell membranes.
• Human VRl receptor-specific affinity beads or human VRl receptor-specific antibodies are used to isolate for example - ⁇ S-methionine labelled or unlabelled human VRl receptor protein.
• Labelled human VRl receptor protein is analyzed by SDS-PAGE.
• Unlabelled human VRl receptor protein is detected by Western blotting, ELISA or RIA assays employing human VRl receptor specific antibodies.
• the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the human VRl receptor sequence but will be capable of hybridizing to human VRl receptor DNA even in the presence of DNA oligonucleotides with mismatches under appropriate conditions. Under alternate conditions, the mismatched DNA oligonucleotides may still hybridize to the human VRl receptor DNA to permit identification and isolation of human VRl receptor encoding DNA.
• DNA encoding human VRl receptor from a particular organism may be used to isolate and purify homologues of human VRl receptor from other organisms.
• the first human VRl receptor DNA may be mixed with a sample containing DNA encoding homologues of human VRl receptor under appropriate hybridization conditions.
• the hybridized DNA complex may be isolated and the DNA encoding the homologous DNA may be purified therefrom.
• this invention is also directed to those DNA sequences that contain alternative codons that code for the eventual translation of the identical amino acid.
• a sequence bearing one or more replaced codons will be defined as a degenerate variation.
• mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
• DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide.
• Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis.
• altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
• a “functional derivative” of human VRl receptor is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of human VRl receptor.
• the term “functional derivatives” is intended to include the “fragments,” “variants,” “degenerate variants,” “analogs” and “homologues” or to “chemical derivatives” of human VRl receptor.
• fragment is meant to refer to any polypeptide subset of human VRl receptor.
• variant is meant to refer to a molecule substantially similar in structure and function to either the entire human VRl receptor molecule or to a fragment thereof.
• a molecule is "substantially similar" to human VRl receptor if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical.
• the term “analog” refers to a molecule substantially similar in function to either the entire human VRl receptor molecule or to a fragment thereof.
• human VRl receptor protein may be recovered to provide human VRl receptor in active form.
• human VRl receptor purification procedures are available and suitable for use. As described above for purification of human VRl receptor from natural sources, recombinant human VRl receptor may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography.
• recombinant human VRl receptor can be separated from other cellular proteins by use of an immunoaffinity column made with monoclonal or polyclonal antibodies specific for full length nascent human VRl receptor, polypeptide fragments of human VRl receptor or human VRl receptor subunits.
• Monospecific antibodies to human VRl receptor are purified from mammalian antisera containing antibodies reactive against human VRl receptor or are prepared as monoclonal antibodies reactive with human VRl receptor using the technique of Kohler and Milstein, Nature 256: 495-497 (1975).
• Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for human VRl receptor.
• Homogenous binding refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the human VRl receptor, as described above.
• Human VRl receptor specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with rabbits being preferred, with an appropriate concentration of human VRl receptor either with or without an immune adjuvant.
• Preimmune serum is collected prior to the first immunization.
• Each animal receives between about 0.1 mg and about 1000 mg of human VRl receptor associated with an acceptable immune adjuvant.
• acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA.
• the initial immunization consists of human VRl receptor in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally (IP) or both.
• SC subcutaneously
• IP intraperitoneally
• Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization.
• Booster injections are given at about three-week intervals until maximal titers are obtained. At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about -20°C.
• Monoclonal antibodies (mAb) reactive with human VRl receptor are prepared by immunizing inbred mice, preferably Balb/c, with human VRl receptor.
• the mice are immunized by the IP or SC route with about 0.1 mg to about 10 mg, preferably about 1 mg, of human VRl receptor in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred.
• the mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 0.1 to about 10 mg of human VRl receptor in a buffer solution such as phosphate buffered saline by the intravenous (IV) route.
• IV intravenous
• Lymphocytes from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art.
• Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions that will allow the formation of stable hybridomas. Fusion partners may include, but are not limited to: mouse myelomas P3/NSl/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being generally preferred.
• the antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. w , at concentrations from about 30% to about 50%.
• Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.
• DMEM Dulbecco's Modified Eagles Medium
• Supernatant fluids are collected from growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using human VRl receptor as the antigen.
• SPIRA solid phase immunoradioassay
• the culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb.
• Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.
• Monoclonal antibodies are produced in vivo by injection of pristane primed Balb/c mice, approximately 0.5 ml per mouse, with about 2 x 10° to about 6 x 10" hybridoma cells about 4 days after priming. Ascites fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
• In vitro production of anti-human VRl receptor mAb is carried out by growing the hybridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb.
• the mAb are purified by techniques known in the art.
• Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of human VRl receptor in body fluids or tissue and cell extracts.
• monospecific antibodies may be utilized to produce antibodies specific for human VRl receptor polypeptide fragments, or full-length nascent human VRl receptor polypeptide, or the individual human VRl receptor subunits. Specifically, it is readily apparent to those skilled in the art that monospecific antibodies may be generated which are specific for only one human VRl receptor subunit or the fully functional receptor.
• Human VRl receptor antibody affinity columns are made by adding the antibodies to Aff ⁇ gel-10 (Bio-Rad), a gel support which is activated with N- hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with IM ethanolamine HCl (pH 8). The column is washed with water followed by 0.23 M glycine HCl (pH 2.6) to remove any non-conjugated antibody or extraneous protein.
• the column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supematants or cell extracts containing human VRl receptor or human VRl receptor subunits are slowly passed through the column.
• the column is then washed with phosphate buffered saline until the optical density (A2g ⁇ ) alls to background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6).
• the purified human VRl receptor protein is then dialyzed against phosphate buffered saline.
• human VRl receptor DNA clones, termed human VRl receptor, are identified which encode proteins that, when expressed in a recombinant host cell, form receptors sensitive to capsaicin.
• the expression of human VRl receptor DNA results in the reconstitution of the properties observed in oocytes injected with human VRl receptor-encoding poly (A) + RNA, including direct activation with the appropriate ligands.
• the present invention is also directed to methods for screening for compounds that modulate the expression of DNA or RNA encoding human VRl receptor as well as the function of human VRl receptor protein in vivo.
• Compounds that modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.
• Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding human VRl receptor, or the function of human VRl receptor protein.
• Compounds that modulate the expression of DNA or RNA encoding human VRl receptor or the function of human VRl receptor protein may be detected by a variety of assays.
• the assay may be a simple "yes/no" assay to determine whether there is a change in expression or function.
• the assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample. Modulators identified in this process are useful as therapeutic agents.
• Kits containing human VRl receptor DNA or RNA, antibodies to human VRl receptor, or human VRl receptor protein may be prepared. Such kits are used to detect DNA that hybridizes to human VRl receptor DNA or to detect the presence of human VRl receptor protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic analyses, diagnostic applications, and epidemiological studies.
• the DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of human VRl receptor DNA, human VRl receptor RNA or human VRl receptor protein.
• the recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of human VRl receptor.
• kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container.
• the carrier would further comprise reagents such as recombinant human VRl receptor protein or anti-human VRl receptor antibodies suitable for detecting human VRl receptor.
• the carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.
• Nucleotide sequences that are complementary to the human VRl receptor encoding DNA sequence can be synthesized for antisense therapy.
• These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-O-alkylRNA, or other human VRl receptor antisense oligonucleotide mimetics.
• Human VRl receptor antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence. Human VRl receptor antisense therapy may be particularly useful for the treatment of diseases where it is beneficial to reduce human VRl receptor activity.
• Human VRl receptor gene therapy may be used to introduce human VRl receptor into the cells of target organisms.
• the human VRl receptor gene can be ligated into viral vectors that mediate transfer of the human VRl receptor DNA by infection of recipient host cells.
• Suitable viral vectors include retrovirus, adenovirus, adeno-associated vims, herpes vims, vaccinia vims, polio vims and the like.
• human VRl receptor DNA can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted DNA transfer using ligand-DNA conjugates or adenovims-ligand-DNA conjugates, lipofection membrane fusion or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo human VRl receptor gene therapy.
• Human VRl receptor gene therapy may be particularly useful for the treatment of diseases where it is beneficial to elevate human VRl receptor activity.
• compositions comprising human VRl receptor DNA, human VRl receptor RNA, or human VRl receptor protein, or modulators of human VRl receptor activity, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, DNA, RNA, or modulator.
• compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders in which modulation of human VRl receptor-related activity is indicated.
• the effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
• the pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
• chemical derivative describes a molecule that contains additional chemical moieties that are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
• Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition of the human VRl receptor or its activity while minimizing any potential toxicity.
• co-administration or sequential administration of other agents may be desirable.
• the present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention.
• compositions containing compounds or modulators identified according to this invention as the active ingredient for use in the modulation of human VRl receptor receptors can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
• the compounds or modulators can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
• ком ⁇ онентs may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
• An effective but non-toxic amount of the compound desired can be employed as a human VRl receptor modulating agent.
• the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per patient, per day.
• the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
• An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day. The range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day.
• the dosages of the human VRl receptor modulators are adjusted when combined to achieve desired effects.
• dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.
• compounds or modulators of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
• compounds or modulators for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
• the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
• the active agents can be administered concurrently, or they each can be administered at separately staggered times.
• the dosage regimen utilizing the compounds or modulators of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed.
• a physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the dmg required to prevent, counter or arrest the progress of the condition.
• Optimal precision in achieving concentrations of dmg within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the dmg's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a dmg.
• the compounds or modulators herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, sy ps and the like, and consistent with conventional pharmaceutical practices.
• carrier suitable pharmaceutical diluents, excipients or carriers
• the active dmg component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
• suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
• suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
• Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
• Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
• the active dmg component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
• suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
• Other dispersing agents include glycerin and the like.
• sterile suspensions and solutions are desired.
• Isotonic preparations which generally contain suitable preservatives, are employed when intravenous administration is desired.
• Topical preparations containing the active dmg component can be admixed with a variety of carrier materials well known in the art, such as, eg., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, eg., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
• carrier materials well known in the art, such as, eg., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, eg., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
• the compounds or modulators of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
• Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines .
• Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
• the compounds or modulators of the present invention may also be coupled with soluble polymers as targetable dmg carriers.
• soluble polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues.
• the compounds or modulators of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a dmg, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
• a class of biodegradable polymers useful in achieving controlled release of a dmg, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
• the compounds or modulators may be administered in capsule, tablet, or bolus form or alternatively they can be mixed in the animals feed.
• the capsules, tablets, and boluses are comprised of the active ingredient in combination with an appropriate carrier vehicle such as starch, talc, magnesium stearate, or di-calcium phosphate.
• suitable carrier vehicle such as starch, talc, magnesium stearate, or di-calcium phosphate.
• These unit dosage forms are prepared by intimately mixing the active ingredient with suitable finely-powdered inert ingredients including diluents, fillers, disintegrating agents, and/or binders such that a uniform mixture is obtained.
• An inert ingredient is one that will not react with the compounds or modulators and which is non-toxic to the animal being treated.
• Suitable inert ingredients include starch, lactose, talc, magnesium stearate, vegetable gums and oils, and the like. These formulations may contain a widely variable amount of the active and inactive ingredients depending on numerous factors such as the size and type of the animal species to be treated and the type and severity of the infection.
• the active ingredient may also be administered as an additive to the feed by simply mixing the compound with the feedstuff or by applying the compound to the surface of the feed. Alternatively the active ingredient may be mixed with an inert carrier and the resulting composition may then either be mixed with the feed or fed directly to the animal.
• Suitable inert carriers include com meal, citrus meal, fermentation residues, soya grits, dried grains and the like. The active ingredients are intimately mixed with these inert carriers by grinding, stirring, milling, or tumbling such that the final composition contains from 0.001 to 5% by weight of the active ingredient.
• the compounds or modulators may alternatively be administered parenterally via injection of a formulation consisting of the active ingredient dissolved in an inert liquid carrier. Injection may be either intramuscular, intraluminal, intratracheal, or subcutaneous.
• the injectable formulation consists of the active ingredient mixed with an appropriate inert liquid carrier.
• Acceptable liquid carriers include the vegetable oils such as peanut oil, cotton seed oil, sesame oil and the like as well as organic solvents such as solketal, glycerol formal and the like.
• aqueous parenteral formulations may also be used.
• the vegetable oils are the preferred liquid carriers.
• the formulations are prepared by dissolving or suspending the active ingredient in the liquid carrier such that the final formulation contains from 0.005 to 10% by weight of the active ingredient.
• Topical application of the compounds or modulators is possible through the use of a liquid drench or a shampoo containing the instant compounds or modulators as an aqueous solution or suspension.
• These formulations generally contain a suspending agent such as bentonite and normally will also contain an antifoaming agent.
• Formulations containing from 0.005 to 10% by weight of the active ingredient are acceptable.
• Preferred formulations are those containing from 0.01 to 5% by weight of the instant compounds or modulators.
• the following examples illustrate the present invention without, however, limiting the same thereto.
• First strand synthesis Approximately 5 ⁇ g of human thalamus mRNA (Clontech) was used to synthesize cDNA using the cDNA synthesis kit (Life Technologies). Two microliters of Not 1 primer adapter was added to 5 ⁇ l of mRNA and the mixture was heated to 70 ° C for 10 minutes and placed on ice. The following reagents were added on ice: 4 ⁇ l of 5x first strand buffer (250mM TRIS-HC1 (pH8.3), 375mM KC1, 15mM MgCl 2 ), 2 ⁇ l of 0.1 M DTT, lOmM dNTP (nucleotide triphosphates) mix and l ⁇ l of DEPC treated water. The reaction was incubated at 42 °C for 5minutes. Finally, 5 ⁇ l of Superscript RT II was added and incubated at 42 °C for 2 more hours. The reaction was terminated on ice.
• 5x first strand buffer 250mM TRIS-HC1 (p
• Second strand synthesis The first strand product was adjusted to 93 ⁇ l with water and the following reagents were added on ice: 30 ⁇ l of 5x 2nd strand buffer (100 mM TRIS-HC1 (pH6.9),450 mM KC1, 23 mM MgCl 2 , 0.75 mM ⁇ -NAD+, 50mM (NH 4 ) 2 SO 4 ), 3 ⁇ l of 10 mM dNTP (nucleotide triphosphates), l ⁇ l R coli DNA ligase (lOunits )l ⁇ l RNase H (2units), 4 ⁇ l DNA pol I (10 units). The reaction was incubated at 16°C for 2 hours.
• 5x 2nd strand buffer 100 mM TRIS-HC1 (pH6.9),450 mM KC1, 23 mM MgCl 2 , 0.75 mM ⁇ -NAD+, 50mM (NH 4 ) 2 SO 4
• the DNA from second strand synthesis was treated with T4 DNA polymerase and placed at 16°C to blunt the DNA ends.
• the double stranded cDNA was extracted with 150 ⁇ l of a mixture of phenol and chloroform (1:1, v:v) and precipitated with 0.5 volumes of 7.5 M NH4OAc and 2 volumes of absolute ethanol.
• the pellet was washed with 70% ethanol and dried down at 37°C to remove the residual ethanol.
• the double stranded DNA pellet was resuspended in 25 ⁇ l of water and the following reagents were added; 10 ⁇ l of 5x T4 DNA ligase buffer, 10 ⁇ l of Sail adapters and 5 ⁇ l of T4 DNA ligase.
• the ingredients were mixed gently and ligated overnight at 16° C.
• the ligation mix was extracted with phenol:chloroform:isoamyl alcohol, vortexed thoroughly and centrifuged at room temperature for 5 minutes at 14,000 x g to separate the phases.
• the aqueous phase was transferred to a new tube and the volume adjusted to 100 ml with water.
• the purified DNA was size selected on a chromaspin 1000 column (Clontech) to eliminate the smaller cDNA molecules.
• the double stranded DNA was digested with Notl restriction enzyme for 3-4 hours at 37° C.
• the restriction digest was electrophoresed on a 0.8 % low melt agarose gel.
• the cDNA in the range of 1-5 kb was cut out and purified using Gelzyme (Invitrogen). The product was extracted with phenol hloroform and precipitated with NH 4 OAc and absolute ethanol. The pellet was washed with 70% ethanol and resuspended in 10 ml of water.
• Ligation of cDNA to the Vector The cDNA was split up into 5 tubes (2 ⁇ l each) and the ligation reactions were set up by adding 4.5 ⁇ l of water, 2 ⁇ l of 5x ligation buffer, l ⁇ l of p-Sport vector DNA (cut with Sal-1 / Notl and phosphatase treated) and 0.5 ⁇ l of T4 DNA ligase. The ligation was incubated at 40° C overnight.
• the ligation reaction volume was adjusted to a total volume of 20 ⁇ l with water.
• the mixture was vortexed thoroughly, and immediately centrifuged at room temperature for 20 minutes at 14000 xg. The pellets were washed in 70% ethanol and each pellet was resuspended in 5 ml of water.
• EXAMPLE 2 Library Screening / human VRl Generation Human thalamus library screening:
• Electromax DH10B cells (Life Technologies). The volume was adjusted to 1 ml with SOC media and incubated for 45 minutes at 37°C with shaking. The library was then plated out on 150cm 2 plates containing LB to a density of 20000 colonies per plate. These cultures were grown overnight at 37°C.
• a human VRl receptor probe was generated by polymerase chain reaction using the following primer pair:
• the probe was generated by PCR using regular PCR conditions using 5' and 3' probe oligos (lOOng each) and 10 ng of diluted miniprep DNA.
• the resulting 274 bp fragment was mn on 1% agarose gel and purified using a QUIAquick Gel extraction kit (Quiagen).
• About 100 ng of the purified probe was labeled with alpha 32P using oligolabeling kit from Pharmacia and the labeled DNA was purified with S-200 columns (Pharmacia).
• the library colonies were lifted on Protran nitrocellulose filters (Scheicher & Schuel) and the DNA was denatured in 1.5 M NaCl, 0.5 M NaOH.
• the filter disks were neutralized with 1.5 M NaCl, 1.0 M Tris-HCl, pH 7.5 and then UV crosslinked to the membrane using a UV-Stratalinker (Stratagene).
• the filters were washed several times in wash solution (1 M Tris-HCl, pH 8.0; 5 M NaCl; 0.5 M EDTA; 20% SDS) at 42°C.
• the disks were washed twice in 2xSSC, 0.2% SDS at room temperature (20 min each) and once in 0.2xSSC, 0.1%SDS at 50C for 30 minutes.
• the membranes were than placed on sheets of filter paper, wrapped in the Saran Wrap and exposed to the film at - 20C overnight.
• the full length clone was generated by PCR with Pfu polymerase using 10 ng of the sequenced library clone as a template and full length oligos with EcoRI (FL 5 'oligo SEQ.ID.NO.3) and Notl (FL 3' oligo SEQ.ID.NO.4) sites.
• FL 5' oligo (SEQ.ID.NO.3): 5 ' AACGTTGAATTCGCC ACC ATGAAGAAATGG AGC AGC AC AGACTTGG
• the PCR product was digested with EcoRI and Notl enzymes and cloned into a pGem HE expression vector. Large-scale preparation of DNA was done using a MEGA prep kit (Quiagen).
• the human VRl receptor cDNAs (collectively referred to as hVRl) were cloned into the mammalian expression vector pcDNA3.1/Zeo(+).
• the cloned PCR product was purified on a column (Wizard PCR DNA purification kit from Promega) and digested with Not I and EcoRI (NEB) to create cohesive ends.
• the product was purified by a low melting agarose gel electrophoresis.
• the pcDNA3.1/Zeo(+) vector was digested with EcoRI and Notl enzymes and subsequently purified on a low melt agarose gel.
• the linear vector was used to ligate to the human VRl cDNA inserts.
• Recombinants were isolated, designated human VRl receptor, and used to transfect mammalian cells (HEK293, COS-7 or CHO-Kl cells) using the Effectene non- liposomal lipid based transfection kit (Quiagen). Stable cell clones were selected by growth in the presence of zeocin. Single zeocin resistant clones were isolated and shown to contain the intact human VRl receptor gene. Clones containing the human VRl receptor cDNAs were analyzed for hVRl protein expression. Recombinant plasmids containing human VRl encoding DNA were used to transform the mammalian COS or CHO cells or HEK293 cells.
• Cassettes containing the human VRl receptor cDNA in the positive orientation with respect to the promoter are ligated into appropriate restriction sites 3' of the promoter and identified by restriction site mapping and/or sequencing.
• These cDNA expression vectors are introduced into fibroblastic host cells for example COS-7 (ATCC# CRL1651), and CV-1 tat [Sackevitz et al., Science 238: 1575 (1987)], 293, L (ATCC# CRL6362)] by standard methods including but not limited to electroporation, or chemical procedures (cationic liposomes, DEAE dextran, calcium phosphate).
• Transfected cells and cell culture supematants are harvested and analyzed for human VRl receptor expression as described herein.
• All of the vectors used for mammalian transient expression can be used to establish stable cell lines expressing human VRl receptor.
• Unaltered human VRl receptor cDNA constructs cloned into expression vectors are expected to program host cells to make human VRl receptor protein.
• the transfection host cells include, but are not limited to, CV-l-P [Sackevitz ej al, Science 238: 1575 (1987)], tk-L [Wigler, et al. Cell 11: 223 (1977)], NS/0, and dHFr- CHO [Kaufman and Sharp, J. Mol. Biol. 159: 601, (1982)].
• Human VRl receptor cDNA constructs are also ligated into vectors containing amplifiable drug-resistance markers for the production of mammalian cell clones synthesizing the highest possible levels of human VRl receptor. Following introduction of these constmcts into cells, clones containing the plasmid are selected with the appropriate agent, and isolation of an over-expressing clone with a high copy number of plasmids is accomplished by selection in increasing doses of the agent.
• the expression of recombinant human VRl receptor is achieved by transfection of full-length human VRl receptor cDNA into a mammalian host cell.
• Xenopus laevis oocytes Characterization of functional protein encoded by pVRlR in Xenopus oocytes Xenopus laevis oocytes were prepared and injected using standard methods previously described and known in the art (Fraser et al., 1993). Ovarian lobes from adult female Xenopus laevis (Nasco, Fort Atkinson, WI) were teased apart, rinsed several times in nominally Ca-free saline containing: 82.5mM NaCl, 2.5mM KC1, ImM MgCl 2 , 5 mM HEPES, adjusted to pH 7.0 with NaOH (OR-2), and gently shaken in OR-2 containing 0.2% collagenase Type 1 (ICN Biomedicals, Aurora, Ohio) for 2-5 hours.
• Stage V and VI oocytes were selected and rinsed in media consisting of 75% OR-2 and 25% ND-96.
• the ND-96 contained: 100 mM NaCl, 2 mM KC1, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES, 2.5 mM Na pymvate, gentamicin (50 ug/ml), adjusted to pH 7.0 with NaOH.
• the extracellular Ca +2 was gradually increased and the cells were maintained in ND-96 for 2- 24 hours before injection.
• pGEM HE Liman et al., 1992
• human VRl was linearized with Nhel and transcribed with T7 RNA polymerase (Promega) in the presence of the cap analog m7G(5')ppp(5')G.
• the synthesized cRNA was precipitated with ammonium acetate and isopropanol, and resuspended in 50 ⁇ l nuclease-free water.
• cRNA was quantified using formaldehyde gels (1% agarose, lxMOPS , 3% formaldehyde) against 1,2 and 5 ⁇ l RNA markers (Gibco BRL, 0.24 - 9.5 Kb).
• Oocytes were injected with 50 nl of the human VRl receptor RNA (0.025-2.5 ng). Control oocytes were injected with 50 nl of water. Oocytes were incubated for 1-10 days in ND-96 before analysis for expression of the human VRl . Incubations and collagenase digestion were carried out at room temperature. Injected oocytes were maintained in 48 well cell culture clusters (Costar; Cambridge, MA) at 18°C. Whole cell agonist-induced currents were measured 1-14 days after injection with a conventional two-electrode voltage clamp (GeneClamp500, Axon Instruments, Foster City, CA) using standard methods previously described and known in the art (Dascal, 1987).
• GeneClamp500 GeneClamp500, Axon Instruments, Foster City, CA
• microelectrodes were filled with 3 M KC1, which had resistances of 1 and 2 M ⁇ .
• Cells were continuously perfused with ND96 at 10 ml/min at room temperature unless indicated.
• Membrane voltage was clamped at -60 mV unless indicated.
• Capsaicin (C; 0.1- 10 ⁇ M) elicited inward currents in oocytes that had been injected with RNA transcribed from the cloned hVRl receptor cDNA as shown in FIGURE 4, a,b,c.
• the inward current (at -60 mV) through hVRl receptors elicited by 1 ⁇ M capsaicin exposure was slowly rising and usually did not show appreciable desensitization over the time studied in BaSOS (FIGURE 4a,b).
• capsaicin-induced response depended on the amount of cRNA injected.
• CPZ produced a dose-dependent block of the capsaicin response as shown in Figure 6.
• Oocytes were challenged with capsaicin (0.6 ⁇ M) and 4 minutes later, preincubated with the indicated concentration of capsazepine (CPZ; Figure 6).
• CPZ dose-dependently blocked a subsequent response to 0.6 ⁇ M capsaicin (in the continued presence of CPZ).
• the IC50 for CPZ was about 100 nM.
• the human VRl expressed from the human VRl -encoding DNA molecule described herein was activated by low pH solutions (ND96, pH 5.5; FIGURE 7).
• the low pH activated current activated with a similar time course as that elicited by capasaicin in the same cell and it was blocked by CPZ (1 ⁇ M; FIGURE 7b).
• Human HEK293 cells were transfected with either human VRl receptor pVRlR or the vector pcDNA3.1/zeo(+).
• Transient fransfections 1 ⁇ g of pVRlR or vector per 10 6 cells per 100 mm dish were performed using the Effectene tranfection kit (Quiagen; 301425).
• Three days after transfection cells were plated onto 96-well plates (Biocoat, poly-D-lysine coated black clear plate; Becton Dickinson part # 354640). After one day, wells were rinsed with F12/DMEM, then incubated in Fluo-4 (2 ⁇ M) with Pluronic acid (20%, 40 ⁇ l used in 20 mis total volume) for 1 hour at room temperature.
• Plates were assayed using the FLIPR (Molecular Devices, FL-101). Cells were challenged with agonists (at 3-fold concentration in 40 ⁇ l added to 80 ⁇ l at a velocity of 50 ⁇ l sec). Transfections with vector alone were tested as controls. Antagonists were included for ⁇ 1-2 minutues, as indicated, prior to the addition of agonist in the presence of antagonist.
• the cells were selected in the presence of zeocin (200 ⁇ g/ml) and grown through three 1:10 dilution passages for approximately two weeks. Individual colonies were picked and grown in 6-well dishes. Cells were then plated onto 96-well plates (Biocoat, poly-D-lysine coated black/clear plate; Becton Dickinson part # 354640). Plates were assayed using the FLIPR (Molecular Devices, FL-101) as described above.
• FLIPR Molecular Devices, FL-101
• HEK293 cells stably expressing hVRl were constmcted and tested for their responsiveness to capsaicin (15 nM) and their sensitivity to mthenium red (FIGURE 9A- B) and CPZ (FIGURE 9C).
• Agonist induced increases in intracellular Ca2+ were observed as an increase in fluorescence intensity measured on the FLIPR and are shown in A.
• the concentration of mthenium red present for 2 min preincubation (as well as together with the stimulus) was increased from 0.1 ⁇ M (second column) to 17 ⁇ M (11 th column).
• the concentrations were 0, 0.1, 0.3, 0.45, 0.7, 1, 1.7, 3, 6, 10, 17 and 0 ⁇ M in columns 1 to 12, respectively.
• the whole cell patch clamp technique (Hamill et al., 1981) was used to record ligand-induced currents from HEK293 stably expressing human VRl receptor maintained for >2 days on 12 mm coverslips. Cells were visualized using a Nikon Diaphot 300 with DIC Nomarski optics. Cells are continuously perfused in a physiological saline ( ⁇ 0.5 ml/min) unless otherwise indicated.
• the standard physiological saline contained: 130 mM NaCl, 4 mM KC1, 1 mM CaCl2, 1.2mM MgCl2, and lOmM hemi-Na- HEPES (pH 7.3, 295-300 mOsm as measured using a Wescor 5500 vapor-pressure (Wescor, Inc., Logan, UT).
• Recording electrodes were fabricated from borosilicate capillary tubing (R6; Gamer Glass, Claremont, CA), the tips were coated with dental periphery wax (Miles Laboratories, South Bend, IN), and had resistances of 1-2 M ⁇ when containing intracellular saline: 100 mM K-gluconate, 25 mM KC1, 0.483 mM CaCl2, 3 mM MgCl2, 10 mM hemi-Na-HEPES and 1 mM K4-BAPTA (lOOnM free Ca +2 ); pH 7.4, with dextrose added to achieve 290 mOsm).
• Liquid junction potentials were -18 mV using standard pipette and bath solutions as determined both empirically and using the computer program JPCalc ((Barry, 1994)). The voltage data shown were not corrected for liquid junction potential (ie., a voltage presented as +10 would be -8 mV after correcting for the junction potential).
• Current and voltage signals were detected and filtered at 2 kHz with an Axopatch ID patch-clamp amplifier (Axon Instmments, Foster City, CA), digitally recorded with a DigiData 1200B laboratory interface (Axon Instmments), and PC compatible computer system and stored on magnetic disk for off-line analysis. Data acquisition and analysis were performed with PClamp software.
• the total membrane capacitance (Cm) was determined as the difference between the maximum current after a 30 mV hyperpolarizing voltage ramp from -68 mV generated at a rate of 10 mV/ms and the steady state current at the final potential (-98 mV) (Dubin et al., 1999).
• V re v Apparent reversal potentials (V re v) of ligand-induced conductance changes were determined using a voltage-ramp protocol (Dubin et al., 1999). Voltage ramps were applied every 1 second and the resulting whole cell ramp-induced currents were recorded. Usually the voltage was ramped from negative to positive to negative values. The current required to clamp the cells at -68 mV was continuously monitored. Ligand-induced conductances were determined from whole-cell currents elicited by a voltage-ramp protocol in the presence and absence of ligand. Voltage ramp-induced currents measured before (control) and in the presence of ligand were compared to reveal the effect of the ligand on the channel to modulate the channel current output. The voltage at which there was no net ligand-induced current was determined (V r ev)-
• Capsaicin caused an increase in conductance in HEK293 cells stably expressing hVRl, consitent with its activation of a flux of non-selective cations.
• Vrev was +13 mV. This value (taking in to account the junction potential, Vrev is -8.4 mV) is similar to that obtained in two electrode voltage clamp studies on oocytes.
• a high level of expression of VRl mediated currents was attained: capsaicin-induced currents measured at +60 mV were 1630 +/- 530 pA.
• the current density was determined by normalizing to the cell capacitance and was 80 +/- 40 pA/pF.
• the response to 100 nM capsaicin was completely blocked by 2 min preincubation in CPZ (1 ⁇ M) (FIGURE 10b). Shown are voltage ramp-induced currents elicited by a steadily depolarizing command potential from -100 to +100 mV over 200 msec. 1 ⁇ M CPZ blocked the response to capsaicin in 3 cells. Partial recovery from CPZ block was observed.
• the potent agonist RTX (160 nM) produced an increase in conductance similar to that observed with capsaicin (FIGURE 11).
• the reversal potential was +10 mV.
• the current density was 26 pA/pF.
• HEK293 cells stably expressing hVRl receptor can be used in -1H-RTX binding assays.
• Equilibrium ligand binding assays can be performed using conventional procedures. Specific -1H-[RTX] binding is observed in membrane preparations from VRl receptor transfected cells.
• Oocytes injected with hVRl in vitro RNA are used in ⁇ H-[RTX] binding assays.
• hVRl RNA (2.5 ng) is injected into oocytes individually and 2 days later the oocytes are disrupted with a dounce homogenizer and yolk proteins removed using standard methods known in the art. Equilibrium ligand binding assays are performed using conventional procedures.
• Oocytes expressing hVRl should bind -1H-[RTX] with high affinity, approximately 1 nM. Specific -1H-[RTX] binding is observed in membrane preparations from hVRl -injected oocytes.
• Oocytes expressing hVRl are used to measure the affinity of binding of test compounds by their ability to modulate 3 H-[RTX] binding.
• the nucleotide sequences of human VRl receptor revealed single large open reading frame of about 2520 human VRl receptor base pairs encoding 839 amino acids.
• the cDNAs have 5' and 3 '-untranslated extensions of about 921 and about 1383 nucleotides for human VRl receptor.
• the first in-frame methionine was designated as the initiation codon for an open reading frame that predicts a human VRl receptor protein with an estimated molecular mass (M r ) of about 95,048 kDa.
• M r estimated molecular mass
• Recombinant human VRl receptor is produced in IL coli following the transfer of the human VRl receptor expression cassette into IL coli expression vectors, including but not limited to, the pET series (Novagen).
• the pET vectors place human VRl receptor expression under control of the tightly regulated bacteriophage T7 promoter.
• expression of human VRl receptor is induced when an appropriate lac substrate (IPTG) is added to the culture.
• IPTG lac substrate
• Baculovims vectors which are derived from the genome of the AcNPV vims, are designed to provide high level expression of cDNA in the Sf9 line of insect cells (ATCC CRL# 1711).
• Recombinant baculoviruses expressing human VRl receptor cDNA is produced by the following standard methods (InVitrogen Maxbac Manual): the human VRl receptor cDNA constmcts are ligated into the polyhedrin gene in a variety of baculovims transfer vectors, including the pAC360 and the BlueBac vector (InVitrogen).
• Recombinant baculo vimses are generated by homologous recombination following co-transfection of the baculovims transfer vector and linearized AcNPV genomic DNA [Kitts, P.A., Nuc. Acid. Res. 18: 5667 (1990)] into Sf9 cells.
• Recombinant pAC360 vimses are identified by the absence of inclusion bodies in infected cells and recombinant pBlueBac vimses are identified on the basis of ⁇ -galactosidase expression (Summers, M. D. and Smith, G. E., Texas Agriculture Exp. Station Bulletin No. 1555). Following plaque purification, human VRl receptor expression is measured by the assays described herein.
• the cDNA encoding the entire open reading frame for human VRl receptor is inserted into the BamHI site of pBlueBacII. Constmcts in the positive orientation are identified by sequence analysis and used to transfect Sf9 cells in the presence of linear AcNPV mild type DNA.
• Active human VRl receptor is found in the cytoplasm of infected cells. Active human VRl receptor is extracted from infected cells by hypotonic or detergent lysis.
• Recombinant human VRl receptor is produced in the yeast S. cerevisiae following insertion of the optimal human VRl receptor cDNA cistron into expression vectors designed to direct the intracellular or extracellular expression of heterologous proteins.
• vectors such as EmBLyex4 or the like are ligated to the human VRl receptor cistron [Rinas, U. si al-, Biotechnology 8: 543-545 (1990); Horowitz B. et al., J. Biol. Chem. 265: 4189-4192 (1989)].
• the human VRl receptor cistron is ligated into yeast expression vectors which fuse a secretion signal (a yeast or mammalian peptide) to the NH terminus of the human VRl receptor protein [Jacobson, M. A., Gene 85: 511-516 (1989); Riett L. and Bellon N. Biochem. 28: 2941-2949 (1989)].
• a secretion signal a yeast or mammalian peptide
• vectors include, but are not limited to pAVEl>6, which fuses the human semm albumin signal to the expressed cDNA [Steep O. Biotechnology 8: 42- 46 (1990)], and the vector pL8PL which fuses the human lysozyme signal to the expressed cDNA [Yamamoto, Y., Biochem. 28: 2728-2732)].
• human VRl receptor is expressed in yeast as a fusion protein conjugated to ubiquitin utilizing the vector pVEP [Ecker, D. J., J. Biol. Chem. 264: 7715-7719 (1989), Sabin, E. A., Biotechnology 7: 705-709 (1989), McDonnell D. P., Mol. Cell Biol. 9: 5517- 5523 (1989)].
• the levels of expressed human VRl receptor are determined by the assays described herein.
• Recombinantly produced human VRl receptor may be purified by antibody affinity chromatography.
• Human VRl receptor antibody affinity columns are made by adding the anti- human VRl receptor antibodies to Affigel-10 (Bio-Rad), a gel support that is pre- activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with IM ethanolamine HCl (pH 8). The column is washed with water followed by 0.23 M glycine HCl (pH 2.6) to remove any non-conjugated antibody or extraneous protein.
• the column is then equilibrated in phosphate buffered saline (pH 7.3) together with appropriate membrane solubilizing agents such as detergents and the cell culture supernatant or cell extract containing solubilized human VRl receptor is slowly passed through the column.
• the column is then washed with phosphate- buffered saline together with detergents until the optical density (A280) falls to background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6) together with detergents.
• the purified human VRl receptor protein is then dialyzed against phosphate buffered saline.
• JPCalc a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements. J. Neurosci Methods 51, 107-116.
• Capsazepine a competitive antagonist of the sensory neuron excitant capsaicin. Br. J. Pharmacol. 107, 544-52.
• the capsaicin receptor a heat-activated ion channel in the pain pathway. Nature fLondon. 389, 816-824.
• Capsaicin activates a nonselective cation channel in cultured neonatal rat dorsal root ganglion neurons. J. Neurosci. 16, 1659- 67.
• Vanilloid receptors new insights enhance potential as a therapeutic target. Pain 68, 195-208.
• Szallasi A., Goso, C, Blumberg, P. M., and Manzini, S. (1993).
• Vanilloid (capsaicin) receptors in the rat distribution in the brain, regional differences in the spinal cord, axonal transport to the periphery, and depletion by systemic vanilloid treatment. Brain Res. 703, 175-83.
• Tyr Asp Arg Arg Ser lie Phe Glu Ala Val Ala Gin Asn Asn Cys Gin 115 120 125
• Leu Leu Glu lie Ala Arg Gin Thr Asp Ser Leu Lys Glu Leu Val Asn 180 185 190
• Ala lie Glu Arg Arg Asn Met Ala Leu Val Thr Leu Leu Val Glu Asn 210 215 220 Gly Ala Asp Val Gin Ala Ala Ala His Gly Asp Phe Phe Lys Lys Thr 225 230 235 240
• Phe Thr lie Gly Met Gly Asp Leu Glu Phe Thr Glu Asn Tyr Asp Phe 645 650 655
• Lys Ala Val Phe lie lie Leu Leu Leu Ala Tyr Val lie Leu Thr Tyr 660 665 670
• Lys lie Ala Gin Glu Ser Lys Asn lie Trp Lys Leu Gin Arg Ala lie 690 695 700

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Neurology (AREA)
• Biomedical Technology (AREA)
• Pulmonology (AREA)
• Neurosurgery (AREA)
• Diabetes (AREA)
• Immunology (AREA)
• Pain & Pain Management (AREA)
• Rheumatology (AREA)
• Gastroenterology & Hepatology (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Hospice & Palliative Care (AREA)
• Emergency Medicine (AREA)
• Dermatology (AREA)
• Hematology (AREA)
• Cell Biology (AREA)
• Obesity (AREA)
• Toxicology (AREA)
• Biophysics (AREA)
• Endocrinology (AREA)
• Biochemistry (AREA)
• Zoology (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Psychiatry (AREA)

Applications Claiming Priority (3)

Publications (2)

Family

ID=23125812

Family Applications (1)

Country Status (5)

Families Citing this family (11)

Citations (3)

• 2000
  • 2000-04-13 CA CA002370570A patent/CA2370570A1/en not_active Abandoned
  • 2000-04-13 EP EP00922143A patent/EP1175504A4/de not_active Withdrawn
  • 2000-04-13 AU AU42374/00A patent/AU4237400A/en not_active Abandoned
  • 2000-04-13 WO PCT/US2000/009897 patent/WO2000063415A1/en not_active Application Discontinuation
  • 2000-04-13 JP JP2000612492A patent/JP2003520022A/ja active Pending

Patent Citations (3)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events