WO2002009736A1 - Procedes et agents servant a traiter une douleur persistante - Google Patents

Procedes et agents servant a traiter une douleur persistante Download PDF

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WO2002009736A1
WO2002009736A1 PCT/US2001/041447 US0141447W WO0209736A1 WO 2002009736 A1 WO2002009736 A1 WO 2002009736A1 US 0141447 W US0141447 W US 0141447W WO 0209736 A1 WO0209736 A1 WO 0209736A1
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nr2b
forebrain
animal
persistent pain
nmda
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Min Zhuo
Joe TSIEN
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Washington University
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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0368—Animal model for inflammation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the field of neurobiology.
  • the invention provides assay methods for identification of substances useful for the treatment of persistent pain, and methods for modulating persistent pain, which relate to down-regulation of NMD A receptor activity in the forebrain.
  • NMDA N-methyl-D-aspartate receptors mediate a slow, voltage- dependent synaptic current at glutamatergic synapses throughout the central nervous system (C ⁇ S).
  • C ⁇ S central nervous system
  • Functional ⁇ MDA receptors contain heteromeric combinations of the ⁇ R1 subunit plus one or more of ⁇ 2A-D, of which the NR2A and NR2B subunits are the major NR2 subtypes found in forebrain structures.
  • NMDA receptors formed by co-expression NR1 and NR2A in heterologous cells mediate currents that decay three to four times faster.
  • Forebrain NMDA receptors are composed almost exclusively of NR1 and NR2B subunits at birth, gradually incorporating more NR2A subunits during postnatal development.
  • EPCs excitatory postsynaptic currents
  • NR2B subunit expression was observed extensively throughout the cerebral cortex, striatum, amygdala, and hippocampus, but not in the thalamus, brainstem, cerebellum or spinal cord (Tang et al., 1999, supra).
  • hippocampal synapses Presumably by increasing the NR2B-.NR2A ratio in NMDA heteromeric complexes, this manipulation led to marked alterations in the physiology of hippocampal synapses (Tang et al., 1999, supra). First, in mature, cultured hippocampal neurons, NMDA receptors mediated more slowly decaying currents and four-fold greater charge transfer in transgenic versus wild-type cells. Second, in hippocampal slices prepared from adult mice, hippocampal CAl synapses exhibited increased susceptibility to long-term potentiation in NR2B transgenic animals.
  • NMDA receptor is also involved in the perception of pain in mammals. Pain can occur following acute or chronic injury to the peripheral or central nervous system, arising from a variety of causes that include traumatic injury or inflammation. Persistent pain can result from either of these causes, and is often more difficult to manage than acute pain, using conventional analgesics.
  • keta ine, dextromethorphan and CPP (3-(2- carboxypiperizin-4-yl)-propyl-l-phosphonic acid) have been reported to produce relief in several neuropathies, including postherpetic neuralgia, central pain caused by spinal cord injury, and phantom limb pain (Kristensen et al., Pain 51: 249-253, 1992; Eide et al., Pain 61: 221-228, 1995; Knox et al., Anaesth. Intensive Care 23:620-622, 1995; Max et al., Clin. Neuropharmacol. 18: 360-368 1995).
  • CP-101,606 has potent analgesic activity in rat hyperalgesia and nociceptive tests at doses that do not cause behavioral abnormality (Taniguchi et al., Br. J. Pharmacol. 122: 809- 812, 1997). It was shown through binding of radiolabeled drug that CP-101,606 was most dense in regions of the central nervous system in which NR2B subunits are selectively expressed (i.,e., the hippocampus). However, the drug was found to be distributed throughout the brain and spinal cord, however, so it was not discerned whether the drug's specific effect on NR2B in the spinal cord or the brain contributed more significantly to the observed analgesic effect.
  • NR2B subunits in addition to their distribution in the brain, are distributed throughout the rat lumbar spinal cord, mainly in fibers in laminae I and JJ of the dorsal horn (Boyce et al., ⁇ 999, supra).
  • NR2B-targeted antagonists appear to be attractive candidates for the treatment and management of pain.
  • the specific mechanisms by which NR2B-specific agonists act in vivo to exert an analgesic effect is not clear; hence a rational basis for testing NR2B-targeted compounds for their antinociceptive effect is presently unavailable. It would be an advance in the art to identify in vivo the means by which NR2B-specific antagonists act as antinociceptive agents, and to utilize this information for the management of pain.
  • the present invention provides assay methods for identification of substances useful for the treatment of persistent pain, and methods for modulating persistent pain, which relate to down-regulation of NMDA receptor activity in the forebrain.
  • a method of reducing or eliminating persistent pain in a patient in need of such treatment comprises modifying NMDA receptors in neural synapses of the patient's forebrain to decrease channel decay time in the NMDA receptors, the modification resulting in reduction or elimination of the persistent pain in the patient.
  • a genetically altered non- human animal which displays increased sensitivity to persistent pain as compared with an equivalent, but unaltered animal.
  • the animal expresses a gene encoding NR2B to a greater extent in its forebrain than does the equivalent, but unaltered animal.
  • a method for identifying compounds that inhibit long-term reaction to a persistent pain stimulus in a subject by decreasing expression of NR2B genes in the forebrain of the subject.
  • This method comprises providing a chimeric DNA construct comprising an NR2B promoter operably linked to a reporter gene, contacting the chimeric DNA construct with a test compound suspected of down-regulating the NR2B promoter, and measuring expression of the reporter gene, a decrease in the expression being indicative that the test compound inhibits long-term reaction to a persistent pain stimulus in the subject.
  • an in vivo assay for identifying compounds that inhibit long-term reaction to a persistent pain stimulus by down-regulating NMDA receptors in the forebrain of a subject comprises: (a) providing a non-human transgenic animal that expresses an NR2B transgene in its forebrain; (b) treating the transgenic animal with a test compound suspected of down-regulating NMDA receptor function; and (c) directly or indirectly measuring NMDA function in the forebrain of the treated animal as compared with an equivalent untreated animal, a decrease in NMDA in the forebrain of the treated animal being indicative that the test compound inhibits long-term reaction to persistent pain by down-regulating NMDA receptor function in the forebrain of the subject.
  • FIG. 1 A Pharmacological inhibition of forebrain NMDA receptors.
  • FIG. 2 A Diagram of a frontal cortical slice taken from an adult mouse, showing the placement of recording and stimulating electrodes in the ACC and insular cortex.
  • FIG. 2B Traces of NMDA receptor-mediated fEPSPs recorded from the ACC and insular cortex in the presence of 20 ⁇ M CNQX (see panel C for scale bars).
  • FIG. 2C Bath application of 100 ⁇ M AP-5 completely (upper traces) and reversibly (lower trace) blocked NMDA receptor-mediated fEPSPs in the ACC.
  • FIG. 2F Traces of EPSPs recorded in the presence of 20 ⁇ M CNQX, after bath application of 100 ⁇ M AP-5 (upper traces) and after washout (lower trace).
  • ACC anterior cingulate cortex
  • SI SI and S2 regions of somatosensory cortex
  • I 2 regions of somatosensory cortex
  • CAl subfield of the hippocampus the periaqueductal gray
  • SCDH spinal cord laminae I- VI
  • FIG. 5B Summarized results from (Fig. 5A). * indicates significant difference from wild-type mice.
  • a “coding sequence” or “coding region” refers to a nucleic acid molecule having sequence information necessary to produce a gene product, when the sequence is expressed.
  • the term “operably linked” or “operably inserted” means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement other transcription control elements (e.g. enhancers) in an expression vector.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • promoter refers generally to transcriptional regulatory regions of a gene, which may be found at the 5' or 3' side of the coding region, or within the coding region, or within introns.
  • a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 1 direction) coding sequence.
  • the typical 5' promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a “vector” is a replicon, such as plasmid, phage, cosmid, or virus to which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.
  • nucleic acid construct or "DNA construct” is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into a vector for transforming a cell. This term may be used interchangeably with the term “transforming DNA” or "transgene”. Such a nucleic acid construct may contain a coding sequence for a gene product of interest, along with a selectable marker gene and/or a reporter gene.
  • selectable marker gene refers to a gene encoding a product that, when expressed, confers a selectable phenotype such as antibiotic resistance on a transformed cell.
  • reporter gene refers to a gene that encodes a product which is easily detectable by standard methods, either directly or indirectly.
  • a "heterologous" region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature.
  • the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • a cell has been "transformed” or “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. If germline cells are stably transformed, the transformation may be passed from one generation of animals arising from the germline cells, to the next generation. In this instance, the transgene is referred to as being inheritable.
  • subject refers to a human subject or a non- human animal subject, or it may refer to any other living organism.
  • patient may be used interchangeably for the term “subject.”
  • acute pain refers to an unpleasant sensation induced by noxious stimuli. It is short-lasting and can occur without any tissue injury.
  • Persistent pain refers long-lasting, unpleasant sensations that are often related to tissue injury. Persistent pain lasts long after the initial noxious stimulus producing acute pain is gone, and may persist from days to years. Other definitions are found in the description set forth below.
  • NMDA receptors play an important role in long-term plasticity in the brain. In persistent pain, central sensitization requires activation of spinal NMDA receptors, but a role for forebrain NMDA receptors heretofore had not been established.
  • intracerebro ventricular injection of an NMDA receptor antagonist inhibits behavioral responses of animal subjects to subcutaneous formalin injection (a standard model for inflammatory pain), leaving acute nociceptive reflexes intact, hi transgenic animals with forebrain-targeted overexpression of the NMDA receptor subunit NR2B (a manipulation previously shown to augment charge transfer through NMDA channels), formalin-induced c-Fos expression was enhanced in pain-related forebrain structures.
  • transgenic animals displayed selectively exaggerated late-phase formalin- induced behavioral responses.
  • the present invention provides a method to reduce or eliminate persistent pain in a patient in need of such treatment.
  • the method involves down-regulating charge transfer through NMDA channels in the forebrain of the patient.
  • NMDA channels in the forebrain of the patient.
  • it has been shown through the forebrain-targeted administration of NMDA antagonists and through the behavioral studies of transgenic animals with augmented NMDA channel charge transfer in the forebrain, that such treatment results in the reduction or alleviation of persistent pain.
  • NMDA receptors in the forebrain can be down- regulated in a variety of ways. It is believed that any means by which NMDA function in the forebrain can be down-regulated will yield the same antinociceptive effect as observed in subjects treated with NR2B antagonists in their forebrains.
  • methods to inhibit NMDA receptor function in the forebrain include, but are not limited to: (1) forebrain-targeted administration of compounds that act directly or indirectly to reduce NMDA receptor function, preferably, but not exclusively, by targeting NR2B; (2) modulating expression of NMDA receptor subunits at the transcriptional (e.g., promoter) and/or translational level (which may include down- regulation of NR2B gene expression or up-regulation of upstream transcription factors for other NR2 subunits, including NR2A, C or D, to reduce the ratio of NR2B to other NR2 subunits in the forebrain); and (3) use of agents that act inside cells of the forebrain at the intracellular domains of the NMDA receptor, to interfere with the interaction between the receptor and its downstream targets.
  • NMDA receptor subunits e.g., promoter
  • translational level which may include down- regulation of NR2B gene expression or up-regulation of upstream transcription factors for other NR2 subunits, including
  • NR2B subunits are targeted for negative regulation. In one embodiment, this is accomplished by forebrain-targeted administration of NR2B-selective antagonists, examples of which are already available.
  • somatic cells of subjects may be stably or transiently transformed with a vector encoding an antisense molecule or ribozyme, or a transcription suppressing protein, for instance, designed to inhibit expression of NR2B.
  • Such "DNA therapy” would comprise targeted administration of an NR2B expression- inhibiting vector which, upon delivery to the target cells, would inhibit the production of NR2B, thus reducing the pool of NR2B subuit available for incorporation into NMDA receptors.
  • DNA therapy to transiently produce such NR2B expression- inhibiting molecules in targeted brain locations is accomplished according to methods well known in the art.
  • the forebrain may be selectively targeted by using a promoter that is specific for gene expression in that region, such as a promoter derived from the CaMKII gene, whose activity has been demonstrated to be restricted to the forebrain region (Mayford et al., Cell 81, 891-904, 1995).
  • the present invention also provides an animal model system useful for the discovery of new substances that can reduce or alleviate persistent pain.
  • This animal model system comprises NR2B transgenic animals. As exemplified by the NR2B transgenic mice described herein, these animals exhibit enhanced late-phase response to a persistent pain stimulus, such as the inflammation that results from injection with formalin or CFA.
  • animal is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. Examples of animals preferred for use in the present invention include, but are not limited to, rodents, most preferably mice and rats, as well as cats, dogs, dolphins and primates.
  • transgenic animal is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus.
  • transgenic animal is not meant to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule, i.e., a "transgene”.
  • transgene refers to any exogenous gene sequence which is introduced into both the somatic and germ cells or only some of the somatic cells of a mammal.
  • This molecule maybe specifically targeted to defined genetic locus, or be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • the term "germline transgenic animal” refers to a transgenic animal in which the transgene was introduced into a germline cell, thereby conferring the ability to transfer the transgene to offspring. If such offspring in fact possess the transgene then they, too, are transgenic animals.
  • the transgene of the present invention includes without limitation, the entire coding region of an NR2B gene, or its complementary DNA (cDNA), or chimeric genes containing part or all of a NR2B coding region, whose expression in the forebrain is driven by a tissue specific promoter. It is preferable, but not essential, that the NR2B coding sequence used in the transgene be of the same species origin as the transgenic animal to be created.
  • Nucleic acid sequences encoding NR2B have been reported for several species. Examples include mouse (GenBank Accession No. U60210), and human (GenBank Accession No. NM 000834).
  • the promoter is comprised of cis-acting DNA sequences capable of directing the transcription of a gene in the appropriate tissue environment and, in some cases, in response to physiological regulators.
  • the promoter preferred for use in the present invention is derived from the aCaMKII gene, whose activity has been demonstrated to be restricted to the forebrain region (Mayford et al., Cell 81 : 891-904, 1995).
  • Other promoters are also known to direct the expression of exogenous genes to specific cell-types in the brain.
  • Promoters useful for stem cell transformation include any promoter whose endogenous genes are expressed in the target cell of interest; e.g., the pkc ⁇ promoter, the telencephalin promoter, the neuronal enolase promoter and the prp promoter.
  • tissue specific promoters may or may not be needed.
  • constitutive promoters such as the CMV promoter or the ⁇ -actin promoter should prove useful for somatic transformation.
  • NR2B transgenic mice by this method has been described (Tsien et al, Cell 87: 1317-26, 1996).
  • Another method for producing germline transgenic mammals utilizes embryonic stem cells.
  • the DNA construct may be introduced into embryonic stem cells by homologous recombination (Thomas et al., Cell 51: 503, 1987; Capecchi, Science 244: 1288, 1989; Joyner, et al, Nature 338: 153, 1989) in a transcriptionally active region of the genome.
  • a suitable construct may also be introduced into the embryonic stem cells by DNA-mediated transfection, such as electroporation
  • the construct may be introduced as a linear construct, as a circular plasmid, or as a viral vector which may be incorporated and inherited as a transgene integrated into the host genome.
  • the transgene may also be constructed so as to permit it to be inherited as an extrachromosomal plasmid.
  • plasmid generally refers to a DNA molecule that can replicate autonomously in a host cell.
  • Transgenic animals also may be obtained by infection of neurons either in vivo, ex vivo, or in vitro with a recombinant viral vector carrying an NR2B gene.
  • Suitable viral vectors include retroviral vectors, adenoviral vectors and Herpes simplex viral vectors, to name a few. The selection and use of such vectors is well known in the art.
  • the present invention also provides a variety of assays and other methods, which utilize the inventors' discovery of the effect of NMDA receptor function on the enhancement of persistent pain.
  • One useful assay is an in vitro assay for identifying compounds that inhibit persistent pain by decreasing expression of NR2B genes. This assay involves the following basic steps: (1) provide a chimeric DNA construct comprising an NR2B promoter operably linked to a reporter gene; (2) contact the chimeric DNA construct with a test compound suspected of down-regulating the NR2B promoter, and (3) measure expression of the reporter gene. A decrease in the expression of the reporter gene in the presence of the test compound indicates that the test compound will be useful in the management of persistent pain by decreasing the expression of NR2B genes.
  • an in vivo assay useful for identifying compounds that negatively affect activation of NMDA receptors in a mammal, comprises the following steps: (1) provide an NR2B transgenic animal; (2) treat the transgenic animal with a test compound suspected of negatively affecting activation NMDA receptors; and (3) directly or indirectly (i.e., biochemically or by behavioral tests) measure a change in activity of the treated animal as compared with the untreated animal, a negative change being indicative that the test compound down-regulates activation of NMDA receptors in the animal.
  • This assay can be extended by measuring behavioral responses to persistent pain stimuli in the transgenic animals, inasmuch as such responses will be more robust in these animals as compared with non-transgenic animals, and differences caused by various test compounds will be more apparent.
  • cultured neuronal or non-neuronal cells may be transformed with a DNA construct for expression of NR2B, optionally together with NR1 subunit or other NR2 subunits (NR2A, NR2C, NR2D), and those cells used for various biochemical and physiological assays to assess the changes resulting from the presence of the transgene.
  • NR2B NR1 subunit or other NR2 subunits
  • cells or tissue slices from NR2B transgenic animals may be utilized for a similar purpose.
  • mice Both wild-type and NR2B transgenic mice were prepared as described by Tang et al., 1999. Adult male mice weighing 15-29 g were used. Behavioral experiments were carried out in a quiet environment and performed during the day. Room temperature was always maintained at 20°C. All behavioral experiments were performed blind.
  • Tail-flick test The spinal nociceptive tail-flick reflex was evoked by focused, radiant heat applied to underside of the tail. The latency to reflexive removal of the tail away from the heat was measured by a photocell timer to the nearest 0.1 sec. The mean tail-flick latency was calculated as the average of 3-4 measurements performed at 10 min intervals.
  • Hot-plate test Mice were placed on a thermally-controlled metal plate (Columbia Instruments; Columbus, Ohio). The time between placement of a mouse on the plate and licking or lifting of a hindpaw was measured with a digital timer. Mice were removed from the hot plate immediately after the first response. The mean hot-plate latency was calculated as the average of 3-4 measurements performed at 10 min intervals.
  • Cold-plate test Mice were placed in a plastic container (21cm diameter x 21cm high) resting on a bed of ice. The temperature of the ice surface was monitored with a digital thermometer and maintained at 0°C by placing additional ice around the container. The time between placement of a mouse on the cold plate and the first jumping or licking of the hindpaw was measured with a digital timer. Mice were removed from the plate after the first response. The mean cold-plate latency was calculated as the average of 3-4 measurements performed at 10 min intervals.
  • Formalin-induced persistent pain Formalin (5%, lO ⁇ l) was injected subcutaneously into the dorsal side of a hindpaw. Mice respond by licking the inj ected hindpaw, and this behavior is typically concentrated in three distinct phases: a first phase (0-10 min), a second phase (10-60 min) and a third, late phase (60-120 min) (Kim et al, Brain Res. 829: 185-189, 1999). The total time spent licking or biting the injected hindpaw was recorded during each 5 min interval over the course of2 hr. Intracerebro ventricular drug injection. Intracerebro ventricular injection of MK-801 was carried out by standard methods.
  • mice were always lightly anesthetized with halothane (2 %); recovery from anesthetic was complete in 2-3 min.
  • the injection was monitored by observing the movement of air behind the solution.
  • the injection sites were confirmed by examining tissue sections prepared after experiments.
  • mice were deeply anesthetized with halothane (3-4% in a gaseous mixture of 70% N 2 O and 30% O 2 ) and perfused through the ascending aorta with 50 ml of saline, followed by 200 ml of cold 0.1 M phosphate buffer containing 4% paraformaldehyde.
  • Cryostat-cut brain sections (30 ⁇ m) were immunocytochemically processed with anti-c-Fos rabbit antibody (1:20,000; Oncogene Science, Uniondale, NY) using Vectastain reagents and developed with DAB/nickel/GOD. Controls, performed by replacing primary antibody with 1% NGS in this protocol, exhibited no staining.
  • Anatomical terminology is based on the atlas of Franklin and Paxinos.
  • the mean number of c-Fos labeled cells in each nucleus of interest was calculated using sections from 4-6 mice. The investigator responsible for plotting and counting the labeled cells was blind to the experimental situation of each animal.
  • mice We examined the responses of awake, adult mice to acute and persistent nociceptive stimuli, testing the effects of manipulating NMDA receptor function through both pharmacological and genetic approaches.
  • acute nociception we recorded the behavioral responses of mice in tail-flick, hot-plate, and cold-plate tests.
  • inflammation-induced persistent pain we tested the responses of mice to peripheral, subcutaneous formalin injection into a single hindpaw.
  • the formalin test is a widely used animal model of tissue injury and inflammatory pain. Nociceptive behavioral responses to formalin, which include licking the injected hindpaw (see Experimental Procedures), occur in three distinct phases in mice: Phase 1 (0-10 min after injection) responses are thought to be due to a peripheral inflammatory response at the site of injection.
  • Phase 2 (10-60 min) occurs after central sensitization in the spinal cord and requires ongoing activity from the periphery.
  • Phase 3 60-120 min responses, which are normally less severe in comparison to the preceding phases (Kim et al., 1999, supra), occur by an unknown mechanism.
  • phase 3 responses could be manipulated independently of phases 1 and 2
  • phase 1 and 2 responses were indistinguishable between these mice and mice not treated with MK-801
  • mice with forebrain-targeted NR2B overexpression were used to test whether prolonging forebrain NMDA receptor-mediated EPSCs and enhancing charge transfer through the channels would affect the nociceptive responses of mice.
  • we made use of mice with forebrain-targeted NR2B overexpression were used to test whether prolonging forebrain NMDA receptor-mediated EPSCs and enhancing charge transfer through the channels would affect the nociceptive responses of mice.
  • NMDA receptor function in two pain-related forebrain areas, the anterior cingulate cortex (ACC) and insular cortex. Brain slices of these areas were prepared from adult mice, and excitatory postsynaptic field potentials (fEPSPs) were recorded upon local electrical stimulation (Fig. 2A). In each region, after blockade of AMP A and kainate receptors by CNQX (20 ⁇ M), a slow fEPSP was observed (Fig. 2B) that could be entirely and reversibly blocked by the NMDA receptor antagonist AP-5 (100 ⁇ M; Fig. 2C).
  • ACC anterior cingulate cortex
  • fEPSPs excitatory postsynaptic field potentials
  • mice Consistent with the high levels of NR2B transgene expression found throughout the cerebral cortex in transgenic mice, these mice, compared to wild-type mice, exhibited enhanced NMDA receptor-mediated fEPSPs in both the ACC and insular cortex (Fig. 2D).
  • c-Fos expression patterns were observed in brain regions both ipsilateral and contralateral to the injected hindpaw, with contralateral c-Fos expression being somewhat more pronounced.
  • formalin injection induced prominent c-Fos staining in the anterior cingulate cortex ACC; Figure 4
  • lateral septal nucleus secondary motor cortex
  • some nuclei in the amygdaloid complex medial, basolateral, and cortical nuclei
  • piriform cortex retrosplenial cortex
  • several midline thalamic nuclei lateral habenular, paraventricular, mediodorsal, centromedial, paracentral, and anterodorsal nuclei
  • various hypothalamic nuclei paraventricular, periventricular, supraoptic, and dorsomedial nuclei.
  • c-Fos expression was observed in the somatosensory cortex (SI, S2, and hindlimb areas) ( Figure 4), the hippocampal CAl ( Figure 4) and CA3 subfields, insular cortex ( Figure 4), accumbens nucleus, and lateral preoptic area.
  • a small amount of formalin-induced c-Fos expression was observed in other thalamic nuclei, including the lateral posterior nucleus, posterior group, ventral lateral, ventral posterolateral, ventral posteromedial, and ventral medial nuclei. No c-Fos expression was observed in the caudate-putamen nucleus.
  • c-Fos expression was also observed in the locus coeruleus, parabrachial nucleus, and rostroventral medulla.
  • NR2B overexpression directly affecting c-Fos expression in response to formalin injection
  • the brain areas in which significantly greater c-Fos expression was observed in transgenic than in wild-type mice were areas in which NR2B overexpression was present (Tang et al., 1999, supra).
  • these areas of greater c-Fos expression encompassed some parts of the limbic system and other forebrain areas known to be important for the central processing of nociceptive information, including, in particular, the ACC, lateral septal nucleus, hippocampus, and insular cortex.

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Abstract

L'invention concerne des procédés et un modèle animal respectivement mis en oeuvre pour permettre de moduler une douleur persistante chez un sujet, et d'identifier de nouveaux agents pouvant réduire cette douleur persistante. Selon l'invention, ces procédés sont fondés sur la découverte selon laquelle une activation stimulée de la fonction des récepteurs de NMDA dans le prosencéphale, en particulier par une expression accrue de la sous-unité NR2B, est associée à un accroissement de la réaction à long terme à un stimulus de douleur persistante.
PCT/US2001/041447 2000-07-27 2001-07-27 Procedes et agents servant a traiter une douleur persistante WO2002009736A1 (fr)

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WO2013156614A1 (fr) 2012-04-20 2013-10-24 Ucb Pharma S.A. Méthodes de traitement de la maladie de parkinson

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US5622952A (en) * 1992-06-22 1997-04-22 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University And The University Of Oregon, Eugene Oregon Glycine receptor antagonists and the use thereof

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US5622952A (en) * 1992-06-22 1997-04-22 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University And The University Of Oregon, Eugene Oregon Glycine receptor antagonists and the use thereof

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JIKKEN IGAKU, vol. 17, no. 19, 1999, pages 2565 - 2568 *
SASNER M. ET AL.: "Distinct N-methyl-D-aspartate receptor 2B subunit gene sequences confer neural and developmental specific expression", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, no. 35, 30 August 1996 (1996-08-30), pages 21316 - 21322, XP001055010 *

Cited By (1)

* Cited by examiner, † Cited by third party
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WO2013156614A1 (fr) 2012-04-20 2013-10-24 Ucb Pharma S.A. Méthodes de traitement de la maladie de parkinson

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