EP1774318A2 - Modeles et procedes de nociception, de transduction de la douleur et de criblage de composes analgesiques - Google Patents

Modeles et procedes de nociception, de transduction de la douleur et de criblage de composes analgesiques

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Publication number
EP1774318A2
EP1774318A2 EP05791319A EP05791319A EP1774318A2 EP 1774318 A2 EP1774318 A2 EP 1774318A2 EP 05791319 A EP05791319 A EP 05791319A EP 05791319 A EP05791319 A EP 05791319A EP 1774318 A2 EP1774318 A2 EP 1774318A2
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subunit
alpha
delta
animal
mice
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Zhigang David Luo
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University of California
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University of California
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • 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
    • 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
    • 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • alpha- 1 adrenergic receptors G-protein coupled transmembrane receptors
  • catecholamines epinephrine, and/or norepinephrine
  • selected cell systems and uses are described in U.S. Pat. No. 6,733,982, and U.S. Pat. App. No. 20040132115.
  • Voltage-gated calcium channels (VGCCs) within sensory neurones are also believed to perform an important role in neuropathic pain.
  • induction of diabetic neuropathy is associated with significant changes in the expression of various VGCC mRNAs (typically an increase in alpha(2)delta(l), alpha(2)delta(2), and alpha(2)delta(3) mRNA levels; see e.g., Yusaf et al. Biochem Biophys Res Commun. 2001 Nov 30;289(2):402-6).
  • downregulation of certain VGCC components was reported by Kim et al (Brain Res MoI Brain Res. 2001 Nov 30;96(l-2):151-6).
  • VGCC alpha(l) gene in the dorsal root ganglion (DRG) following chronic constriction injury (CCI) and axotomy of the rat sciatic nerve resulted in decreased alpha(lC), alpha(lD), alpha(lH), and alpha(ll) mRNA expression at 7 days in the ipsilateral DRG, to approximately 34-50% of the contralateral side, hi still further published studies, novel splice isoforms of the Ca V2.2. alpha subunit of N-type calcium channels (preferentially expressed in a subset of nociceptive neurons) containing exon 37a were implicated in neuropathic pain as described in U.S. Pat. App. No. 20040214238.
  • Neuropathic pain diagnostics has therefore looked at a variety of target molecules and an exemplary molecular diagnostic test is described in U.S. Pat. App. No. 20030216341, in which expression of various sequence tags (representing genes differentially expressed under neuropathic pain in the models of Bennett, Seltzer and Kim & Chung (Bennett & Xie 1988 Pain 33:87-107, Seltzer et al. 1990 Pain 43:205-218; Kim & Chung 1992 Pain 50:355-363)) is quantitatively monitored. Similarly, Brooksbank et al. describe in U.S. Pat. App. No.
  • 20030138803 a system in which sequences associated with streptozocin-induced diabetes and implicated with pain are analyzed and used for various models.
  • the inventor has discovered that overexpression of voltage-gated calcium channel alpha-2-delta-l subunit in neural tissue correlates in vivo with pain responses in transgenic animals otherwise observed with nerve injury-induced neuropathic pain. Significantly, such transgenic animals can be employed in animal models for nerve injury-induced neuropathic pain without inflicting nerve injury, which often interferes with test results. Similarly, the inventor discovered that neural cells obtained from transgenic animals or cell transformed to overexpress the alpha-2-delta-l subunit exhibit physiological parameters remarkably similar to those of neural tissue obtained from animals thought to have nerve injury-induced neuropathic pain.
  • the inventor contemplates a model for peripheral nerve injury-induced neuropathic pain in which a transgenic non-human animal preferentially expresses the alpha-2-delta-l subunit of a voltage gated calcium channel in a neuronal tissue in an amount sufficient to produce non-injurious tactile allodynia and/or thermal hyperalgesia, preferably while retaining normal pain reaction to inflammatory pain (as compared to a control animal).
  • the alpha-2-delta-l subunit is expressed in the hippocampus, the cortex, the spinal cord, and the dorsal root ganglion, and is substantially not expressed in the heart, skeletal muscles, the lung, kidneys, the spleen, and/or the intestines.
  • the expression may be facilitated using various manners, however, it is generally preferred that expression is driven from the Thy- 1.2 gene promoter.
  • preferred transgenic animals include mammals, and most preferably mice.
  • a method of testing a pharmaceutically active compound for use in treatment of peripheral nerve injury-induced neuropathic pain will preferably include a step in which the compounds is administered in vivo to the transgenic animal (or recombinant cell, which may or may not be isolated from the transgenic animal).
  • administration is parenteral administration of the compound at a dosage effective to reduce the non-injurious tactile allodynia and/or thermal hyperalgesia, and most typically intrathecal inj ection.
  • Suitable compounds for reduction of neuropathic pain will include those that reduce expression and/or activity of the alpha-2-delta-l subunit or a component that is functionally associated with the alpha-2-delta-l subunit.
  • contemplated animal models will allow the investigator to determine metabolic effects of a drug in vivo (e.g., the animal metabolizes the compound to thereby generate an active metabolite that is effective to reduce the allodynia and/or hyperalgesia).
  • contemplated methods will include a step of measuring paw withdrawal threshold and a measurement of thermal escape latency.
  • Figures IA and IB depict a Western blot and a graph illustrating Ca V a 2 O 1 expression in neuronal tissues from transgenic mice according to the inventive subject matter.
  • Figures 2A-2F are graphs depicting specific pain behaviors under defined conditions in transgenic and wild type mice.
  • Figure 3 is a set of micrographs depicting Ca V (X 2 O 1 immunohistostaining in spinal dorsal horn (DH) sections taken from lumbar spinal cord of adult transgenic mice (TG) and wild-type (WT) littermates.
  • Figures 4A and 4B are graphs depicting gabapentin dose-dependent reversal of tactile allodynia in TG mice without significant alteration of paw sensitivity to mechanical stimulation in WT mice.
  • Figures 5A-5C are graphs depicting results for various parameters of whole-cell voltage-clamp experiments in acutely isolated DRG neurons from TG and WT mice.
  • Figures 6A and 6B are graphs depicting significant increased activation rates of voltage gated calcium channels in cells with Ca V a 2 O 1 over-expression.
  • Figures 7A and 7B are graphs depicting voltage-dependent changes in deactivation rates of voltage gated calcium channels.
  • Figures 8A and 8B are graphs depicting gabapentin dose-dependent attenuation of high threshold voltage-gated barium currents in TG sensory neurons.
  • the inventor has discovered that expression of voltage-gated calcium channel alpha- 2-delta-l subunits is implicated in various aspects of neuropathic pain.
  • the inventors have now discovered that increased expression of the voltage-gated calcium channel alphas- delta- 1 subunit, and especially increased expression in spinal cord and dorsal root ganglia in transgenic mice correlates in vivo with typical nerve injury-induced nociceptive responses to innocuous mechanical and thermal stimulation (tactile allodynia and thermal hyperalgesia).
  • pain perception due to tissue injury and/or inflammation was not affected to a measurable degree in such transgenic animals.
  • contemplated animal models advantageously lend themselves to investigate the specific role of the voltage-gated calcium channel alpha-2-delta-l subunits in various aspects of treatment and research of neuropathic pain.
  • responses to certain stimuli attributable to prior injury of the animals (which was needed in previously known models to generate the neuropathic pain model) can now be excluded.
  • the inventors have observed that in transgenic mice over-expressing the calcium channel alpha-2-delta-l subunit, the abnormal sensations that mimic the pain states after nerve injury indicate that the alpha-2-delta subunit is a molecular determinant of abnormal sensations. This observation was supported by the finding that abnormal sensations in such transgenic mice can be normalized in a dose-dependent manner by gabapentin, a drug binds to the alpha-2-delta subunit, but not by ketorolac, a non-steroidal anti-inflammatory drug.
  • the prepared transgenic mice overexpressing the calcium channel alpha- 2-delta-l subunit in neurons have hypersensitivity to mechanical and thermal stimuli, mimicking pain states after injuries such as nerve injuries, neuropathies and inflammation involving neural tissue. Consequently, the presently contemplated injury- free transgenic mouse model provides an excellent tool for screening compounds related to the development and evaluation of pain medications (e.g., anti-hyperalgesic agents, general analgesic agents, etc.), and for mechanistic studies of pain transduction mechanisms.
  • pain medications e.g., anti-hyperalgesic agents, general analgesic agents, etc.
  • transgenic animals may be made in which the gene for the voltage-gated calcium channel alpha-2-delta-l subunit is constitutively overexpressed.
  • inducible, repressible, and/or temporal overexpression is also deemed suitable. Consequently, suitable overexpression may be performed using viral transfection, chemical transfection, ballistic gene transfer, electroporation, cell fusion, and transgenic animals.
  • suitable overexpression may be performed using viral transfection, chemical transfection, ballistic gene transfer, electroporation, cell fusion, and transgenic animals.
  • the amount of overexpressed alpha-2-delta-l subunit maybe either variable, or constant. Nucleic acid constructs for regulated expression are well known in the art and all of them are deemed suitable for use herein.
  • the expression is driven from a promoter and/or other regulatory element that is tissue specific to neuronal tissue, and/or that transgenic expression is inducible/controllable by exogenously added agents.
  • the alpha-2-delta-l subunit gene is expressed in the hippocampus, cortex, spinal cord, and/or dorsal root ganglion.
  • suitable alphas- delta- 1 subunit genes are expressed from a cDNA construct.
  • expression constructs may also include one or more introns and/or other non-coding (preferably regulatory) elements.
  • the alpha-2-delta-l subunit gene is homologously expressed in the animal (e.g., murine gene in transgenic mouse), and that the gene is the wildtype form for that particular animal.
  • the animal e.g., murine gene in transgenic mouse
  • heterologous expression may also be advantageous (e.g., for immunological distinction over wildtype, or for kinetic analyses of the human subunit expressed in a rodent).
  • the overexpressed alpha-2- delta- 1 subunit gene may be modified to provide one or more advantages over the unmodified form.
  • modified forms may include affinity tags for labeling and/or isolation, or may have deletions or alterations to remove or alter a particular biochemical parameter (e.g., degree of glycosylation, degree of interaction with components up- or downstream in signal transduction, drug and/or ligand binding, or association with other subunits).
  • modifications may also include base substitutions to modify the primary sequence of the overexpressed protein to obtain desired physical properties. Therefore, all at least partially functional, and even non- functional homologues of the alpha-2-delta-l subunit gene are specifically contemplated herein.
  • suitable animals include those in which the voltage- gated calcium channel alpha-2-delta-l subunit is naturally expressed as a component in nociception and/or pain transduction. Therefore, contemplated animals include numerous non-human mammals, and preferably rodents for which suitable test protocols are already well established. Alternatively, it is also contemplated that suitable animals are chosen from a group that does not naturally express the voltage-gated calcium channel alpha-2-delta-l subunit as a component in nociception and/or pain transduction.
  • contemplated animals may be selected from invertebrates (e.g., cnidaria or nematoda, where relatively simple neuronal systems are desired) or from arthropods (e.g., where large progeny in relatively short period are desired).
  • invertebrates e.g., cnidaria or nematoda, where relatively simple neuronal systems are desired
  • arthropods e.g., where large progeny in relatively short period are desired.
  • Such animals may provide particularly desirable advantages as a particular signal transduction chain that may be engineered into such animals.
  • activation of the voltage gated calcium channel may be coupled with beta 2 ( ⁇ 2) adrenergic receptors (Davare et al, 2001), which may be detected by numerous methods well known in the art.
  • expression of the alpha-2-delta-l subunit may also be performed in yeast.
  • expression systems may use Pichia, Saccharomyces, or other suitable species.
  • Such animal models may be useful in numerous experimental settings, and especially preferred uses include those in which the animal is a transgenic animal for in vivo study of nociception, and especially of neuropathic pain.
  • it should be recognized that such animals may also be useful for numerous other abnormal and/or pathologic conditions in which the alpha-2-delta-l subunit is overexpressed relative to a normal and/or non-pathologic condition.
  • contemplated models may be useful for investigation of various aspects related to ischemic stroke, epilepsy, neoplastic diseases, etc. (see below).
  • the inventor contemplates a model for peripheral nerve injury-induced neuropathic pain that comprises a transgenic non-human animal that preferentially expresses (i.e., at least two times, more typically at least three times, and most typically at least five times more than in non-neuronal tissue) an alpha-2-delta-l subunit of a voltage gated calcium channel in a neuronal tissue and in an amount sufficient to produce at least one of non-injurious tactile allodynia and non- injurious thermal hyperalgesia while the animal retains normal pain reaction to tissue injury and inflammatory pain.
  • a transgenic non-human animal that preferentially expresses (i.e., at least two times, more typically at least three times, and most typically at least five times more than in non-neuronal tissue) an alpha-2-delta-l subunit of a voltage gated calcium channel in a neuronal tissue and in an amount sufficient to produce at least one of non-injurious tactile allodynia and non- injurious thermal
  • the expression is driven by a tissue specific promoter or other construct such that the preferential expression is observed in the hippocampus, the cortex, the spinal cord, and/or the dorsal root ganglion.
  • preferential expression will provide substantially no recombinant expression (i.e., less than 10% of neuronal tissue) in the heart, skeletal muscles, the lung, kidneys, the spleen, and/or the intestine.
  • a tissue specific promoter or other construct such that the preferential expression is observed in the hippocampus, the cortex, the spinal cord, and/or the dorsal root ganglion.
  • preferential expression will provide substantially no recombinant expression (i.e., less than 10% of neuronal tissue) in the heart, skeletal muscles, the lung, kidneys, the spleen, and/or the intestine.
  • the alpha-2-delta-l subunit is expressed under the control of a Thy- 1.2 gene promoter as exemplified below.
  • contemplated animal models may be provided for research and development, as well as a commercial product together with an information to use the animal for testing of a compound for use in treatment of pain, and especially peripheral nerve injury-induced neuropathic pain.
  • the compound to be tested is administered in vivo.
  • Such models will therefore allow not only to study the direct interaction of certain pharmaceutical compounds in their role as modifiers of the alphas- delta- 1 subunit mediated pain, but also allow for pharmacokinetic and pharmacodynamic studies in which the role of the animal's metabolism is observed. Consequently, suitable models also allow identification of active metabolites, studies of prodrug activation, passage of the drug to the target cells, and clearance of the drug and/or metabolite from the animal's system.
  • administration of the compound maybe parenterally (intravenously, intraperitoneally, intrathecally, etc.), orally, or any reasonable combination thereof.
  • Suitable compounds will typically include those that bind (reversibly or irreversibly) to the alpha-2- delta- 1 subunit, those that reduce activity of the alpha-2-delta-l subunit or a component functionally associated with the alpha-2-delta-l subunit, and/or those that interfere with expression of the alpha-2-delta-l subunit (see below).
  • eukaryotic cells or microorganisms may be employed.
  • refinement of the compound e.g., to increase affinity to the subunit, to reduce expression of the subunit
  • exemplary microorganisms include various yeasts (e.g., Pichia spec).
  • calcium flux may be directly observed by change in fluorescence (e.g., using fluorescent calcium indicator Fura-2) or other physico-chemical parameter.
  • fluorescence e.g., using fluorescent calcium indicator Fura-2
  • the quantity of recombinant subunit may be significantly increased.
  • heterologous expression of the alpha-2-delta-l subunit in numerous non-neuronal cells may be especially advantageous as no intrinsic background expression is expected.
  • a pharmaceutical compound in which in one step a pharmaceutical compound is administered to a tissue and/or a cell of a transgenic animal in vitro.
  • the compound may also be administered to a recombinant cell over-expressing the alpha-2-delta-l subunit.
  • suitable tissues especially include hippocampus, cortex, spinal cord, and dorsal root ganglion or cells therefrom.
  • the transgenic animal or recombinant cell may or may not naturally express the alpha-2-delta-l subunit.
  • alpha-2-delta-l subunit natural expression of the alpha-2-delta-l subunit may be modulated (and most typically down-regulated or even abrogated) in animals, and particularly human suffering from neuropathic pain.
  • therapeutically active small molecules can be identified that interfere with the biological function of the alphas- delta- 1 subunit.
  • antibodies (or fragments thereof) maybe at least temporarily used to reduce biological activity of alpha-2-delta-l subunit in a patient.
  • anti-sense RNA and/or siRNA may be employed to reduce or even completely abolish functional expression of the alpha-2-delta-l subunit gene in corresponding cells.
  • anti-sense RNA and/or siRNA There are numerous procedures for in vivo use of anti-sense RNA and/or siRNA known in the art, and all of such uses are deemed suitable for use herein.
  • alpha-2-delta-l subunit may be employed in an analytic and/or diagnostic manner in which the subunit is detected in vivo or in vitro using methods well known in the art.
  • peptide expression may be monitored using immunoscintygraphy (e.g., using technetium- labeled antibodies) in vivo.
  • peptide expression may be quantified ex vivo from biopsy specimen using western blot or other immunographic methods.
  • Contemplated diagnostic applications also include a determination of the sequence (nucleic acid and/or peptide) of the alpha-2-delta-l subunit to identify and/or characterize mutations that may be present.
  • components in pain transduction may be identified that functionally cooperate with the alpha-2-delta-l subunit. Such components may then be employed as molecular targets for further therapeutic use, and most preferably for reduction in pain perception of an individual.
  • alpha-2-delta-l subunit in pain, and particularly neuropathic pain
  • numerous compounds and compositions may be prepared that have therapeutic use in treatment and/or prevention of neuropathic pain.
  • inhibitors of the alpha-2-delta-l subunit maybe identified that act as modulating agents, reduce expression of the alpha-2-delta-l subunit or functionally associated component, and/or reduce pain perception of an individual.
  • Transgenic mice over-expressing the voltage-gated calcium channel alpha-2- delta-1 subunit (Ca v ⁇ 2 ⁇ i) gene were generated substantially following a protocol as described previously 13 .
  • a transgene vector was used containing the mouse brain Ca V a 2 O 1 cDNA (Genbank accession number U73484) that was cloned into the vector down-stream of a 6.5 kb murine thy- 1.2 gene extending from the promoter region to the intron after exon 4 without exon 3 and its flanking introns. Deletion of exon 3 and its flanking introns has been shown to abolish expression in non-neuronal cells 14 .
  • Frozen tissues were pulverized and extracted with lysis buffer (50 mM Tris-HCl buffer, pH 7.5, containing 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA) containing protease inhibitors. Equal amounts of total protein extracts in gel loading buffer from each sample were applied to electrophoresis in NuPAGE Tris-acetate gels under reducing conditions (0.05 M dithiothreitol), then electrophoretically transferred to the nitrocellulose membranes (Schleicher & Schuell, Keene, NH).
  • lysis buffer 50 mM Tris-HCl buffer, pH 7.5, containing 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA
  • Equal amounts of total protein extracts in gel loading buffer from each sample were applied to electrophoresis in NuPAGE Tris-acetate gels under reducing conditions (0.05 M dithiothreitol), then electrophoretically transferred to the
  • the membranes were incubated with monoclonal antibodies against the Ca V a 2 O 1 subunit in phosphate-buffered saline containing 0.1% Tween 20 for 1 hr at room temperature or overnight at 4 0 C after blocking nonspecific binding with 5% non-fat milk for 1 hr at room temperature.
  • the antigen-antibody complexes were detected by incubating the membranes with horseradish peroxidase (HRP) labeled secondary antibody for 1 h at room temperature followed by washing and addition of HRP substrate. Under reducing conditions, the 6-peptide separates from the ⁇ 2 subunit so the positive bands detected by the primary antibody reflect the ⁇ 2 subunit only.
  • HRP horseradish peroxidase
  • the blots were stripped and re-blotted with primary antibodies against house-keeping protein glyceraldehyde-3 -phosphate dehydrogenase (GAPDH, Ambion, Austin, TX) that was not changed due to Ca V a 2 S 1 over-expression and other manipulations on the mice.
  • GPDH house-keeping protein glyceraldehyde-3 -phosphate dehydrogenase
  • mice were placed into individual enclosures on the glass surface of the hot box maintained at 30 °C and allowed to acclimatize for at least 30 min. After acclimation, the escape latencies to a thermal stimulation were measured in both hindpaws of the WT and TG mice.
  • the source of radiant thermal stimulus underneath the glass surface was aligned to the planter surface of the hindpaw.
  • Activation of the light source activated a timer, and paw withdrawal from the light source or 20 s of light stimulation turned off the light bulb and timer.
  • averaged escape latency (in seconds) from both sides of hindpaws was used for comparing sensitivities between WT and TG mice, or before and after systemic and intrathecal drug treatments.
  • mice were placed into individual transparent cylinders for acclimation at least 1 hr before the formalin injection.
  • 20 ⁇ l of 2% formalin was injected subcutaneously into the plantar surface of right hindpaw of WT and TG mice.
  • the flinching responses were recorded automatically in an automated nociception analyzer (University of California, San Diego) and lifting/licking was recorded by a digital camcorder and counted manually.
  • the number of flinching/lifting and licking was recorded from 0 to 60 min.
  • the numbers of flinching/min or lifting/licking per a 5 min interval were compared between groups.
  • mice were placed into a clear plastic cage with a wire mesh bottom for acclimation at least 1 hr before carrageenan injections. Behavioral base lines were determined immediately before carrageenan injections.
  • 20 ⁇ l of 1% (w/v) carrageenan was injected intraplantarly into the right hindpaw of WT and TG mice.
  • the paw withdrawal thresholds to mechanical stimulation were measured at designated time points post injection.
  • the degree of inflammation induced by carrageenan was determined by measuring the thickness of right hindpaws before and at designated time points post injection with a caliper.
  • mice Tissue preparations and sectioning from TG and WT mice were performed simultaneously to minimize experimental variations. Mice were deeply anesthetized with 3% — 4% isofluorane and the L5/L6 level spinal cord segments were dissected and fixed in 4% paraformaldehyde. Paraffin-embedded spinal cord samples were sectioned (5 ⁇ M) using a microtome and mounted onto Superplus precleaned slides (Fisher Scientific, Pittsburg, PA) and kept at room temperature.
  • Immunohistological staining was used to localize Ca V a 2 O 1 expression in paraffin sections of spinal cord from the WT and TG mice. Briefly, tissue sections were treated with a citrate buffer (pH 6.0) followed by 3% H 2 O 2 in PBS, washed and blocked with 1% bovine serum albumin and 10% normal goat serum, then incubated with the monoclonal antibodies against the Ca V a 2 O 1 subunit (Sigma, Saint Louis, MO) overnight at 4 °C. After thorough rinsing, sections were incubated with biotinylated secondary antibody conjugated to horseradish peroxidase and then with avidin-biotin complex solution (Vectastain Elite ABC kit, Vector Laboratories). After several rinses, sections were developed in diaminobenzidine- H 2 O 2 solution, washed, and mounted on slides, air-dried, dehydrated and coverslipped with Permount. The positive staining in sections was examined and images were taken under a microscope.
  • Sensory neurons were obtained from the lumbar dorsal root ganglia of adult mice. Ganglia were enzymatically treated and mechanically dispersed as described 21 , except that the ganglia were bubbled in carbogen (5% CO 2 , 95% O 2 ) during the 20 minute collagenase treatment. DRG neurons were plated onto laminin/ornithine-coated glass coverslips and incubated for 2 hrs in MEM containing 10% fetal bovine serum at 37°C, 90% humidity, and 3% CO 2 . Neurons were then transferred to an L- 15 based medium containing 10 % fetal bovine serum, and stored at room temperature before recording. AU recording was performed within 8 hours of harvesting ganglia.
  • Voltage-clamp recordings were performed using an Axopatch 200B amplifier (Axon Instruments, Union City, CA) in the whole-cell patch configuration as described. Data were filtered with a 4-pole Bessel filter and digitized. Series resistance ⁇ 12 MD was compensated (>80%) by using amplifier circuitry. Only data obtained from neurons in which uncompensated series resistance resulted in voltage-clamp errors of less than 5 mV were used. A P/4 protocol was used for leak subtraction.
  • Ba 2+ was used as the charge carrier.
  • the bath solution contained 130 mM choline chloride, 5 mM BaCl 2 , 0.6 mM MgCl 2 , 10 mM Hepes, and 10 mM glucose (pH was adjusted to 7.4 with Tris base and osmolality was adjusted with sucrose to 325 milliosmolar).
  • the electrode solution contained 110 mM Cs-Methansulfonate, 30 mM TEA-Cl, 1 mM CaCl 2 , 5 mM MgCl 2 , 11 mM EGTA, 10 mM Hepes, 2 mM Mg-ATP, and 1 mM Li-GTP (pH was adjusted to 7.2 with Tris base and osmolality was adjusted with sucrose to 310 milliosmolar). Patch pipettes filled with electrode solution had resistances of 1.5-3 M ⁇ .
  • Ba 2+ currents were evoked from a holding potential of -70 mV.
  • Conductance- voltage curves were constructed for each neuron from I-V curves generated by assessing peak current evoked with 40-ms voltage steps between -80 and +60 mV taken at every 5 mV. Reversal potential was determined by interpolating between inward and outward currents. Conductance was determined by dividing peak current by driving force.
  • Conductance (g) gMax/(l+exp(-( Vm- V ⁇ 2 )Zk)); where gMax is maximal conductance, Vm is membrane potential, Vy 2 is the potential at which conductance is half of maximal and k is a slope factor.
  • Transgenic mice overexpressing the mouse Ca V O 2 O 1 cDNA were generated under the control of a mutant thy-1 promoter.
  • Thy-1 is a member of the immunoglobulin superfamily that is expressed in both neuronal and non-neuronal tissues, including thymocytes 15 .
  • the mutant used in the present study involved the deletion of a particular intron in the thy-1 gene, which produces the neuronal specificity of this promoter to drive down-stream target gene expression 13 ' 14 .
  • Data from Western blot analysis indicated that Ca V a 2 O 1 protein levels were elevated in forebrain, cortex, hippocampus, cerebellum, spinal cord, and DRG of the TG mice compared with their WT littermates.
  • Figures IA and IB depict enhanced Ca V a 2 O 1 expression in neuronal tissues from the transgenic mice.
  • Figure IA illustrates representative Western blot data from three independent determinations showing selective increases of Ca v ⁇ 2 ⁇ i subunit expression in neuronal tissue samples of adult transgenic mice. 1 - wild-type mice, 2 - transgenic mice.
  • Figure IB illustrates summarized Western blot data presented as the mean ⁇ SEM from three independent determinations. WT — wild-type mice, TG — transgenic mice.
  • Hindpaw withdrawal thresholds to mechanical stimulation were significantly reduced (tactile allodynia). Importantly, this reduction in mechanical threshold was similar to that seen in spinal nerve injured WT mice as can be seen from Figure 2A. Paw withdrawal latencies to noxious thermal stimulation were also significantly reduced (thermal hyperalgesia) in TG mice compared with WT littermates as can be seen from Figure 2B. The tactile allodynia state lasted for six months, the longest time tested (see Figure 2C).
  • FIG. 1 A shows that Ca V a 2 O 1 overexpression in the TG mice induced tactile allodynia (shown as reduced paw withdrawal thresholds to mechanical stimulation).
  • the tactile allodynia state in the transgenic mice was similar to that observed in mice with one- week L5 spinal nerve ligation injury, and reversed by systemic gabapentin, but not ketorolac, treatment.
  • the inventor compared the efficacies of gabapentin, morphine, and ketorolac on the allodynic and thermal hyperalgesic states of the TG mice.
  • Intraperitoneal administration of gabapentin reversed the tactile allodynia (see e.g., Figure 2A) and thermal hyperalgesia (see e.g., Figure 2B) in the TG mice.
  • the effects of gabapentin were dose- dependent with an estimated EC50 value -13 mg/kg (see Figure 4B).
  • Intraperitoneal ketorolac cyclooxygenase inhibitor used to treat inflammation and related pain conditions
  • Intraperitoneal ketorolac cyclooxygenase inhibitor used to treat inflammation and related pain conditions
  • Intraperitoneal morphine could only partially reverse tactile allodynia at the dose of 1 mg/kg, and failed to further reduce allodynia at a higher dose (5 mg/kg) (Figure 4B).
  • the tested drugs did not affect significantly the baseline tactile responses in the WT animals.
  • the sensitivity of TG mice to tactile stimulation has a pharmacological profile similar to that observed following nerve injury with the efficacies of gabapentin > morphine > cyclooxygenase inhibitors.
  • conductance (g) I/(Vt e st-V r ev) 5 where I is membrane current, V test , is the potential at which current was evoked and V rev is the current reversal potential), derived conductance- voltage relationship was compared to that based on tail currents (i.e., an instantaneous G-V); both methods yielded similar results (data not shown).
  • the increased Ca V a 2 O 1 subunit also caused a significant increase in the activation rate as can be seen from Figure 6A (assessed with an exponential fit of the increasing phase of voltage-gated I ⁇ a following membrane depolarization), compared with that in I ⁇ a from WT neurons, at voltages > -20 mV.
  • current activation was fitted with a single exponential equation in order to determine the activation time constant. Both traces were normalized to the peak inward current obtained during a 40 ms test pulse to -10 mV. Barium currents in TG neurons activated more rapidly than those in WT neurons.
  • fea activation rate became voltage dependent in TG neurons in contrast to the minimal voltage-dependence observed in fea from WT neurons as shown in Figure 6B.
  • the deactivation rate was slowed by increased membrane depolarization in I ⁇ a evoked in TG neurons, but increased with membrane depolarization in I ⁇ a evoked from WT neurons as can be seen from Figure 7B.
  • I ⁇ a inactivation was similar in DRG neurons from TG and WT mice (data not shown).
  • AU data are presented as the mean ⁇ SEM. The increases in current and maximal conductance are consistent with results from heterologous expression systems where the Ca V a 2 S 1 subunit has been shown to increase membrane expression of Ca V a 1 subunits 22 as well as increase open channel probability 23 .
  • Figure 8A depicts raw current evoked at -5 mV from a TG neuron before (control) and after application of increasing concentrations of gabapentin.
  • the transgenic mice were generally indistinguishable from their wild-type littermates and had normal motor functions, appearance, growth rate, and fertility.
  • the TG mice exhibited hypersensitivity to light touch (tactile allodynia) and thermal stimulation (thermal hyperalgesia).
  • the pharmacological sensitivity of the nociceptive changes was similar to that for neuropathic pain states.
  • inflammation-induced nociception and its pharmacological sensitivity were similar between WT and TG mice.
  • Intrathecal administration of gabapentin reversed dose-dependently the tactile allodynia state in the transgenic mice.
  • VGCC in DRG neurons from TG mice displayed altered kinetics, voltage-dependence of activation, increased densities of high threshold VGCC and gabapentin sensitivity.
  • modulation of VGCC activity at the spinal level by elevated calcium channel Ca V a 2 O 1 subunit is the molecular mechanism underlying the development and maintenance of certain types of abnormal sensations.
  • Gabapentin a compound with efficacy in neuropathic pain treatment 24 " 26 , can attenuate tactile allodynia in the TG mice without affecting the tactile threshold in normal animals, while other analgesics such as morphine and ketorolac have less or little efficacy in allodynia reversal 27 > 28 .
  • a Ca V a 2 O 1 subunit-mediated increase in low threshold T-type currents may contribute to bursting activity in DRG and spinal neurons following nerve injury as T-type currents have been shown to mediate depolarizing after- potential in DRG neurons 30 .
  • an increase in high threshold VGCC currents and excitability at the pre-synaptic terminals may contribute to an increase in transmitter release 31 ' 32 .
  • an increase in inward calcium currents through high threshold VGCC in inhibitory dorsal horn neurons may make the inhibitory neurons less excitable by activating calcium-activated potassium channels 33 , thus contributing to neuropathic pain-like behaviors.
  • These pathological changes may not be mutually exclusive, and their roles in the abnormal sensations remain to be determined.
  • Rat dorsal root ganglia express distinctive forms of the alpha2 calcium channel subunit. Neuroreport 11 , 3449-3452 (2000).
  • Hargreaves, K., Dubner, R., Brown, F., Flores, C. & Joris, J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77- 88. (1988).

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Abstract

Selon l'invention, la sous-unité alpha-2-delta-1 du canal calcique dépendant du potentiel est de préférence surexprimée dans un modèle animal transgénique non humain de douleur neuropathique. Les animaux transgéniques du type précité offrent l'avantage de présenter une allodynie tactile et/ou une hyperalgésie thermique non nocives tout en conservant une réaction à la douleur normale lors de lésions tissulaires et de douleurs inflammatoires. Par conséquent, et en opposition significative avec les modèles animaux de douleur neuropathique connus jusqu'à présent qui requièrent une lésion afin de provoquer la douleur neuropathique, le comportement réactionnel de l'animal à un stimulus peut être clairement attribué à la surexpression de la sous-unité alpha-2-delta-1.
EP05791319A 2004-07-13 2005-07-12 Modeles et procedes de nociception, de transduction de la douleur et de criblage de composes analgesiques Withdrawn EP1774318A2 (fr)

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AR061728A1 (es) * 2006-06-30 2008-09-17 Pfizer Prod Inc Composicion para tratamiento usando compuestos selectivos alfa-2-delta-1
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