WO2004028448A2 - Procede d'attenuation de la douleur par inhibition de la synthese des neurotransmetteurs - Google Patents

Procede d'attenuation de la douleur par inhibition de la synthese des neurotransmetteurs Download PDF

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WO2004028448A2
WO2004028448A2 PCT/US2003/028701 US0328701W WO2004028448A2 WO 2004028448 A2 WO2004028448 A2 WO 2004028448A2 US 0328701 W US0328701 W US 0328701W WO 2004028448 A2 WO2004028448 A2 WO 2004028448A2
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inhibitor
inflammation
glutamate
effective amount
pain
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PCT/US2003/028701
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English (en)
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WO2004028448A3 (fr
WO2004028448A9 (fr
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Kenneth E. Miller
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Miller Kenneth E
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Priority claimed from PCT/US2002/029108 external-priority patent/WO2003022261A1/fr
Priority claimed from US10/660,093 external-priority patent/US7504231B2/en
Application filed by Miller Kenneth E filed Critical Miller Kenneth E
Priority to AU2003294221A priority Critical patent/AU2003294221A1/en
Publication of WO2004028448A2 publication Critical patent/WO2004028448A2/fr
Publication of WO2004028448A3 publication Critical patent/WO2004028448A3/fr
Publication of WO2004028448A9 publication Critical patent/WO2004028448A9/fr

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    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • 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
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids

Definitions

  • the present invention generally relates to methods of alleviating pain, and more particularly, but not by way of limitation, to a method of alleviating chronic pain by regulation of neurotransmitter synthesis.
  • the present invention generally relates to methods of alleviating pain, and more particularly, but not by way of limitation, to a method of alleviating chronic pain by regulation of neurotransmitter synthesis.
  • Chronic inflammatory pain is a debilitating condition causing suffering, loss of work and loss of revenue.
  • Several methods of relieving pain from chronic inflammatory conditions such as rheumatoid arthritis, muscle damage, and osteoarthritis are known in the art.
  • the prior art methods of relieving pain have several unpleasant or serious side effects and require multiple daily administrations to be effective.
  • narcotics can be used for refractory chronic pain, but administration of narcotics has many side effects, including respiratory depression as well as the possibility of abuse.
  • another current method for relief of peripheral pain is topical application of capsaicin cream. This method may be effective for several days but produces severe acute pain in many patients.
  • some pain conditions such as myofascial pain and neuropathies due to nerve injury or disease currently do not have any effective therapies for alleviating pain associated therewith.
  • the present invention is related to a method of alleviating chronic pain in a subject for an extended period of time, as well as to a composition having analgesic effects that provides alleviation of chronic pain in a subject for an extended period of time.
  • the method of alleviating chronic pain of the present invention includes administration of an effective amount of at least one inhibitor of neurotransmitter synthesis into an inflammatory field.
  • Such inhibitor of neurotransmitter synthesis may be a glutamine synthetase inhibitor, a glutamine cycle inhibitor, a glutamate dehydrogenase inhibitor, a pyruvate carboxylase inhibitor, a glial cell tricarboxylic acid cycle inhibitor, or combinations thereof.
  • Pain is a major complication in arthritis and other disorders, and it is difficult to treat effectively for long periods of time. Persistent stimulation of sensory nerves in the area of inflammation is one of the contributors to chronic pain.
  • One stimulator of sensory nerve fibers is glutamate produced by the sensory nerve fibers themselves. Glutamate is a neurotransmitter utilized in signaling by the sensory neurons, and glutamate causes sensitization of surrounding sensory nerves, thereby producing the feeling of pain.
  • the present invention discloses that during experimental arthritis in rats, the sensory nerve cells increase production of glutaminase (GT), the neuronal enzyme that produces glutamate from glutamine.
  • GT glutaminase
  • Elevated amounts of glutaminase are shipped to the sensory nerve endings in the skin and joints, thereby causing increased amounts of glutamate to be produced (see FIG. 1).
  • the skin and joints from control rats have little to no detectable glutamate or glutaminase, so this enzyme and neurotransmitter have not been considered previously as possible therapeutic targets for pain relief via peripheral inhibition.
  • the method of the present invention includes local administration of an effective amount of at least one inhibitor of neurotransmitter synthesis, such as a glutaminase inhibitor, to a subject suffering from chronic pain at a site of inflammation, and the administration of the inhibitor of neurotransmitter synthesis results in a reduction in hociceptive responses, such as thermal and mechanical pain responses, at the site of inflammation for a period of at least two days without any resulting acute pain behavior.
  • at least one inhibitor of neurotransmitter synthesis such as a glutaminase inhibitor
  • rats were injected in the hindpaw with Complete Freund's adjuvant (heat killed Mycobacterium) to create an experimental arthritis. Rats with this type of chronic inflammation have increased sensitivity to pressure and temperature. After several days . of inflammation, some rats were injected with a glutaminase inhibitor or an inhibitor of neurotransmitter synthesis, such as but not limited to, 6-diazo-5- oxo-L-norleucine (DON), N-ethylmaleimide (NEM), dicoumarol (DC), bromothymol blue (BB), Palmitoyl CoenzymeA (P-CoA), methioninesulfoximine (MSO) and fluoroacetate (FA).
  • DON 6-diazo-5- oxo-L-norleucine
  • NEM N-ethylmaleimide
  • DC dicoumarol
  • BB bromothymol blue
  • P-CoA Palmitoyl CoenzymeA
  • MSO methi
  • the animal's sensitivities to pressure and temperature were brought to more normal values for many days, and these results were seen after only a single injection of the glutaminase inhibitor or inhibitor of neurotransmitter synthesis.
  • the present invention also includes a method of alleviating both acute and chronic pain in a subject for an extended period of time.
  • the method includes administration of a combination therapy of an effective amount of at least one compound having analgesic effects that provides substantially immediate relief of acute pain in combination with an effective amount of at least one inhibitor of neurotransmitter synthesis to a subject suffering from acute and chronic pain at a site of inflammation.
  • Such combination therapy will provide relief of both acute and chronic pain and results in a substantially immediate reduction of nociceptive responses at the site of inflammation that last for a period of at least two days without any resulting acute behavior.
  • Fig. 1 is a diagrammatic representation of the effects of Glutamate and glutaminase on peripheral sensory nerve stimulation and exacerbation of pain responses.
  • Inflammatory mediators in the skin and joints stimulate the release of glutamate and other agents that sensitize peripheral sensory nerve fibers (1).
  • Initial activation of the glutamine cycle to increase glutamate production as a response to acute pain occurs in the glutamine cycle enzymes via flux control or signal transduction pathways.
  • a retrograde signal (2) possibly nerve growth factor (NGF) causes the DRG cell body (3) to increase production of glutaminase (GT).
  • NGF nerve growth factor
  • DRG satellite cells (3) also are activated and increase production of glutamine synthetase (GS), glutamate dehydrogenase (GDH), pyruvate carboxylase (PC), and glutamine (GLN). Increased amounts of GTand glutamate are transported peripherally (4) producing elevated levels in peripheral primary afferent nerve terminals (5). Elevated levels of glutamate are released causing peripheral terminals to remain sensitized and exacerbates pain responses (1). Blockade of glutaminase with glutaminase metabolic inhibitors stops glutamate production and release and decreases pain.
  • GS glutamine synthetase
  • GDH glutamate dehydrogenase
  • PC pyruvate carboxylase
  • GNN glutamine
  • Increased amounts of GTand glutamate are transported peripherally (4) producing elevated levels in peripheral primary afferent nerve terminals (5). Elevated levels of glutamate are released causing peripheral terminals to remain sensitized and exacerbates pain responses (1). Blockade of glut
  • Fig.2 is a model regarding glutamate production in primary sensory neurons during chronic inflammation. Inflammatory mediators (lightning bolts) activate and sensitize peripheral afferent terminals. This leads to the release of glutamate (GLU) and other substances from peripheral terminals causing further sensit ⁇ zat ⁇ on (arrow). Inflammation stimulates keratinocytes to increase production of nerve growth factor (NGF).
  • GLU glutamate
  • NGF nerve growth factor
  • NGF is taken up and retrogradely transported to the neuronal cell body where it stimulates increased production of glutaminase (GT).
  • Increased production of GT occurs from stabilization of GT mRNA via zeta-crystallin:quinone oxidoreductase (ZC).
  • ZC quinone oxidoreductase
  • Increased amounts of GT are shipped to the periphery causing elevated glutamate production and release, further primary afferent sensit ⁇ zation, and exacerbation of no ⁇ ceptive responses.
  • Fig. 3 are photomicrographs illustrating the effects of fixation onglutam ⁇ nase (GT) ⁇ mmunoreactivity (IR) in the rat dorsal root ganglia (DRG).
  • DRG sections were processed simultaneously with a mouse monoclonal GT antibody (A, C) or a rabbit polyclonal GT antiserum (B, D).
  • Some DRG's (A,B) were fixed with 4% paraformaldehyde and others (C,D) were fixed with 70% picric acid and 0.2% paraformaldehyde.
  • intense GT-IR was restricted to small sized DRG neurons (long arrows) with both GT antibodies (A,B). Large to medium sized neurons (short arrows) were lightly stained (A,B).
  • FIG. 4 are photomicrographs illustrating Glutaminase (GT) immunoreactivity (IR) in rat dorsal root ganglia (DRG) following 7 days of CFA inflammation in the right hindpaw.
  • GT Glutaminase
  • IR immunoreactivity
  • DRG dorsal root ganglia
  • A In control sections, GT-IR was light to moderate in all neuronal cell sizes, small (long arrows) and medium to large (short arrows).
  • B Increased GT-IR intensity was observed in small (long arrows) and medium to large neurons (short arrows) in the left (contralateral) DRG following right hindpaw inflammation.
  • FIG. 5 is a graphic illustration of an image analysis of glutaminase (GT) immunoreactivity (IR) in L4DRG neurons after 7 days of CFA inflammation in the right paw. Data are presented as intensity divided by the area of the cell. DRG neurons were categorized into three area size groups: (A) small - 100 - 600 ⁇ m2,(B) medium - 600 - 1200 ⁇ mz, (C) large - >1200 ⁇ r . (A) Small sized neurons in the left DRG contained a significantly greater immunoreactive signal (*, p ⁇ 0.05) than controls. Neurons in the right DRG were more intensely stained than left DRG or controls (**, p ⁇ 0.01).
  • GT glutaminase
  • IR immunoreactivity
  • FIG. 6 is a graphic illustration of GT enzyme activity in the L» DRG at 7 days following CFA inflammation in the right hindpaw. GT activity from the right DRG (2.83 + 0.30 moles/kg/hr) was elevated (*, p ⁇ 0.05) over control values (2.20 + 0.18 moles/kg/hr). The left (contralateral) DRG (2.61 + 0.20 moles/kg/hr) was not significantly different from controls or the right (ipsilateral) DRG.
  • Fig. 7 is a grap c representation or the effects of inhibition of glutaminase on thermal and mechanical pain.
  • the hindpaw responses to thermal stimulation (FIG. 7A) and pressure sensitivity (FIG. 7B) were determined for a control rat, a control rat following glutaminase inhibition with 6-diazo-5-oxo-L-norleucine (DON), a rat after CFA inflammation, and a rat after CFA inflammation and following glutaminase inhibition with DON.
  • Fig. 8A is a graphic representation illustrating the efficacy of DON to provide long term pain relief from pressure (mechanical stimulation). After administration of DON at day three following CFA inflammation, pain relief occurred for several days with three different doses of DON (0.1- 10 ⁇ Moie/25 ⁇ l).
  • Fig. 8B is a graphic representation representing the DON dose response for pain relief from pressure stimulation. The area under the curve for each dose was determined from Day 3 to Day 5. No differences in the amount of pain relief were determined for the doses tested (0.1 - 10 ⁇ Mole/25 ⁇ l).
  • Fig. 9A is a graphic representation illustrating the efficacy of DON to provide long term pain relief to heat. After administration of DON at day three following CFA inflammation, pain relief occurred for several days with three different doses of DON (0.1 - 10 ⁇ Mole/25 ⁇ l).
  • Fig. 9B is a graphic representation illustrating the DON dose response for pain relief from thermal stimulation. The area under the curve for each dose was determined from Day 3 to Day 5. Pain relief was most efficacious at the higher doses (1 - 10 ⁇ Mole/25 ⁇ l).
  • Fig. 10 are graphic representations illustrating that intraplantar injection of DON into the hindpaw of normal rats does not affect pressure or thermal senstivities.
  • DON was injected (10 ⁇ Mole/25 ⁇ l) on day three. Both the pressure (Fig. 10A) and thermal (Fig. 10B) sensitivities in DON-treated rats were the same as saline controls.
  • Fig. 11A is a graphic representation demonstrating the efficacy of N-ethylmaleimide (NEM) to provide long term pain relief to pressure (mechanical stimulation). After administration of NEM (10 mM/25 ⁇ l) at day three following CFA inflammation, pain relief occurred for several days.
  • Fig. 1 IB is a graphic representation illustrating the efficacy of NEM to provide long term pain relief from heat. After administration of NEM (10 mM/25 ⁇ l) at day three following CFA inflammation, pain relief occurred to near normal levels at days 4 and 6.
  • FIG. 12 are photomicrographs illustrating glutamate immunoreactivity in tissue sections from the hindpaw skin of a control rat (FIG. 12A), a rat after CFA inflammation (FIG. 12B), and a rat after CFA inflammation and following glutaminase inhibition with NEM (FIG. 12C).
  • FIG. 12A very little glutamate immunoreactivity is detected in sensory nerves (arrows) in normal skin.
  • FIG. 12B after CFA inflammation, sensory nerve fibers contain elevated amounts of glutamate (arrows), in FIG. 12C, following CFA inflammation and glutaminase inhibition with NEM or DON, glutamate levels in sensory nerve fibers (arrows) are reduced to near normal levels. Similar results in all three conditions occur for glutaminase immunoreactivity in sensory nerves.
  • Fig. 13A is a graphic representation demonstrating the use of two inhibitors at regulatory sites on glutaminase and their efficacy to provide long term pain relief to pressure (mechanical stimulation). After administration of Palmitoyl Coenzyme A (P-CoA, 2 mM/25 ⁇ l) or bromothymol blue (BB, 200 ⁇ M/25 ⁇ l) at day three following CFA inflammation, pain relief occurred for several days.
  • P-CoA Palmitoyl Coenzyme A
  • BB bromothymol blue
  • Fig. 13B is a graphic representation illustrating the efficacy of P-CoA and BB to give long term pain relief to heat.
  • P-CoA 2 mM/25 ⁇ l
  • BB 200 ⁇ M/25 ⁇ l
  • Fig. 14 are photomicrographs illustrating that glutaminase production in many cells is regulated by zeta-crystallin:quinone ox ⁇ doreductase (ZC).
  • ZC quinone ox ⁇ doreductase
  • ZC-immunoreact ⁇ vity was examined in the rat L 4 DRG during inflammation at an early and later time point (2, 6 days).
  • ZC-IR in DRG neurons of control rats shows a moderate staining of the cytoplasm of all neurons.
  • ZC-IR is elevated in the cytoplasm and now appears in the nuclei of many neurons (arrows).
  • ZC-IR remains elevated at 6 days of inflammation and occurs mainly in the cytoplasm although some nuclei (arrows) contain light ZC-IR.
  • Fig. 15 is a graphic representation that illustrates that dicoumarol, a ZC inhibitor, disrupts increased glutaminase production during chronic inflammation and decreases the prolonged hyperalgesia of chronic inflammation.
  • Inflammation was initiated with complete Freund's adjuvant (CFA) at Day 0, and dicoumarol (15 ⁇ l @ 500 ⁇ M) or saline was administered intrathecally on days 0, 1 and 2.
  • Thermal latencies and pressure responses (not shown) were recorded, and both the groups with inflammation (CFA) and inflammation plus dicoumarol (CFA + DC) experienced hyperalgesia and allodynia during acute inflammation (Day 1).
  • CFA + DC rats experienced hyperalgesia and allodynia during acute inflammation (Day 1).
  • the responses of CFA + DC rats became less hyperalgesic and allodynic.
  • the DRG's from the rats were collected and processed for glutaminase and ZC-IR, as shown in Fig. 16.
  • Fig. 16 are photomicrographs illustrating that dicoumarol inhibits ZC and glutaminase production.
  • ZC-IR was elevated (A) in rats with inflammation, but the ZC-IR (B) from rats treated with DC during inflammation was similar to controls.
  • ZC-IR was found in the cytoplasm and nuclei (arrows) from rats with inflammation, whereas in rats treated with DC during inflammation, the nuclei (arrows) were not stained and ZC-IR was found primarily in the cytoplasm.
  • glutaminase-IR was observed at moderate levels from controls (C), elevated following inflammation (D), and similar to controls in rats treated with DC during inflammation (E).
  • FIG. 17 are photomicrographs illustrating representative immunohistochemical controls.
  • A Rabbit anti-glutamine absorption control in sciatic nerve. Compare with an adjacent section stained for glutamine (see FIG. 19A).
  • B Rabbit anti-pyruvate carboxylase absorption control in DRG with rabbit anti-pyruvate carboxylase. Compare with an adjacent sectiono stained for pyruvate carboxylase (see FIG. 18C).
  • C Omission of primary antiserum and subsequent processing with horse anti-mouse IgG and FITC-Avidin. No specific staining is observed in these controls.
  • FIG. 18 are photomicrographs illustrating glutamine and enzyme immunoreactivity in DRG satellite cells.
  • A Intensely labeled glutamineimmunoreactive satellite cells (arrows) surround the DRG cell bodies (*).
  • B Satellite cells immunoreactive for glutamine synthetase surround DRG cell bodies (*). As with glutamine, GS immunoreactivity appears to have a cytoplasmic appearance.
  • C Pyruvate carboxylase-immunoreactivity found in satellite cells (arrows) was punctuate in appearance. This section was adjacent to the absorption control shown in FIG. 17B.
  • D Confocal micrograph of glutamate dehydrogenase immunoreactivity in satellite cells (arrows). GDH and PC immunoreactivities were punctuate in the cells and presumably are mitochondria (see detailed description herein below).
  • FIG. 19 are photomicrographs illustrating immunoreactivity in Schwann cells.
  • A GLutamine immunoreactivity was found along the course of the sciatic nerve in long immunoreactive cellular processes. GLNimmunoreactive cell bodies (arrows) were apparent, also. This section was adjacent to the absorption control shown in FIG. 17A.
  • B Confocal micrograph of glutamine synthetase immunoreactivity in Schwann cells. Arrows point to a node of Ranvier and arrowheads point to a Schwann cell body immunoreactive for GS.
  • C Pyruvate carboxylase immunoreactivity occurred throughout the course of the sciatic nerve in long immunoreactive cellular processes and Schwann cell bodies (arrows).
  • FIG. 20 are photomicrographs illustrating double immunofluorescence for glia and neurons.
  • satellite cells green
  • neurons red
  • GS appeared to stain all satellite cells.
  • Small DRG neurons ⁇ 600 ⁇ m 2
  • medium 600-1200 ⁇ m 2
  • large asterisks, >1200 ⁇ m 2
  • DRG neurons were surrounded by three to seven cells (long arrows) in 20 ⁇ m thick sections.
  • FIG. 21 is a diagrammatic represenation illustrating that glial cell metabolism is intricately related to neuronal metabolism.
  • This diagram illustrates that glutamine, glutamine synthetase, glutamate dehydrogenase, and pyruvate carboxylase are located in the peripheral nervous system in satellite cells of the DRG and Schwann cells of the peripheral nerve. These enzymes could have major roles in supporting peripheral neuronal metabolism and neurotransmission.
  • Glial cells take up glutamate from the extracellular milieu via transporters (GLAST, GLT-1) and GS converts it to glutamine.
  • Glutamine can be shuttled out of glial cells by the SN1 glutamine transporter and taken up by neurons via the SAT/ATA glutamine transporters for use by glutaminase (GT) in the glutamine cycle.
  • glutamine is an important branch point substrate for purine synthesis via GPATase.
  • Glutamate dehydrogenase is a bidirectional enzyme that can either add glutamate for GS in the glutamine cycle or convert glutamate to 2-oxoglutarate for the TCA cycle.
  • 2-Oxoglutarate and other TCA intermediates such as malate can be shuttled from glia for use in neurons. Malate also can be converted to pyruvate via malic enzyme (ME).
  • FIG. 22 are graphic representations of the effects of inhibition of glutamine synthetase on thermal and mechanical pain.
  • the hindpaw responses of rats to pressure sensitivity (FIG. 22A) and thermal senstiv ⁇ ty (FIG. 22B) were determined for a control rat, a rat after CFA inflammation, and a rat after CFA inflammation and following glutamine synthetase inhibition with methionine sulfoximine (MSO).
  • MSO methionine sulfoximine
  • FIG. 23 is a graphic representation illsutrating the effects of intrathecal injection of MSO, DON or fluoroacetate (FA) on pressure sensitivity in the hindpaw of rats following CFA inflammation.
  • FIG. 24 are photomicrographs illustrating that satellite (glial) cells in the dorsal root ganglia (DRG) increase 'glutamine cycle' enzymes and products during chronic inflammation. Inflammation was induced with intraplantar CFA in the right hindpaw. In normal DRG's, glutamine synthetase (A; GS) and glutamine (C; the product of GS) immunoreactivity is located in satellite cells surrounding DRG neuronal cell bodies. After 3 days of inflammation, increased immunoreactivity for GS and glutamine is observed in most satellite cells.
  • DRG dorsal root ganglia
  • the method of the present invention includes administration of an effective amount of at least one inhibitor of neurotransmitter synthesis to a subject suffering from chronic pain at a site of inflammation.
  • the inhibitor of neurotransmitter synthesis is a glutaminase inhibitor.
  • glutaminase inhibitors or "GT inhibitors” as used herein will be understood to include inhibitors that affect the activity of the glutaminase enzyme, such as inhibitors that may affect binding of glutamine, glutamate or various cofactors to the enzyme. That is, a GT inhibitor may block binding of the substrate glutamine to glutaminase, inhibit release of the product glutamate from glutaminase, or block cofactor binding and therefore slow the catalytic rate of the enzyme.
  • GT inhibitors examples include nonspecific inhibitors such as amidotransferase inhibitors and long chain fatty acids.
  • specific inhibitors of glutaminase activity which may be utilized in the method of the present invention include 6-diazo-5-oxo-L-norleucine (DON), N-ethylmaleimide (NEM),/?-chloromercuriphenylsulfonate (pCMPS), L-2-amino- 4-oxo-5-chloropentoic acid, DON plus ⁇ -carbamoyl-L-serine, acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid],azaserine, palmitoyl coenzyme A (palmitoyl CoA), stearoyl coenzyme A (stearoyl CoA), bromothymol blue, and combinations or
  • glutaminase inhibitors or ⁇ GT inhibitors will also be understood to include inhibitors of glutaminase production.
  • Inhibitors of glutaminase production include, but are not limited to, inhibitors of transcription of the gene encoding glutaminase as well as inhibitors of regulatory proteins involved in transcription of the glutaminase gene.
  • Inhibitors of glutaminase production also include, but are not limited to, inhibitors of translation of the glutaminase mRNA and inhibitors of stabilization of the glutaminase mRNA as well as compounds which increase degradation of the glutaminase mRNA. For example, as shown in FIG.
  • NGF nerve growth factor
  • ZC quinone oxidoreductase
  • GT inhibitor a compound capable of neutralizing or inhibiting ZC or NGF also falls within the scope of the terms "glutaminase inhibitor” or "GT inhibitor”.
  • DC dicoumarol
  • Glutaminase inhibitor is shown herein to inhibit ZC activity and thus inhibit GT production, thereby relieving pain. Therefore, the terms "glutaminase inhibitor”, “inhibitor of glutaminase enzyme activity” and “inhibitor of glutaminase synthesis” can all be used interchangeably herein.
  • an inhibitor of neurotransmitter synthesis will also include compounds that inhibit, either directly or indirectly, the synthesis of a substrate that is converted to a neurotransmitter.
  • glutaminase converts glutamine to the neurotransmitter glutamate, and therefore inhibitors of enzymes which are directly or indirectly involved in synthesis of glutamine, such as but not limited to pyruvate carboxylase, glutamate dehydrogenase, glutamine synthetase, and various known enzymes of the tricarbpxylic acid (TCA) cycle, also fall within the scope of the term “inhibitor of neurotransmitter synthesis", as used in accordance with the present invention.
  • TCA tricarbpxylic acid
  • Examples of pyruvate carboxylase inhibitors that may be used in accordance with the present invention include, but are not limited to, phenyl acetic acid (PAA), phenylacetyl Coenzyme-A, phenylacetyl Co-A ester, oxamate, and combinations and derivatives thereof.
  • Examples of glutamine synthetase inhibitors that may be used in accordance with the present invention include, but are not limited to, methion ⁇ ne-S-sulfoximine (MSO), phosphinothr ⁇ cin (PPT), 4-N-hydroxy-L-2,4-d ⁇ aminobutyric acid (NH-DABA), Delta-hydroxylysine, and combinations and derivatives thereof.
  • glutamate dehydrogenase inhibitors examples include, but are not limited to, bromofuroate, Palmitoyl- Coenzyme-A (Palm ⁇ toyl-Co-A), vanadium compounds (including, but not limited to, orthovanadate, vanadyl sulphate, vanadyl acetylacetonate, and combinations thereof), glutarate, 2-oxoglutarate ( -ketoglutarate), estrogen, estrogen analogues, pyridine-2,6-dicarboxylic acid, and derivatives thereof as well as combinations thereof, such as, but not limited to, 2-oxoglutarate and vanadyl sulphate.
  • bromofuroate Palmitoyl- Coenzyme-A
  • Palm ⁇ toyl-Co-A vanadium compounds
  • vanadium compounds including, but not limited to, orthovanadate, vanadyl sulphate, vanadyl acetylacetonate,
  • glial cell TCA cycle inhibitors examples include, but are not limited to, fluoroacetate, fluorocitrate, and combinations and derivatives thereof.
  • the term "inhibitor of neurotransmitter synthesis" will also include two or more of the inhibitors listed above from two or more different classes, for example, but not by way of limitation, the combination of a glutamine synthetase inhibitor and a glial cell TCA cycle inhibitor.
  • the method of alleviating chronic pain of the present invention results in pain relief (both thermal and mechanical) for several days by way of peripheral glutaminase inhibition without any resulting acute pain behavior, as observed by the prior art methods, such as application of capsaicin cream. While the initial experiments described herein have utilized injection of an inhibitor of neurotransmitter synthesis, the inhibitor of neurotransmitter synthesis should also be amenable to topical or oral application. For example, an oral inhibitor of neurotransmitter synthesis given as a prodrug or with limited to substantially no penetration into the central nervous system would also be effective in producing widespread pain relief.
  • the method of alleviating chronic pain of the present invention is not limited to injection of an inhibitor of neurotransmitter synthesis but also includes other methods of application of such inhibitor(s), such as, but not limited to, oral, topical, transdermal, parenteral, subcutaneous, intranasal, intramuscular and intravenous routes, including both local and systemic applications.
  • the formulations containing at least one inhibitor of neurotransmitter synthesis described herein may be designed to provide delayed or controlled release using formulation techniques which are well known in the art. Using such methods of delayed or controlled release would provide an even longer period of pain relief.
  • subject as used herein will be understood to include a mammal, that is, a member of the Mammalia class of higher vertebrates.
  • mammal as used herein includes, but is not limited to, a human.
  • method of alleviating pain as used herein will be understood to include a reduction, substantial elimination or substantial amelioration of the condition of pain, including nociceptive behavior in response to mechanical or thermal stimuli.
  • nociceptive responses as used herein will be understood to refer to responses that occur in reaction to pain, such as mechanical or thermal stimuli.
  • pain as used herein will be understood to refer to all types of pain, including acute pain and chronic pain.
  • chronic pain as used herein will be understood to include, but is not limited to, pain associated with rheumatoid arthritis or osteoarthritis, neuropathic pain, pain associated with muscle damage, myofascial pain, chronic lower back pain, pain resulting from burns, and the like.
  • the present invention also includes a method of alleviating both acute and chronic pain in a subject for an extended period of time.
  • the method includes administration of a combination therapy of an effective amount of at least one compound having analgesic effects that provides substantially immediate relief of acute pain in combination with an effective amount of at least one inhibitor of neurotransmitter synthesis to a subject suffering from acute and chronic pain at a site of inflammation.
  • Such combination therapy will provide relief of both acute and chronic pain and results in a substantially immediate reduction of nociceptive responses at the site of inflammation that last for a period of at least two days without any resulting acute behavior.
  • Compounds having analgesic effects that may be utilized in such a method are known to those of ordinary skill in the art and include, but are not limited to, benzocaine, lidocaine, novocaine, and the like.
  • compounds which function as glutamate inhibitors or inhibitors of glutamate binding to glutamate receptors on peripheral sensory nerves may also be utilized as the compound having analgesic effects in the above-described combination therapy.
  • Other compounds having analgesic effects that may be utilized in the method of the present invention include aspirin, acetaminophen, paracetamol, indomethacin, cholinergic analgesics, adrenergic agents, nonsteroidal anti-inflammatory drugs, and other like compounds known in the art.
  • bradykinin, serotonin, prostaglandins, ATP, H+ and glutamate activate and/or sensitize the afferent limb of primary sensory neurons by increasing spontaneous activity, lowering activation threshold, and increasing or prolonging firing to stimuli [Benton et al., 2000; Millan, 1999; Wood and Mederty, 1997; Zhou et al., 1996].
  • Sensory neurons respond chronically to inflammation by increasing tachykinin (substance P [SP]) and calcitonin generelated peptide (CGRP) expression and content in dorsal root ganglia (DRG) [Calza et al., 1998; Donaldson et al., 1992; Garrett et al., 1995; Hanesch et al., 1993; Hanesch et al., 1995; Noguchi et al., 1988; Smith et al., 1992] and enhanced immunoreactivity in the spinal dorsal horn [Marlier et al., 1991], skin and joints [Ahmed et al., 1995; Nahin and Byers, 1994].
  • SP tachykinin
  • CGRP calcitonin generelated peptide
  • peptide containing neurons also are glutamatergic [Battag ⁇ a and R ⁇ stioni, 1988; DeBiasi and Rustion ⁇ , 1988; Miller et al., 1993; Miller et al., 2002], using glutaminase (GT) as the synthetic enzyme for neurotransmitter glutamate production.
  • GT glutaminase
  • GT glutaminase
  • glutamateimmunoreactive fibers in the spinal cord increase 30% at 4 hr. and nearly 40% at 8 hr. [Sluka et al., 1992].
  • extracellular levels of spinal glutamate in rats are 150% above controls [Yang et al., 1996] indicating a possible prolonged, activity-dependent recruitment of glutamate release from central primary afferents.
  • Pressure sensitivities determined with von Frey hairs are expressed as gm force. Pressure and thermal control values for each day were compared with CFA values with a Student's t test. * p ⁇ 0.01, ** p ⁇ 0.0001
  • GT-IR in the DRG was evaluated with 2 fixatives and 2 antibodies.
  • a 4% PFA fixative small (100-600 ⁇ m 2 ) neuronal cell bodies were labeled intensely with GT-IR (Fig. 3A, 3B).
  • the 70% PA, 0.2% PFA fixative the majority of DRG neuronal cell bodies were labeled with both GT antibodies (Fig. 3C, 3D).
  • the PA-PFA fixative was used for the remainder of the experiments described herein.
  • the seven day rat immunohistochemistry images were analyzed with the SCION image analysis program in order to quantify the GT-IR intensities of three different sizes of DRG cell bodies (Fig. 5),
  • the small (100- 600 ⁇ m 2 ) DRG cell bodies showed the greatest amount GT-IR/area and the largest differences in intensities among control, left, and right cell bodies of the three different DRG cell sizes.
  • the small DRG cell bodies had intensities of 484.6 ⁇ 2.0/ ⁇ m 2 for controls, 532.6 ⁇ 1.7/ ⁇ m 2 for the left DRG from CFA rats, and 585.6 ⁇ 7.7/ ⁇ m 2 for the right DRG from CFA rats (Fig. 5A).
  • the GT-IR intensities for the medium (600-1200 ⁇ m 2 ) DRG cell bodies were 469.3 + 4.9/ ⁇ m 2 for the control, 509.6 + 8.9/ ⁇ m 2 for the left DRG from CFA rats, and 556.9 ⁇ 7.7/ ⁇ m 2 for the right DRG from CFA rats (Fig. 5B).
  • the GT-IR intensities for the large (>1200 ⁇ m 2 ) DRG cell bodies were 431.6 + 12.2/ ⁇ m 2 for the control, 448.5 + 10.7/ ⁇ m 2 for the left DRG from CFA rats, and 491.0 ⁇ 5.8/ ⁇ m 2 for the right DRG from CFA rats (Fig. 5C).
  • GT levels were elevated at the neuronal cell body and peripheral fibers and in response to chronic inflammation, several GT inhibitors were examined for their ability to alleviate nociceptive responses to thermal and mechanical stimuli.
  • Several compounds inhibit GT enzyme activity (Shapiro et al., 1978, 1979; Kvamme et al., 1975, 1991; Kvamme &.
  • DON 6-diazo-5-oxo-L-norleucine
  • NEM N-ethylmaleimide
  • Intraparenchymal or ICV injection of DON inhibits GT and causes a decrease in glutamate and GT for several days in rat brain until neurons synthesize new GT (Bradford et al., 1989; Kaneko et al., 1992; Conti & Minelli, 1994). Therefore, DON and NEM were administered peripherally during chronic inflammation to observe the effect of GT enzyme inhibition on nociceptive responses.
  • CFA complete Freund's adjuvant
  • DRG neurons increase glutaminase (GT) production for shipment to peripheral terminals causing elevated glutamate (GLU) levels in skin and joints. Increased glutamate release may be responsible for maintaining thermal hyperalgesia and/or mechanical allodynia.
  • GT inhibitors including 6-diazo-5-oxo- Lnorleucine (DON) and N-ethylmale ⁇ mide (NEM)
  • DON 6-diazo-5-oxo- Lnorleucine
  • NEM N-ethylmale ⁇ mide
  • Fig. 8A the efficacy of DON to provide long term pain relief to pressure (mechanical stimulation) was determined by using three different doses of DON (0.1 - 10 ⁇ Mole/25 ⁇ l). After administration of DON at day three following CFA inflammation, pain relief occurred for several days with all three doses of DON.
  • a dose response curve was constructed, as shown in Fig. 8B.
  • the area under the curve for each dose was determined from Day 3 to Day 5. No differences in the amount of pain relief were determined for the doses tested (0.1 - 10 ⁇ Mole/25 ⁇ l).
  • the efficacy of DON to provide long term pain relief to heat was determined for the same three doses of DON (0.1 - 10 ⁇ Mole/25 ⁇ l). After administration of DON at day 3 after CFA inflammation, pain relief occurred for several days with all three doses of DON.
  • a dose response curve was constructed, as shown in Fig. 9B.
  • the area under the curve for each dose was determined from Day 3 to Day 5. Pain relief was most efficacious at the higher doses (1 - 10 ⁇ Mole/25 ⁇ l).
  • Fig. 10 illustrates DON controls.
  • DON was injected (10 ⁇ Mole/25 ⁇ l) on day 3, and such injection of DON does not affect thermal or pressure sensitivities.
  • Both the pressure (Fig. 10A) and thermal (Fig. 10B) sensitivities in DON treated rats were the same as saline controls.
  • NEM N-ethylmaleimide
  • P-CoA (2 mM/25 ⁇ l) or BB (200 ⁇ M/25 ⁇ l) was administered at day three following CFA inflammation, and both were shown to be effective in providing long term pain relief to pressure (mechanical stimulation, as shown in Fig. 13A) and heat (thermal stimulation, as shown in Fig. 13B).
  • P-CoA (• line) provided pain relief from Days 4 -7
  • BB ( ⁇ line) gave pain relief on Day 5.
  • P-CoA provided pain relief to near normal levels from Days 4 - 7, while BB provided pain relief from Days 5 -7 and at near normal levels from Days 6 and 7.
  • FIG 14 illustrates that glutaminase production in many cells is regulated by zeta-crystalIin:quinone oxidoreductase (ZC).
  • ZC zeta-crystalIin:quinone oxidoreductase
  • ZC-IR remains elevated at 6 days of inflammation and occurs mainly in the cytoplasm, although some nuclei (arrows) contain light ZCIR.
  • ZC stabilizes glutaminase mRNA
  • inhibition of ZC should not allow neurons to increase glutaminase production during inflammation.
  • Intrathecal (i.t.) cannulae were implanted to the L4 DRG, and rats recovered several days. Inflammation was initiated with complete Freund's adjuvant (CFA) at Day 0 and dicoumarol (15 ⁇ f @ 500 ⁇ M) or saline was administered i.t. on days 0, 1 and 2. Thermal latencies (Fig. 15) and pressure responses (not shown) were recorded. Both the groups with inflammation (CFA) and inflammation plus dicoumarol (CFA + DC) experienced hyperalgesia and allodynia during acute inflammation (Day 1).
  • sensory neurons respond chronically by modifying neuropeptide, receptor, and ion channel production [Calza et al., 1998; Donaldson etal., 1992; Garrett tal., 1995; Gould etal., 1998; Hanesch etal., 1993, 1995; Miilan, 1999; Mulder et a!., 1997, 1999; Nahin and Byers, 1994; Noguchi et al., 1988; Seybold et al., 1995; Smith et al., 1992; Tate et al., 1998; Zhang et al., 1998].
  • DRG neuronal cell bodies have an altered phenotype that maintains or exacerbates inflammatory sensitization [Donnerer et al., 1992; Hanesch et al., 1993; Nah ⁇ n and Byers, 1994; Ahmed et al., 1995; Garrett et al., 1995] and since most DRG neurons are glutamatergic [Miller et al., 1993, 2002a], it was necessary to determine if long-term alterations occur in glutamate metabolism of primary sensory neurons in chronic inflammation. Indeed, it has been shown herein that long-term elevated GT levels occur in DRG neurons during chronic inflammation. In the present invention, the largest long term increase of GT IR occurred in small and medium sized DRG neuronal cell bodies.
  • Neurons of these sizes commonly are considered to include nociceptive neurons that give rise to unmyelinated C and lightly myelinated A-delta fibers [Cameron et al., 1986; Garry et al., 1989; Harper and Lawson, 1985; Willis and Coggeshall, 1991]. Elevated amounts of GT are likely to lead to increased production of glutamate in nociceptive, primary afferent nerve terminals in the spinal cord. SP and CGRP are found along with glutamate in primary afferent terminals [Merighi et al., 1991], and the co-release of glutamate and these neuropeptides generate hypersensitivity of spinal neurons [Besson etal., 1999]. Therefore, an increase in the amount of GT during chronic inflammation may lead to increased production and release of glutamate along with substance P and CGRP. Increased production and release of these substances could sustain spinal hypersensitivity maintaining a state of chronic pain.
  • a cycle therefore, of increased glutamate production and release, elevated numbers of axons with glutamate receptors, and maintenance of sensitization of peripheral nerve terminals would further exacerbate the process of chronic pain from the periphery.
  • long-term changes due to inflammation include an increase in glutaminase in the rat DRG cell body. This increase in glutaminase will lead to elevated production and release of glutamate at both the peripheral and central processes of primary afferents.
  • An increase in glutamate metabolism in primary sensory neurons may be partly responsible for heightened nociceptive sensitivity in tonic pain models.
  • Prevention of increased glutaminase production or inhibition of glutaminase enzyme activity therefore, may reduce or block some nociceptive responses in inflammatory models.
  • NGF neurotrophic factor
  • NGF causes an increase in mRNA for growth-associated protein 43 and preprotachykinin A [SP] in DRG neurons, and anti-NGF prevents these increases [Malcangioetal., 1997; Reinert et al., 1998], These DRG neurons also are glutamatergic, but the influence of NGF on glutamate metabolism in chronic inflammation has not been investigated.
  • NGF influences GT expression in DRG neurons in ⁇ tero and in oculo [McDougal et al., 1981; Miller et al., 1999], and preliminary data indicate that NGF influences GT expression in the DRG and peripheral primary afferents similar to inflammation [Miller et al., 2001]. Therefore, it is believed that by inhibiting NGF's role on modifying glutamate metabolism in DRG neurons during chronic inflammation, GT expression and therefore glutamate levels can be reduced, thereby reducing nociceptive responses.
  • the rate of GT transcription is unaffected by these conditions, but the level of total and translatable GT mRNA is increased by stabilization of GT mRNA [Tong et al., 1987; Curthoys and Watford, 1995; Curthoys and Gstraunthaler, 2001]. Stabilization occurs by the binding of a cytosolic protein to an eight-base AU sequence repeat within the 3'-nontranslated region of the GT mRNA [Hansen et al., 1996; Laterza et al., 1997; Laterza and Curthoys, 2000; Porter et al., 2002].
  • This stabilizing protein is zeta-crystallin:q ⁇ inone oxidoreductase [ZC; Tang and Curthoys, 2001; Curthoys and Gstraunthaler, 2001]. Since nervous system GT is similar or identical to kidney GT [Curthoys and Watford, 1995; Holcomb et al., 2000], it is possible that a similar mechanism exists in primary sensory neurons. Therefore, it is important to determine the role ZC has in increased GT production in DRG neurons during chronic inflammation.
  • ZC is inhibited by several classes of compounds [al-Hamidi et al., 1997; Rabbani and Duhaiman, 1998; Winsk ⁇ et al., 2001; Bazzi et al., 2002].
  • Dicoumarol [DC] is a potent, competitive inhibitor of ZC, binding to the pyridi ⁇ e nucleotide site [Hollander and Ernster, 1975; Hosada et al., 1974, Jaiswal, 2000] and has been used as the traditional inhibitor of ZC in many studies [Cross et al., 1999; Winski et al., 2001]. Therefore, DC was administered to DRG neuronal cell bodies during chronic inflammation to disrupt ZC's regulation of GT production.
  • nociceptors are categorized into Adelta fibers that evoke a rapid, acute pain sensation and C fibers that produce a later, 'dull' pain [Campbell, 1987].
  • primary hyperalgesia typified by increased sensitivity to mechanical, heat, and chemical stimuli.
  • a secondary hyperalgesia in nearby undamaged areas is thought to be due to central spinal mechanisms [review, Millan, 1999].
  • Sensitizing substances released during acute inflammation include: 5-HT, histamine - mast cells; prosta-glandins (PG) - fibroblasts, Schwann cells; cytokines, H+, nitric oxide (NO) - macrophages; ATP, H+- damaged cells; 5-HT - platelets; ATP, NO - blood vessels; bradykinin, other kinins - blood; PG, neuropeptide Y, ATP - sympathetic terminals. There also is a neurogenic component of inflammation due to the release of bioactive substances from peripheral primary afferent terminals.
  • Substance P SP
  • CGRP calcitonin generelated peptide
  • SP stimulated terminals or via axon reflexes (collateral fibers) further sensitizing surrounding afferent terminals and tissues.
  • These algogenic substances influence primary afferents to increase Ca2+ and Na+ permeability, decrease K+ permeability, increase intracellular Ca2+concentration, NO and PG production, and adenylate cyclase and phospholipase C activities [Millan, 1999].
  • the peripheral primary terminal therefore, is acutely sensitized producing primary hyperalgesia.
  • Glutamate also is involved in neurogenic inflammation.
  • EAA antagonists sensitize peripheral afferents and produce acute nociceptive reflexes/hyperalgesia that can be blocked by EAA antagonists [Ault and Hildebrand, 1993a,b; Jackson etal., 1995; Zhou et al., 1996; Davidson et al., 1997; Law and et al., 1997; Wang etal., 1997; Carlton et al., 1998; Ushida et al., 1999; Bhave et al., 2001]. Fibers of the Ab type also contain EAA receptors [Coggeshall and Carlton, 1997; Wood and Mederty, 1997] and may be involved in mechanical ailodynia [Millan, 1999].
  • glutamate metabolism is altered for weeks in rat primary sensory neurons during chronic inflammation. Elevated levels of glutamate and glutaminase (GT), its synthetic enzyme, occur in the neuronal cell bodies of dorsal root ganglia (DRG) followed by increases in the peripheral afferents of skin and joints. Chronic increase in production and release of glutamate can stimulate glutamate receptors on sensory afferents to produce hyperalgesia and ailodynia. Therefore, elevated peripheral levels of glutamate cause exaggerated nociceptive responses during chronic inflammation.
  • GT glutamate metabolism is altered for weeks in rat primary sensory neurons during chronic inflammation. Elevated levels of glutamate and glutaminase (GT), its synthetic enzyme, occur in the neuronal cell bodies of dorsal root ganglia (DRG) followed by increases in the peripheral afferents of skin and joints. Chronic increase in production and release of glutamate can stimulate glutamate receptors on sensory afferents to produce hyperalgesia and
  • ZC zeta-crystallin:quinone oxidoreductase
  • NGF nerve growth factor
  • ZC is a stabilizer of GT mRNA, allowing increased GT translation during times of cellular stress.
  • An effective amount of a ZC inhibitor can be administered to the DRG to disrupt GT mRNA stabilization and reduce nociceptive responses during the development of chronic inflammation.
  • (3) glutamate metabolism in primary sensory neurons can be modified by NGF. NGF has been implicated in chronic alterations of DRG neurons. Administration of NGF to na ⁇ ve rats and NGF neutralization in chronic inflammation should have a similar effect as a ZC inhibitor on nociceptive behavior and glutamate metabolism in primary sensory neurons. DETAILED DESCRIPTION OF FIGS.
  • the present invention also includes compounds that inhibit, either directly or indirectly, the synthesis of a substrate that is converted to a neurotransmitter.
  • glutaminase converts glutamine to the neurotransmitter glutamate, and therefore inhibitors of enzymes which are directly or indirectly involved in the synthesis of glutamine, such as but not limited to, pyruvate carboxylase, glutamate dehydrogenase, glutamine synthetase, and various known enzymes of the tricarboxyl ⁇ c acid (TCA) and glutamine cycles, also fall within the scope of the present invention.
  • TCA tricarboxyl ⁇ c acid
  • astrocytes within the central nervous system (CNS), astrocytes contain several glial-specific enzymes related to the tricarboxylic acid (TCA) and glutamine cycles.
  • TCA tricarboxylic acid
  • PC pyruvate carboxylase
  • GDH Glutamate dehydrogenase
  • GDH serves as a link between the TCA and glutamine cycles by reversibly converting 2-oxoglutarate into glutamate.
  • Glutamine synthetase is one of two integral enzymes of the glutamine cycle and converts glutamate into glutamine.
  • GDH enzyme activity has been described in DRG, dorsal roots, and peripheral nerves, although lower than in CNS regions.
  • the Schwann cells of the giant squid nerve have 10 times the amount of GDH enzyme activity compared to nerve fiber axoplasm.
  • DRG, dorsal roots, and peripheral nerves contain glutamine levels comparable to glutamate concentrations due to the high amount of GS activity found in dorsal roots and peripheral nerves.
  • GS is localized in satellite and Schwann cells based on uptake studies of radiolabeled glutamate. In these studies, glutamate quickly enters satellite and Schwann cells and rapidly is converted to glutamine.
  • FIG. 17A and B When sections were incubated in antiserum and respective antigen, no or weak immunoreactivity was observed (Fig. 17A and B). When the primary antisera were omitted, no or weak immunoreactivity was observed (Fig. 17C).
  • Use of the immunoperoxidase reaction with the omission of the primary antiserum caused some satellite cells in the DRG to appear (data not shown). These cells appeared when the reaction was allowed to proceed for several minutes after stopping the regularly stained immunoperoxidase sections. These data are similar to a previous paper describing endogenous peroxidase activity in glial cells. This type of staining did not appear in control sections stopped at the same time as regularly stained sections.
  • GDH and PC are localized to mitochondria, and a previous immunohistochemical study of GDH in the CNS demonstrated that the immunoreactive puncta are mitochondria.
  • IR Schwann cells were best observed around large diameter, myelinated axons (Figs. 19B,D and 20B). Immunoreactivity was most intense in three areas of these Schwann cells: cell body (perinuclear) cytoplasm, nodes of Ranvier, and the rim of cytoplasm outside of the myelin (Figs. 19B,D and 20B). The myelin sheath was not immunoreactive for any of the four substances (Fig. 20B).
  • DRG satellite and sciatic nerve Schwann cells contain specific enzymes related to the TCA and glutamine cycles.
  • Pyruvate carboxylase is ah anaplerotic enzyme that catalyzes the fixation of CO ⁇ to pyruvate to form oxaloacetate for entry into the TCA cycle [L. Hertz et al., 1999].
  • PC is important for the synthesis of glutamine and glutamate [W.C. Gamberino et al.1997; R.P. Shank et al.1985], and PC- immunoreactivity has been localized to astrocytes [M Cesar and B. Hamprecht, 1995; R.P. Shank et al., 1985].
  • the presence of PC in satellite cells may explain uptake and metabolism studies in sensory ganglia [J.L.
  • [ 14 C]-Glucose is taken up preferentially by DRG neurons and radiolabel is found in glutamate and alanine within minutes followed by a small amount of rad ⁇ olabeled glutamine after 1 h [J.L. Johnson, 1976; M.C.W. Minchin and P.M. Beart, 1975].
  • [2- 14 C]-pyruvate or NaH 14 COs DRGs contain significant amounts of radiolabeled glutamine and glutamate at 15 and 60 min [J.L. Johnson, 1976; M.C.W.
  • Cerebellar granule cells intercultured in the absence of astrocytes and incubated in [l- 14 C]pyruvate are capable of pyruvate carboxylation [B. Hassell and A. -Brathe, 2000].
  • neurons may increase production of enzymes that are not expressed or expressed at low levels under normal conditions.
  • studies with striatal injections of radiolabeled pyruvate indicate that pyruvate carboxylation can occur in vivo in neurons [B. Hassell and A. Brathe, 2000].
  • a weak PC IR in DRG neurons suggests a low level of PC expression in vivo in DRG neurons.
  • the low amount of PC IR staining in the DRG neurons and the apparent large amount of pyruvate carboxylation in the rat striatal neurons may indicate heterogenous expression of PC in different neuronal areas.
  • the rat PC gene has 19 coding exons and at least two alternate promoters to produce multiple PC transcripts [S. Jitrapakdee et al., 1996; S. Jitrapakdee et al., 1997].
  • the putative PC expressed by neurons [B. Hassell and A. Brathe, 2000] may be a PC isoform with antigenic sites not recognized by the antisera used in the present study.
  • [OIOO] A link between the TCA and glutamine cycles has been observed in the DRG.
  • [ 14 C]-Acetate is taken up preferentially by satellite cells [M.C.W. Minchin and P.M. Beart, 1975], possibly via a transport mechanism similar to CNS astrocytes [R.A. Waniewsk ⁇ and D.L. Martin, 1998].
  • [ 1 C]-label is incorporated rapidly in glutamate and glutamine [J.L. Johnson, 1974; P. Keen and P.J. Roberts, 1996; M.C.W. Minchin and P.M. Beart, 1975; P.J. Roberts and P. Keen, 1974].
  • GDH appears to be enriched in glial cells, but neurons also have been implicated to have this enzyme.
  • Neuronal GDH enzyme activity has been detected in CNS synaptosomes [C. Arce et al., 1990; N. Kuo et al., 1994; M. Yudkoff et al., 1991], although it is difficult to determine the amount of astrocytic contamination from such preparations.
  • Immunohistochemical studies in CNS typically have localized GDH to astrocytes [T. Kaneko et al., 1987; T. Kaneko et al., 1988; J.E. Madl et al., 1988], but some studies have noted weak to light immunostaining in neurons [C.
  • GDH activity in both the cytoplasm and nucleus contained GDH activity in both the cytoplasm and nucleus [T. Kato and O.H. Lowry, 1973].
  • the presence of GDH activity in the nucleus may indicate an alternative role for GDH such as a mRNA-binding protein, e.g. cytochrome c oxidase transcript-binding protein (COLBP) [T. Preiss et at., 1993; T. Preiss et al., 1995].
  • COLBP cytochrome c oxidase transcript-binding protein
  • Schmitt and Kugler (1999) showed very low GDH staining in satellite cells of rat cervical DRGs.
  • GDH activity or staining whereas the present study used perfusion fixed tissue.
  • GDH may exist in multiple forms with different biophysical properties [S.W. Cho et al., 1995; S.W. Cho et al., 1996; A.D. Colon et al., 1986; J. Lee et al., 1995] and detection of these forms via diverse methods may give rise to the differences observed in the various DRG studies.
  • Glutamine transfer in the PNS between glia and neurons might use similar glutamine transporters as in the CNS (SNl— glia; SAT/ATA— neurons) [S. Br ⁇ er and N. Brookes, 2001].
  • the glutamine cycle is described as a phenomenon occurring at the synaptic terminal and astrocytic process for production and degradation of glutamate as a neurotransmitter [G.J. Siegel et al., 1999].
  • the uptake of glutamine and conversion to glutamate for eventual synaptic use may also occur in the cell bodies and axons of DRG neurons [J.L. Johnson, 1974; J.L. Johnson, 1974; P.J. Roberts and P.
  • glutamine is the branch point substrate for multiple metabolic paths [A.J.L. Cooper, 1988] (Fig. 21) and the localization of glutamine-related enzymes in satellite cells surrounding neuronal cell bodies and Schwann cells associated with axons denotes a larger role than neurotransmitter regulation [P.R. Laming, 1998; S.R. Robinson et al., 1998].
  • GS is important for shuttling carbon in the form of glutamine from astrocytes to be used in the neuronal TCA cycle [D.L. Martin and R.A. Wan ⁇ ewski, 1996].
  • GDH can convert glutamate to 2-oxoglutarate for release and neuronal energy use, along with related metabolites, malate, pyruvate, and lactate [G.C. Leo et al., 1993; D.L. Martin and R.A. Waniewski, 1996; L. Pellerin et al., 1998; R.P. Shank and D.J. Bennett, 1993; N. Westergaard et al., 1994].
  • glutamine and glutamate are used as amino acids in most proteins and glutamine is a primary source for purine biosynthesis [A.J. L. Cooper, 1988].
  • Glutamine phosphoribosylpyrophosphate amidotransferase (GPATase; EC 2.4.2.14) represents the first and key regulatory enzyme for de novo purine synthesis [S. Li et al., 1999; H. Zalkin and J.L. Smith, 1998].
  • Glutamine concentrations and GPATase activity limit the rate of de novo purine synthesis [J.H. Kim et al., 1996; L.J. Messenger and H. Zalkin, 1979; J.L. Smith, 1998] and are linked closely to cellular activity, e.g., increased transcriptional requ ⁇ rements and augmented ATP levels for elevated energy demands [J. Allsop and R.W. Watts, 1980; S.
  • glutamine-related enzyme metabolism would increase along with overall general satellite and Schwann cellular activity (e.g., Refs. [R.W. Leech, 1967; B. Stevens et ai., 1998]).
  • glutamine, GS, GDH, and PC are enriched in DRG satellite cells and peripheral nerve Schwann cells. Glutamine and related enzymes in these cells may facilitate glutamate production in DRG neurons for synaptic transmission in the spinal dorsal horn.
  • the glutamine cycle' is a set of enzymes that are responsible for the production and degradation of the neurotransmitter glutamate in the central nervous system.
  • the glial TCA cycle is intimately associated with the glutamine cycle.
  • Enzymes associated with the "glutamine cycle' are present in glial cells in the peripheral nervous system, including glutamine synthetase, glutamate dehydrogenase, and pyruvate carboxylase, and these glial enzymes are elevated after the induction of experimental arthritis in rats. This allows primary sensory neurons to increase glutamate production in their cell bodies and peripheral nerve fibers. The neuronal cell bodies and nerve terminals, therefore, have increased amounts of glutamate.
  • the "glutamine cycle' had not been adequately described in the peripheral nervous system until the present invention, so these enzyme have not previously been considered as possible therapeutic targets for pain relief via peripheral inhibition.
  • FIGS. 22A and 22B the hindpaw responses of rats to pressure and thermal sensitivity were determined several days prior to the start of the experiment.
  • two groups were formed: 1. Control group with saline injection into the hindpaw; 2. Inflammation group with injection of complete Freund's adjuvant into hindpaw.
  • a glutamine synthetase inhibitor methionine sulfox ⁇ mine (MSO) was injected into half of the rats with inflammation.
  • the other rats received a saline injection at Day 3.
  • Rats were tested for 4 days (pressure) or 6 days (thermal) following glutamine synthetase inhibition. Prior to inflammation, rats responded to ⁇ 70 gms of pressure and at ⁇ 9 sec for thermal stimulation.
  • rats were implanted with intrathecal (i.t.) cannulae to the lumbosacral spinal cord and sensory ganglia.
  • the hindpaw responses of rats to pressure sensitivity were determined several days prior to the start of the experiment.
  • rats were injected with saline injection into the hindpaw or with injection of complete Freund's adjuvant into hindpaw.
  • a glutamine synthetase inhibitor methionine sulfoximine (MSO), glutaminase inhibitor, 6-diazo-5-oxo-L-norleucine (DON), or glial TCA cycle inhibitor, fluoroacetate (FA)
  • MSO methionine sulfoximine
  • DON 6-diazo-5-oxo-L-norleucine
  • FA glial TCA cycle inhibitor
  • Rats were tested for 4 days following initiation of inflammation. Prior to inflammation, rats responded to ⁇ 70 gms of pressure. Following inflammation, pressure responses dropped to ⁇ 10 gms, whereas rats with intrathecal inhibitors were able to maintain near normal pressure responses for several days.
  • FIG. 24 illustrates that satellite (glial) cells in the dorsal root ganglia (DRG) increase 'glutamine cycle' enzymes and products during chronic inflammation. Inflammation was induced with intraplantar CFA in the right hindpaw. In normal DRG's, glutamine synthetase (A; GS) and glutamine (C; the product of GS) immunoreactivity is located in satellite cells surrounding DRG neuronal cell bodies. After 3 days of inflammation, increased immunoreactivity for GS and glutamine is observed in most satellite cells. [0110] In summary, the present invention provides pain relief (thermal and mechanical) for several days by way of "glutamine cycle' or glial TCA cycle inhibition.
  • A glutamine synthetase
  • C glutamine
  • the present invention provides pain relief (thermal and mechanical) for several days by way of "glutamine cycle' or glial TCA cycle inhibition.
  • pyruvate carboxylase inhibitors that may be used in accordance with the present invention incJude, but are not limited to, phenyl acetic acid (PAA) [Farfari et al., 2000; Bahl et al., 1997], phenylacetyl Coenzyme-A [Bahl et al, 1997], phenylacetyl Co-A ester, oxamate [Martin-Requero etal., 1986; Attwood etal, 1992], and combinations and derivatives thereof.
  • PAA phenyl acetic acid
  • glutamine synthetase inhibitors examples include, but are not limited to, methionine-S-sulfoximine (MSO) [Sellinger, 1967; Ronzio et al, 1969], phosphinothricin (PPT) [Fushiya et al, 1988; Gill et al, 2001], 4-N-hydroxy-L- 2,4-diaminobutyric acid (NH-DABA) [Fushiya etal, 1988], Delta-hydr ⁇ xylysine [Dranoff et al, 1985], and combinations and derivatives thereof.
  • MSO methionine-S-sulfoximine
  • PPT phosphinothricin
  • NH-DABA 4-N-hydroxy-L- 2,4-diaminobutyric acid
  • NH-DABA 4-N-hydroxy-L- 2,4-diaminobutyric acid
  • Dranoff et al, 1985 Delta-hydr ⁇ xylysine
  • glutamate dehydrogenase inhibitors examples include, but are not limited to, bromofuroate [Matsuno et al, 1986; Vorschi et al, 1977], Paimitoyl-Coenzyme-A (Palmitoyl-Co-A) [Fang et al, 2002; Lai et al, 1993], vanadium compounds (including, but not limited to, orthovanadate, vanadyl sulphate, vanadyl acetylacetonate, and combinations thereof) [Kiersztan et al, 1998], glutarate, 2-oxoglutarate ( ⁇ -ketoglutarate) [Caughey et al, 1957], estrogen and estrogen analogues [Pons et al, 1978], pyridine-2,6-dicarboxylic acid [Broeder et al, 1994], and derivatives thereof as well as combinations thereof, such as, but not limited to, 2-ox
  • the L*DRG was examined for the following reason.
  • the tibial nerve a branch of the sciatic nerve, innervates the majority of the plantar surface of the rat hindpaw [Swett and Woolf, 1985].
  • Approximately, 99% of the tibial DRG neuronal perikarya of rats are located in the Lt- DRG's, and the L 4 DRG contains more than twice the number than L_ [Swett et al, 1991].
  • Two to three days prior to and for the days following CFA injection, rats were tested for pressure sensitivity with von Frey hairs (Semmes-Weinstein monofilaments; Stoelting, Inc.).
  • Rats were allowed to acclimate for five to ten minutes in a plastic box (25x25x25cm) with 6 mm holes spaced every 6 mm [Pitcher et al, 1999a,b]. Monofilaments calibrated for specific forces were inserted through the holes underneath the box to probe the plantar surface of the hindpaw, 5 times in 3-4 sec intervals in different places on the plantar surface. Filaments with light force were used first, followed by filaments of increasing force. A filament was slowly applied perpendicularly to the plantar surface until bending of the filament occurred. If the paw did not retract three out of five times, the next larger filament was used. The threshold force was defined as the filament (force) that caused the foot retraction without bending the monofilament three out of five times. Using a conversion table for the filaments, thresholds were reported as gram force.
  • Additional control rats ⁇ n 3) were perfused transcardially with 4% PFA in 0.1M Sorenson's phosphate buffer, pH 7.4. DRG's were removed and placed in fixative overnight at 4°C. All tissues were transferred to 20% sucrose in 0.1M Sorenson's phosphate buffer, pH 7.4 for 24-96 hr. at 4°C. The tissue was frozen, sectioned at 20 ⁇ m in a cryostat, thaw mounted onto gelatin coated slides, and dried for 1 hr. at 37 C C. Sections were washed three times for 10 min.
  • mouse anti-glutaminase IgM MAb 120, 1:500 - 5mg/ml; gift from Dr. T. Kaneko, Kyoto Univ., Kyoto, Japan
  • mouse anti-glutamate 1:3000; gift from Dr. J. Madl, Colo. St. Univ., Ft. Collins, CO
  • the tissue was washed three times in PBS and incubated in biotinylated goat anti-rabbit IgG or biotinylated goat anti-mouse IgM secondary antibody (5 ⁇ g/ml; Vector) in PBST for 1 hr.
  • tissue sections were washed two times in PBS following secondary antibody incubation, washed in sodium carbonate buffered saline (SCBS), pH 8.5, incubated in fluorescein-avidin (1.5mg/ml; Vector) in SCBS for 1 hr., and washed three times in PBS. Coverslips were apposed with Vectashield mounting media (Vector) to retard fading of immunofluorescence.
  • Other sections were washed three times in PBS following secondary antibody, incubated in avidin-biotin-peroxidase (Vector), and washed three times in Trisbuffered saline, pH 7.6.
  • Sections were incubated in diaminobenzidine (DAB) solution (0.5mg/ml DAB, 0.003% Hz ⁇ 2in Tris-sal ⁇ ne) for 1-5 minutes. Sections were dehydrated in an ascending series of ethanols, cleared in xylenes, and coverslips were apposed with Pro-Texx (Baxter Diagnostics). [0118] Aseries of dilutions (1:200 - 1:6000) of the rabbit ant ⁇ -glutaminase antiserum was used to determine an optimal dilution (1:3000) for evaluating alterations in immunohistochemical staining intensity.
  • DAB diaminobenzidine
  • DRG's were evaluated qualitatively for 3, 7 and 10 day groups, and the 7 day group was chosen for quantitative densitometric analysis.
  • Immunofluorescent images from 7 day DRG's were captured using the CCD camera and saved as uncompressed TIFF files. Exposures were adjusted and pre-set by using experimental (CFA) images for baseline exposure.
  • CFA experimental
  • the glutaminase-immunoreactive DRG images were analyzed using the SCION Image program (Scion Co., Frederick, MD). Individual DRG neurons were circumscribed, and the area, pixel number, and intensity were recorded. The data were recorded as intensity divided by the area of the cell.
  • Neuronal cell bodies in the DRG were distributed into the following three sizes for analysis: 100-600 ⁇ m 2 (small), 600-1200 ⁇ m 2 (medium), and >1200 ⁇ m 2 (large) [Willis and Coggeshall, 1991]. Differences in the intensity per area were analyzed with ANOVA followed by a Student-Newman-Keuls post hoc test (p ⁇ 0.05 for significance) using InStat biological statistics program (GraphPad Software, Inc.).
  • Enzyme assays for GT were performed according to the method of Curthoys and Lowry (1973). Five to six randomly selected sections of right and left DRG from rats with CFA and from control rats were placed individually in a 40 : l volume of reaction mixture containing: 20 mM glutamine, 100 mM
  • NAD + Reduction of NAD + was measured using a fluorometer (Farrand Inc.) with an excitation wavelength of 365 nm and emission at 340 nm. Quantitation of NADH production was accomplished by reacting multiple concentrations of glutamate standards in the indication reaction. The GT activity from each DRG section was ascertained and a mean activity for each DRG was determined. Differences in GT activity from the left and right L? DRG's of CFA rats and DRG's from control rats were analyzed with ANOVA followed by a Student- Newman-Keuls post hoc test (p ⁇ 0.05 for significance) using InStat biological statistics program (GraphPad Software, Inc.).
  • fixatives were used at pH 7.4: (1) 4% paraformaldehyde, 0.3% glutaraldehyde in 0.1 M Sorenson's phosphate buffer; (2) 4% glutaraldehyde, 0.2% picric acid in 0.1 M Sorenson's phosphate buffer [J.E. Madl, et al., 1988]; (3) 0.2% photoparaformaldehyde, 70% picric acid in 0.1 M phosphate buffer [K.E. Miller, et al., 1993]. Lumbar DRGs and sciatic nerves from the mid-thigh were removed and placed in fixative at 4°C overnight. The paraformaldehyde concentration of fixative #3 was increased to 2% for post-fixation [K.E. Miller, 1993].
  • Tissues were transferred to 20% sucrose in 0.1 M Sorenson's phosphate buffer, pH 7.4, for 24-96 h.
  • the tissue was frozen, sectioned at 20 ⁇ m in a cryostat, thaw mounted onto gelatin-coated slides, and dried for 1 h at 37°C. Sections were washed three times for 10 min in phosphate buffered saline (PBS) and incubated in 10% normal goat serum, 10% normal horse serum, 10% fetal bovine serum, 2% BSA, and 1% polyvinylpyrolidone in PBS with 0.3% Triton (PBS-Triton).
  • PBS phosphate buffered saline
  • Sections were incubated overnight at 4°C in: rabbit anti- glutamine (1:1000; Chemicon International, Temecula, CA, USA); mouse anti- glutamine synthetase (1: 1000; G45020; Transduction Laboratories, Lexington, KY, USA); mouse anti-glutamate dehydrogenase (1:10,000; J. Madl, Colorado St. Univ., Ft. Collins, CO, USA); rabbit anti-pyruvate carboxylase (1:100; J. Wallace, Univ. Sydney, Sydney, SA, Australia); mouse anti-pyruvate carboxylase (1:300; B. Pfe ⁇ ffer, Physiol. -Chem. Inst. Univ. Tubingen, Tubingen, Germany).
  • the tissue was washed three times in PBS and incubated in biotinylated secondary antibody (3 mg/ ml; Vector), either goat anti-rabbit IgG or horse anti-mouse IgG, for 1 hr.
  • biotinylated secondary antibody 3 mg/ ml; Vector
  • For immunoperoxidase staining sections were washed three times in PBS, incubated 1 hr. in avidin-biotin-peroxidase (Vector), washed two times in PBS, washed in Tris buffered saline (TBS), pH 7.6, and reacted in 0.5 mg/ml diaminobenzidine, 0.03% H ⁇ O ⁇ in TBS for 1-4 min. The reaction was stopped by washing the tissue three times in PBS.
  • Sections were dehydrated in an ascending series of ethanols, cleared in xylenes, and coverslips apposed with Pro-Texx permanent mounting media (Baxter).
  • Pro-Texx permanent mounting media Baxter.
  • sections were washed two times in PBS following secondary antibody incubation, washed in sodium carbonate buffered saline, pH 8.5, incubated in fluorescein-avidin (1.5 mg/ ml; Vector) for 1 hr., and washed three times in PBS.
  • Coverslips were apposed with Vectashield mounting media (Vector) to retard fading of immunofluorescence.
  • Vector Vectashield mounting media
  • Sections were incubated overnight in mouse anti-GS with rabbit anti-glutaminase (GT, 1: 1000, N. Curthoys, Colorado St. Univ.) or rabbit anti-protein gene product 9.5 (PGP-9.5, 1:500, Chemicon).
  • GT rabbit anti-glutaminase
  • PGP-9.5 rabbit anti-protein gene product 9.5
  • Cy3-labeled donkey anti-rabbit IgG (1:1000, Jackson Laboratories, West Grove, PA, USA) was incubated with the biotinylated horse anti-mouse IgG. The remainder of the immunofluorescence protocol was the same as described above.
  • Immunoperoxidase stained sections were observed and photographed in brightfield or differential interference contrast with an OlympusProvis AX70 microscope. Immunofluorescent sections were observed and photographed with epifluorescence microscopy using an Olympus Provis AX70 microscope or with confocal microscopy using a Leica TCS NT confocal microscope (OUHSC/Warren Foundation Flow and Image Cytometry Laboratory).
  • compositions having sustained pain- relieving properties such that the composition may be administered to a subject to alleviate chronic pain.
  • the composition includes an effective amount of at least one inhibitor of neurotransmitter synthesis.
  • a method for alleviating chronic pain in a subject for an extended period of time is also disclosed, in which the compound is administered to a subject suffering from chronic pain at a site of inflammation such that the administration of the compound results in a reduction in at least one of thermal and mechanical pain responses at the site of inflammation for a period of at least two days without any resulting acute pain behavior.
  • the composition may further include an effective amount of at least one compound having analgesic effects such that the composition also alleviates acute pain.
  • Lumbar dorsal root ganglia of the cat a quantitative sudy of peptide immunoreactivity and cell size. J. Comp. Neurol. 284:36-47.
  • Kiersztan A Jarzyna R, Bryla J, Inhibitory effect of vanadium compounds on glutamate dehydrogenase activity in mitochondria and hepatocytes isolated from rabbit liver. Pharmacol. Toxicol. 82:167-172, 1998.
  • Glutaminase immunoreactive neurons in the rat dorsal root ganglion contain calcitonin generelated peptide (CGRP). Neurosci. Lett. 160: 113-116.
  • Islet amyloid polypeptide and calcitonin gene-related peptide expression are upregulated in lumbar dorsal root ganglia after unilateral adjuvant-induced inflammation in the rat paw. Brain Res. Mol. Brain Res. 50:127-135.
  • mRNA-binding protein COLBP is glutamate dehydrogenase.
  • Ronzio RA Rowe WB, Meister A, Studies on the mechanism of inhibition of glutamine synthetase by methionine sulfoxim ⁇ ne. Biochemistry 8(3): 1066-75, 1969.
  • AMPA/kainate antagonist LY293558 reduces capsaicin-evoked hyperalgesia but not pain in normal skin in humans. Anesthesiology 89: 1060-1067.
  • NAD(P)H quinone oxidoreductase activity is increased in hippocampal pyramidal neurons of patients with Alzheimer's disease.

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Abstract

L'invention concerne une composition à propriétés d'atténuation durable de la douleur susceptible d'être administrée pour l'atténuation de douleur chronique. Cette composition comprend une quantité efficace d'au moins un inhibiteur de synthèse des neurotransmetteurs. L'invention concerne également un procédé d'atténuation de douleur chronique pendant une durée étendue, avec administration du composé sur un site d'inflammation, de manière à réduire au moins l'une des réponses de douleur thermique et mécanique sur le site d'inflammation, pendant une durée d'au moins deux jours, sans composante de douleur aiguë. La composition peut aussi renfermer une quantité efficace d'au moins un composé à effets analgésiques, de manière à atténuer la douleur aiguë.
PCT/US2003/028701 2002-09-13 2003-09-12 Procede d'attenuation de la douleur par inhibition de la synthese des neurotransmetteurs WO2004028448A2 (fr)

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US41131102P 2002-09-13 2002-09-13
US10/245,098 2002-09-13
PCT/US2002/029108 WO2003022261A1 (fr) 2001-09-13 2002-09-13 Methode pour soulager la douleur
US60/411,311 2002-09-13
US10/245,098 US7288246B2 (en) 2001-09-13 2002-09-13 Method of alleviating chronic pain via peripheral glutaminase regulation
USPCT/US02/29108 2002-09-13
US10/660,093 2003-09-11
US10/660,093 US7504231B2 (en) 2001-09-13 2003-09-11 Method of alleviating chronic pain via peripheral inhibition of neurotransmitter synthesis

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1752143A1 (fr) * 2005-08-08 2007-02-14 NewThera Nouvelle utilisation d'agents visant la synthétase de glutamine
WO2007058612A1 (fr) * 2005-11-15 2007-05-24 Entress Ab Medicament a utiliser en rapport avec la deterioration de cartilage
US7655232B2 (en) 2002-12-24 2010-02-02 Pfizer Inc. Anti-NGF antibodies and methods using same
JP2018528261A (ja) * 2015-07-31 2018-09-27 ザ・ジョンズ・ホプキンス・ユニバーシティー グルタミン類似体のプロドラッグ
US10323086B2 (en) 2002-12-24 2019-06-18 Rinat Neuroscience Corp. Methods for treating osteoarthritis pain by administering a nerve growth factor antagonist and compositions containing the same
CN111139270A (zh) * 2019-12-23 2020-05-12 浙江大学 一种用于生产l-草铵膦的酶组合和l-草铵膦生产方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6013672A (en) * 1997-12-18 2000-01-11 Uab Research Foundation Agonists of metabotropic glutamate receptors and uses thereof
US6291523B1 (en) * 1997-08-28 2001-09-18 Novartis Ag Certain 5-alkyl-2-arylaminophenylacetic acids and derivatives

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6291523B1 (en) * 1997-08-28 2001-09-18 Novartis Ag Certain 5-alkyl-2-arylaminophenylacetic acids and derivatives
US6013672A (en) * 1997-12-18 2000-01-11 Uab Research Foundation Agonists of metabotropic glutamate receptors and uses thereof

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7655232B2 (en) 2002-12-24 2010-02-02 Pfizer Inc. Anti-NGF antibodies and methods using same
US8088384B2 (en) 2002-12-24 2012-01-03 Rinat Neuroscience Corp. Anti-NGF antibodies and methods using same
US10323086B2 (en) 2002-12-24 2019-06-18 Rinat Neuroscience Corp. Methods for treating osteoarthritis pain by administering a nerve growth factor antagonist and compositions containing the same
US11008386B2 (en) 2002-12-24 2021-05-18 Rinat Neuroscience Corp. Anti-NGF antibodies and methods using same
EP1752143A1 (fr) * 2005-08-08 2007-02-14 NewThera Nouvelle utilisation d'agents visant la synthétase de glutamine
WO2007017768A2 (fr) * 2005-08-08 2007-02-15 Newthera Nouvelles utilisations pour medicaments ciblant la glutamine synthetase
WO2007017768A3 (fr) * 2005-08-08 2008-10-30 Newthera Nouvelles utilisations pour medicaments ciblant la glutamine synthetase
WO2007058612A1 (fr) * 2005-11-15 2007-05-24 Entress Ab Medicament a utiliser en rapport avec la deterioration de cartilage
RU2454999C2 (ru) * 2005-11-15 2012-07-10 Энтресс Аб Лекарственное средство, применяющееся при поражениях хряща
JP2018528261A (ja) * 2015-07-31 2018-09-27 ザ・ジョンズ・ホプキンス・ユニバーシティー グルタミン類似体のプロドラッグ
CN111139270A (zh) * 2019-12-23 2020-05-12 浙江大学 一种用于生产l-草铵膦的酶组合和l-草铵膦生产方法

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