WO2011111355A1 - Agent prophylactique/thérapeutique pour neuropathie périphérique comprenant de la l-sérine - Google Patents

Agent prophylactique/thérapeutique pour neuropathie périphérique comprenant de la l-sérine Download PDF

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WO2011111355A1
WO2011111355A1 PCT/JP2011/001303 JP2011001303W WO2011111355A1 WO 2011111355 A1 WO2011111355 A1 WO 2011111355A1 JP 2011001303 W JP2011001303 W JP 2011001303W WO 2011111355 A1 WO2011111355 A1 WO 2011111355A1
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serine
peripheral neuropathy
therapeutic agent
prophylactic
paclitaxel
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PCT/JP2011/001303
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English (en)
Japanese (ja)
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知之 川股
友洋 木谷
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北海道公立大学法人札幌医科大学
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Priority to JP2012504316A priority Critical patent/JP5773392B2/ja
Publication of WO2011111355A1 publication Critical patent/WO2011111355A1/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
    • A61K31/185—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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids

Definitions

  • the present invention relates to a prophylactic / therapeutic agent for peripheral neuropathy and a screening method for a prophylactic / therapeutic agent for peripheral neuropathy.
  • Peripheral nerves are all nerves other than the central nervous system, that is, all nerves other than the brain and spinal cord, and include motor nerves and sensory nerves.
  • peripheral nerves When peripheral nerves are affected by internal factors such as ischemia, administration of anticancer drugs, diabetes, infections, or external factors such as trauma caused by an accident, peripheral nerve dysfunction (peripheral neuropathy) occurs, sensory disorders, Peripheral neuropathy symptoms such as paralysis, pain, muscle weakness, muscle atrophy, deep tendon reflexes, and vasomotor symptoms occur. The symptoms of peripheral neuropathy often persist for long periods of time, reducing the patient's quality of life (QOL).
  • QOL quality of life
  • peripheral neuropathies those caused by administration of anticancer drugs and diabetes are particularly problematic because symptoms can be serious and the number of patients suffering from the disorder is large.
  • paclitaxel Texol (registered trademark)
  • peripheral neuropathy mechanical allodynia, cold allodynia, continuous burning pain, tingling, numbness
  • symptomatic treatments such as analgesics, vitamins (vitamin B agents), steroids and the like are performed, but sufficient effects cannot be obtained.
  • peripheral neuropathy is a dose-limiting factor for paclitaxel and often causes chemotherapy interruption.
  • Patent Document 1 describes a peripheral neuropathy improving agent containing phosphatidylserine as an active ingredient.
  • Patent Document 2 describes a peripheral neuropathy alleviating agent by administration of a drug comprising a pituitary adenylate cyclase activating polypeptide as an active ingredient.
  • L-serine (S) -2-amino-3-hydroxypropionic acid) is a kind of non-essential amino acid. L-serine is important in metabolism because it is involved in the biosynthesis of purines, pyrimidines, cysteines and the like. In vivo, L-serine is converted from 3-phosphoglycerate, an intermediate of glycolysis, to 3-phosphoglycerate dehydrogenase (EC (1.1.1.95), phosphoserine aminotransferase (EC 2.6.1.52), and It is synthesized through the action of three enzymes, phosphoserine phosphatase (EC 3.1.3.3).
  • Non-Patent Document 1 describes that L-serine promotes neurite outgrowth of dorsal root ganglia in chicken embryo cells in vitro.
  • Non-Patent Document 2 describes that L-serine supports the survival of rat fetal cell-derived hippocampal neurons in vitro.
  • Non-Patent Document 3 describes that L-serine promotes the survival and differentiation of rat fetal cerebellar Purkinje neurons in vitro.
  • All of Non-Patent Documents 1 to 3 only confirmed in vitro what action L-serine exerts on the central nervous system during development.
  • An object of the present invention is to provide a practical preventive / therapeutic agent for peripheral neuropathy and to provide a screening method for a practical prophylactic / therapeutic agent for peripheral neuropathy.
  • paclitaxel does not decrease the L-serine concentration in the sciatic nerve or spinal cord, but decreases the L-serine concentration in the dorsal root ganglion (DRG), which is a peripheral nerve.
  • DRG dorsal root ganglion
  • paclitaxel reduces the expression of 3PGDH (3-phosphoglycerate dehydrogenase) in DRG.
  • C According to immunohistochemical staining, 3PGDH is localized not in DRG neurons but in satellite cells.
  • Peripheral neuropathy induced by paclitaxel is improved by administration of L-serine.
  • E Peripheral neuropathy induced by oxaliplatin (anticancer agent) is improved by administration of L-serine.
  • F Peripheral neuropathy induced by streptozotocin (diabetes-inducing agent) is prevented or improved by administration of L-serine.
  • L-serine improves various peripheral neuropathies in vivo, and completed the present invention.
  • the present invention relates to (1) a prophylactic / therapeutic agent for peripheral neuropathy containing L-serine, and (2) the peripheral neuropathy is a peripheral neuropathy caused by administration of an anticancer agent or a peripheral neuropathy caused by diabetes.
  • the present invention relates to the prophylactic / therapeutic agent for the described peripheral neuropathy.
  • the present invention also includes (4) a method for screening a prophylactic / therapeutic agent for peripheral neuropathy, comprising the following steps (A) to (D): (A) a peripheral neuropathy model non-human mammal (B) A step of measuring L-serine concentration in dorsal root ganglia of a peripheral neuropathy model non-human mammal: (C) A measured value of L-serine concentration in step (B) Comparing the measured value of L-serine concentration when the test substance was not administered with (D) when the measured value of L-serine concentration in step (B) was not administered with the test substance The step of evaluating the test substance as a prophylactic / therapeutic agent for peripheral neuropathy when the L-serine concentration is higher than the measured value of L-serine: or (5) the following steps (a) to (d) A screening agent for the prevention and treatment of peripheral neuropathy, Ninging method: (a) Step of administering test substance to peripheral neuropathy model non-human mammal: (b) 3-
  • peripheral neuropathy can be prevented or treated.
  • the effect of preventing and treating peripheral neuropathy in the present invention has been demonstrated in vivo, it is extremely practical.
  • the present invention is used for peripheral neuropathy caused by an anticancer agent, it is possible to expand the range of selection of the anticancer agent or to secure a sufficient dose of the anticancer agent. As a result, the survival rate and prognosis of cancer patients can be ensured. Improvement can be expected.
  • a prophylactic / therapeutic agent for peripheral neuropathy can be efficiently screened.
  • FIG. 1A is a diagram showing the results of a von Frey test.
  • A1 shows the result using 4 g von Frey filament
  • A2 shows the result using 8 g von Frey filament
  • A3 shows the result using 15 g von Frey filament.
  • FIG. 1B is a diagram showing the results of a sensory nerve conduction velocity test.
  • * P ⁇ 0.05.
  • the numerical value D represents the number of days elapsed from the start date of the paclitaxel process or the vehicle process.
  • FIG. 2A The left graph shows the results of the standard solution containing L-serine and D-serine, and the right graph shows the results of the DRG sample.
  • FIG. 2B shows the measurement result of the amount of L-serine in the spinal cord.
  • FIG. 2C shows the measurement results of the amount of L-serine in the sciatic nerve.
  • FIG. 2D shows the measurement result of L-serine amount in DRG.
  • the numerical value of D represents the elapsed days from the start date of a paclitaxel process or a vehicle process. It is a figure which shows the result of the Western blot with respect to 3PGDH in DRG of a paclitaxel processing group (Paclitaxel) or a vehicle processing group (Vehicle). Upper panel: shows a band obtained as a result of Western blotting. Lower panel: shows the relative intensity (relative expression level) obtained by standardizing the signal intensity of the 3PGDH band with the signal intensity of ⁇ -actin.
  • the numerical value of D represents the elapsed days from the start date of a paclitaxel process or a vehicle process.
  • FIG. 4A shows an image of DRG tissue sections triple-stained for 3PGDH, PGP9.5 (neuronal marker), and CD31 (blood vessel marker).
  • FIG. 4B1 shows an image stained for 3PGDH.
  • FIG. 4B2 shows an image stained for S100 ⁇ .
  • FIG. 4B3 An image obtained by superimposing the images of FIG. 4B1 and FIG. 4B2.
  • burr in FIG. 4A or 4B1 represents 20 micrometers.
  • FIG. 5A shows the results of the von Frey test.
  • A1 shows the result using 4 g von Frey filament
  • A2 shows the result using 8 g von Frey filament
  • A3 shows the result using 15 g von Frey filament.
  • FIG. 5B is a diagram showing the results of a sensory nerve conduction velocity test.
  • the numerical value of D represents the elapsed days from the start date of a paclitaxel process or a vehicle process.
  • the preventive / therapeutic agent for peripheral neuropathy of the present invention (hereinafter also simply referred to as “the preventive / therapeutic agent of the present invention”) is not particularly limited as long as it is a composition containing L-serine,
  • inventions that are technically equivalent to the present invention include inventions for methods for preventing and treating peripheral neuropathy and methods for using L-serine.
  • the method for preventing and treating peripheral neuropathy is not particularly limited as long as L-serine is administered to a subject, and L-serine is used for preventing peripheral neuropathy as a method for using L-serine. -It will not be restrict
  • the “prophylactic / therapeutic effect on peripheral neuropathy” means the effect of suppressing (preventing) the onset of peripheral neuropathy and / or improving (treating) peripheral neuropathy, It preferably includes the effect of preventing and / or treating mechanically induced allodynia, hyperalgesia and decreased sensory nerve conduction velocity (SNCV).
  • L-serine in the present invention a commercially available product can be used, which may be derived from a natural product or synthesized.
  • the content of L-serine in the preventive / therapeutic agent of the present invention is not particularly limited, but is, for example, 0.01 to 100% by mass, preferably 0.5 to 100% by mass with respect to the total amount of the prophylactic / therapeutic agent. More preferably, 5 to 100% by mass, further preferably 20 to 100% by mass, more preferably 50 to 100% by mass, and still more preferably 70 to 95% by mass can be exemplified.
  • a suitable ratio (%) of the number of moles of L-serine to the number of moles of all amino acids (or amino acid residues) contained in the preventive / therapeutic agent of the present invention is, for example, 10% or more, preferably 20% or more. More preferably 40% or more, still more preferably 60% or more, more preferably 80% or more, still more preferably 90% or more, more preferably 95% or more, still more preferably 99% or more, and most preferably other than L-serine. It is possible to suitably illustrate that the amino acid is not contained.
  • a suitable ratio (%) of the number of moles of L-serine to the number of moles of D-serine in the preventive / therapeutic agent of the present invention is, for example, 40% or more, preferably 50% or more, more preferably 60%. More preferably, 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, and still more preferably 99% or more can be suitably exemplified. Most preferably, it does not contain serine.
  • peripheral neuropathy such as sensory disorder, paralysis, pain, muscle weakness, muscle atrophy, deep tendon reflex reduction, vasomotor symptoms, etc.
  • a known method for confirming whether the symptoms are reduced can be used.
  • the prophylactic / therapeutic agent of the invention As a suitable degree of the preventive / therapeutic effect of peripheral neuropathy in the prophylactic / therapeutic agent of the present invention, 2 mg / kg paclitaxel was intraperitoneally injected four times every other day (D1, D3, D5, D7).
  • the percentage of the pull-in response to von Frey stimulation (4 g, 8 g, or 15 g) at D29 is determined by administering the prophylactic / therapeutic agent of the present invention 20% or more, preferably 30% or more, more preferably 40% or more, still more preferably 50% or more, more preferably 60% or more, and even more preferably 70% or more, compared with the ratio in the absence This can be preferably exemplified.
  • 2 mg / kg paclitaxel is administered four times every other day (D1, D3, D5, D7) intraperitoneally.
  • a preferable example is that the latency is decreased by 4% or more, preferably 6% or more, more preferably 8% or more, compared to the latency when the preventive / therapeutic agent of the present invention is not administered. be able to
  • the prophylactic / therapeutic agent of the present invention may contain any component such as a prophylactic / therapeutic agent for other peripheral neuropathy in addition to L-serine as long as the desired prophylactic / therapeutic effect for the peripheral neuropathy is obtained. Good.
  • L-serine contained in the preventive / therapeutic agent of the present invention can be made into an appropriate preparation by a conventional method.
  • the dosage form of the preparation may be a solid preparation such as a powder or a granule, but from the viewpoint of obtaining an excellent preventive / therapeutic effect on peripheral neuropathy, a liquid such as a solution, an emulsion, or a suspension is used. It is preferable.
  • a method for producing a liquid agent for example, a method of mixing L-serine with a solvent and a method of further mixing a suspending agent or an emulsifier can be exemplified.
  • an appropriate pharmaceutically acceptable carrier such as an excipient, a binder, a solvent, a solubilizing aid is used as necessary in the preparation.
  • An optional component such as a diluent can be blended.
  • the method for administering the prophylactic / therapeutic agent of the present invention is not particularly limited as long as the desired prophylactic / therapeutic effect for peripheral neuropathy is obtained, and can be exemplified by intravenous administration, intraperitoneal administration, oral administration, etc. Especially, intravenous administration and oral administration can be illustrated suitably. Further, the dose, frequency and dose of the preventive / therapeutic agent of the present invention can be appropriately adjusted according to the state of peripheral neuropathy to be administered, the body weight of the subject to be administered, etc.
  • L-serine for example, 0.001 nmol / kg to 1 mol / kg, preferably 0.1 nmol / kg to 100 mmol / kg, more preferably 1 nmol / kg to 10 mmol / kg, still more preferably 10 nmol / kg to 5 mmol. / Kg can be administered.
  • peripheral neuropathy to which the prophylactic / therapeutic agent of the present invention is applied is not particularly limited, and it may be peripheral neuropathy caused by internal factors such as administration of an anticancer drug, diabetes, ischemia, infection, etc. It may be a peripheral neuropathy caused by an external factor such as trauma due to an accident, but preferably a peripheral neuropathy caused by an internal factor such as administration of an anticancer drug, diabetes, ischemia or infection Among them, the administration of an anticancer agent and peripheral neuropathy caused by diabetes can be exemplified more preferably, and the peripheral neuropathy caused by administration of an anticancer agent can be exemplified more preferably.
  • Such an anticancer agent is not particularly limited as long as it is an anticancer agent that can cause peripheral neuropathy by administration, but is a taxane anticancer agent such as paclitaxel or docetaxel; a platinum complex anticancer agent such as oxaliplatin, cisplatin or carboplatin; a vinca alkaloid such as vincristine Preferred examples include anticancer agents; alkylating agent anticancer agents such as ifosfamide; antimetabolite anticancer agents such as methotrexate, fluorouracil, cytarabine; antibiotic anticancer agents such as doxorubicin and bleomycin; and taxanes such as paclitaxel and docetaxel.
  • Anti-cancer agents such as platinum complex anti-cancer agents such as oxaliplatin, cisplatin, carboplatin, etc. can be more preferably exemplified, and paclitaxel and oxaliplatin are particularly preferably exemplified. Door can be.
  • Other preferable peripheral neuropathies to which the prophylactic / therapeutic agent of the present invention is applied include peripheral neuropathies involving a decrease in L-serine concentration in DRG.
  • the administration target of the prophylactic / therapeutic agent of the present invention is not particularly limited as long as it is a mammal, and humans, monkeys, mice, rats, hamsters, guinea pigs, cows, pigs, horses, rabbits, sheep, goats, cats, dogs Etc. can be preferably exemplified, and among these, humans can be more suitably exemplified.
  • the L-serine administration method and the like in the method for preventing and treating peripheral neuropathy of the present invention are the same as those described above for the agent for preventing and treating the present invention.
  • the method for preparing L-serine into the prophylactic / therapeutic agent of the present invention is the same as described above for the prophylactic / therapeutic agent of the present invention.
  • step (A) a step of administering a test substance to a peripheral neuropathy model non-human mammal: (B) after a peripheral neuropathy model non-human mammal Step of measuring L-serine concentration in root ganglia: (C) The measured value of L-serine concentration in step (B) is compared with the measured value of L-serine concentration when the test substance was not administered.
  • a peripheral neuropathy model non-human mammal (b) peripheral neuropathy model non-human mammal 3-ho in the dorsal root ganglia of
  • the peripheral neuropathy model non-human mammal in the above screening method may be a non-human mammal that naturally developed peripheral neuropathy, an anticancer agent (preferably paclitaxel, oxaliplatin) or a diabetes inducer (preferably May be a non-human mammal in which peripheral neuropathy is artificially developed by administering a peripheral neuropathy inducer such as streptozotocin).
  • an anticancer agent preferably paclitaxel, oxaliplatin
  • a diabetes inducer preferably May be a non-human mammal in which peripheral neuropathy is artificially developed by administering a peripheral neuropathy inducer such as streptozotocin.
  • the measurement of the L-serine concentration in the above step (B) can be performed using HPLC or the like, and the expression level of 3PGDH in the above step (b) is measured by Western blot or the like for dorsal root ganglion cells. Can be performed.
  • the average body weight of the rats at the start of the experiment was 167 g and 175 g in the vehicle treatment group and the paclitaxel treatment group, respectively.
  • the rats in the vehicle-treated group and the paclitaxel-treated group continued to gain weight as usual, and the average weight of the rats at the end of the experiment period (D43) was 403 g and 407 g in the vehicle-treated group and the paclitaxel-treated group, respectively. It was. There was no significant difference in the average weight gain between the two groups during the 43 days of the experiment. All rats survived until the end of the experiment without showing health problems including alopecia and diarrhea.
  • paclitaxel solution for administration by dissolving paclitaxel (Sigma) at 6 mg / mL in Cremophor EL / anhydrous ethanol and then diluting with saline to a final concentration of 2 mg / kg / mL did. Further, without using paclitaxel, a vehicle (control solution) was prepared by mixing Cremophor EL / anhydrous ethanol and physiological saline at the same ratio as the above-mentioned paclitaxel solution.
  • the paclitaxel-treated rats prepared in Example 1 were treated with the paclitaxel solution described above, and the vehicle-treated rats were treated with the aforementioned vehicle according to the method described in Polomano et al (Pain 2001; 94: 293-304). Injected intraperitoneally four times every other day (D1, D3, D5, D7). The body weight of each rat was measured and recorded before and after the start of paclitaxel treatment or vehicle treatment.
  • the wire mesh floor was arranged to have a certain height. On this wire mesh floor, each rat was housed and left for 20 minutes to adapt to this environment. Next, three von Frey filaments having bending forces of 4 g, 8 g, and 15 g were prepared, and each von Frey filament was applied to the skin of the sole of the rat's hind limb for 5 seconds at a time. Irritation was given. The hindlimb withdrawal response to the von Frey filament was measured, and the number of hindlimb withdrawal responses was expressed as a percentage of the number of applied stimuli.
  • the response to 4 g induced by paclitaxel is generally called allodynia, the response to 15 g is called hypersensitivity, and the response to 8 g is between them (Pain 2004; 109: 150-161) .
  • the results of this von Frey test are shown in FIG. 1A. As can be seen from the results in FIG.
  • the rats were lightly anesthetized by intraperitoneal injection with pentobarbital. A recording needle electrode was then injected into the 2 cm and 8 cm positions from the tail anus. The stimulation electrode was placed at a position 12 cm from the anus. After nerve stimulation, the potential latencies (from peak to peak) recorded at two locations were measured, thereby calculating SNCV. The rat skin temperature was maintained at 36-37 ° C. during the experiment. The calculated SNCV result is shown in FIG. 1B. As can be seen from the results in FIG. 1B, at D0, the SNCV was comparable between the paclitaxel treated group and the vehicle treated group.
  • the SNCV decreased in the paclitaxel-treated group compared to the vehicle-treated group (FIG. 1B).
  • D15 (44.6 ⁇ 0.7 vs 38.4 ⁇ 0.9 m / s, P ⁇ 0.05)
  • D29 (39.5 ⁇ 0.9 vs 42.8 ⁇ 1.1 m / s, P ⁇ 0.05)
  • D36 (41.6 ⁇ 0.5 vs 43.0 ⁇ 0.6 m / s, P ⁇ 0.05)
  • the paclitaxel treated group had a significant SNCV compared to the vehicle treated group (FIG. 1B).
  • peripheral neuropathy model rats were produced by paclitaxel treatment.
  • the rats were deeply anesthetized with urethane at D0 before measurement of von Frey filament and SNCV measurement, and at D8, D15, D22, D29 and D43 after measurement, and the rats were decapitated.
  • the spinal cord, sciatic nerve and L3 / 4 / 5DRG were rapidly removed from the rat and frozen at -80 ° C.
  • Samples of spinal cord, sciatic nerve and DRG were homogenized with methanol on ice. The amount of methanol added was 10 times the sample weight. The homogenate was centrifuged at 15,000 xg for 20 minutes at 4 ° C.
  • the supernatant was diluted with 10 times the amount of distilled water, and 1/4 volume (v) of 5 mM orthophthalaldehyde (OPA) / N-acetylcysteine (NAC) solution was added. After allowing to stand at room temperature for 2 minutes, 20 ⁇ L of the reaction solution was injected into a high performance liquid chromatography (HPLC) system.
  • HPLC high performance liquid chromatography
  • the HPLC system consists of two EP-300 pumps (Eicom Corporation), DG-300 mobile phase degasser (Eicom Corporation), ATC-300 column oven (Eicom Corporation) and FLD-370 fluorescence detector. (Eicom Corporation).
  • Samples and standard solutions were automatically derivatized and injected into the MPLC system using an M-231SL autosampler (manufactured by Eicom Corporation).
  • the diastereomeric derivatives of D-serine and L-serine in OPA / NAC were separated by SC-5ODS reverse phase column (150 ⁇ 3 mm id, particle size 5 ⁇ m; manufactured by Eicom Corporation), and the excitation wavelength of 340 nm and Monitoring was performed at an emission wavelength of 445 nm.
  • a mixture (86:14, v / v) of methanol containing 5 mg / L EDTA-2Na and 0.1 M phosphate buffer (pH 6.0) was used as the mobile phase.
  • FIG. 2A shows the HPLC results for the standard solution containing L-serine and D-serine
  • the right graph in FIG. 2A shows the HPLC results for the DRG sample.
  • FIG. 2B shows the measurement result of the L-serine amount in the spinal cord
  • FIG. 2C shows the measurement result of the L-serine amount in the sciatic nerve
  • FIG. 2D shows the measurement result of the L-serine amount in DRG.
  • 3PGDH is an initial enzyme in L-serine biosynthesis from 3-phosphoglycerate and an enzyme that generates 3-phosphohydroxypyruvic acid from 3-phosphoglycerate.
  • the protein concentration in the supernatant was measured by a DC protein assay (Bio-Rad Laboratories) using bovine serum albumin as a standard. Equal amounts of protein were separated by sodium laurate sulfate-polyacrylamide electrophoresis (4%) and transferred to a polyvinylidene fluoride membrane (Millipore). The membrane was incubated overnight at 4 ° C. with guinea pig anti-3PGDH antibody (1 ⁇ g / mL) in PBS containing 10% skim milk. Next, the concentration of 3PGDH in DRG was measured by detecting the guinea pig anti-3PGDH antibody in the membrane by an immune reaction.
  • the concentration of actin which is a housekeeping protein, was also measured using a rabbit anti-actin antibody (1: 5,000; A2066, manufactured by Sigma).
  • the immune reaction was visualized using a chemiluminescence amplification method and a chemiluminescence detection system (Amersham Biosciences).
  • the intensity of the band was determined using an image densitometer (NIH Image 1.63; manufactured by National Institutes of Health), and standardized with the intensity of ⁇ -actin.
  • the results of these Western blots are shown in FIG.
  • the 3PGDH protein level of DRG (3PGDH / ⁇ -actin) in the paclitaxel-treated group is D15 (2.5 ⁇ 0.2 vs 3.9) compared to that level in the vehicle-treated group.
  • Significant decreases were shown in ⁇ 0.3, P ⁇ 0.05) and D22 (3.0 ⁇ 0.3 vs 4.6 ⁇ 0.2, P ⁇ 0.05).
  • This time course of 3PGDH expression in DRG was consistent with the time course of L-serine concentration in DRG (FIG. 2D).
  • Guinea pig anti-3PGDH antibody (1.0 ⁇ g / mL; provided by Hokkaido University), rabbit antiprotein gene product 9.5 (PGP9.5) (1: 4000, RA95101; manufactured by Ultraclone), mouse anti-CD31 antibody (1: 100 10R-CD31gRT; manufactured by Fitzgerald) and rabbit anti-S-100 ⁇ antibody (1: 100, RY330; manufactured by Yanaihara Institute).
  • PGP9.5 rabbit antiprotein gene product 9.5
  • mouse anti-CD31 antibody (1: 100 10R-CD31gRT; manufactured by Fitzgerald
  • rabbit anti-S-100 ⁇ antibody (1: 100, RY330; manufactured by Yanaihara Institute).
  • the specificity of the above anti-3GPDH antibody has been confirmed by previous studies (J. Neurosci 2001; 21: 7691-704; Arch Histol Cytol2003; 66: 429-36).
  • Rats were injected intraperitoneally with 50 mg / kg ketamine, and after deep anesthesia, phosphate buffer (96 mM NaH 2 PO 4 -12H 2 O and 25 mM Na 2 HPO) containing 4% paraformaldehyde (PFA). It was subjected hearts perfused with an aqueous solution) containing 4 -2H 2 O. The L4 DRG on the left side was removed, and fixed by being immersed in a phosphate buffer solution (PB) containing 4% paraformaldehyde (PFA) for 2 hours.
  • PB phosphate buffer solution
  • the DRG is then placed in PBS containing 25% sucrose (9.6 mM NaH 2 PO 4 -12H 2 O, 2.5 mM Na 2 HPO 4 -2H 2 O and 136 mM NaCl in water) overnight at 4 ° C. Cryoprotected.
  • the DRG was placed in a Tissue-Tek embedding medium (Sakura) and then quickly frozen. The frozen section was cut to 20 ⁇ m using a sliding cryostat (Sakura) and thawed on a gelatin-coated slide.
  • This tissue section was washed in PBS and incubated for 1 hour at room temperature with a blocking solution consisting of PBS (PBS-T) containing 10% normal donkey serum and 0.2% TritonX-100 (Sigma). did. The sections were then incubated overnight in the primary antibody mixture. The section was rinsed with PBS-T, and then the section was incubated with Alexa Fluor 488-, Alexa 597-, and Alexa 647-labeled species-specific secondary antibody (Invitrogen) solution diluted 501 times with PBS-T at room temperature. Incubated for 2 hours. The fluorescence image of the secondary antibody was obtained with a confocal laser scanning microscope (Digital Eclipse C1; manufactured by Nikon). One stack was obtained for each image by line-by-line continuous scanning to prevent bleeding and cross-excitation of fluorophore molecules.
  • PBS-T PBS-T
  • TritonX-100 TritonX-100
  • FIG. 4A shows an image obtained by triple-staining a tissue section of DRG for 3PGDH, PGP9.5 (a neuronal marker), and CD31 (a blood vessel marker).
  • a strong immunostaining of 3PGDH was observed as a large ring-like structure surrounding many of the DRG neuron somatic cells (FIG. 4A).
  • DRG tissue sections were double-stained for 3PGDH, S100 ⁇ (satellite cell marker).
  • 4B1 shows an image stained with 3PGDH
  • FIG. 4B2 shows an image stained with S100 ⁇
  • FIG. 4B3 shows an image obtained by superimposing the images of FIGS. 4B1 and 4B2.
  • Example 2 The same method as described in Example 2 except that 1 mmol / kg L-serine solution (1 mL) or vehicle (0.9% physiological saline) (1 mL) was further administered intraperitoneally.
  • the method was von Frey test and sensory nerve conduction velocity test.
  • a 0.1 mmol / kg L-serine solution (1 mL) was further orally administered once daily for 28 consecutive days from the day when the paclitaxel solution was injected into the rat.
  • the von Frey test and the sensory nerve conduction velocity test were performed in the same manner as described in Example 2 except that the drug was inserted into and administered.
  • FIG. 5A The results of these von Frey tests are shown in FIG. 5A, and the results of sensory nerve conduction velocity tests are shown in FIG. 5B.
  • FIG. 5A the 0.1 mmol / kg intraperitoneal administration group of L-serine and the 0.1 mmol / kg oral administration group of L-serine were mechanical allodynia compared to the intraperitoneal administration group of vehicle. / Hypersensitivity improved.
  • the ratio of withdrawal reaction to 4 g of von Frey stimulation in D22 and D29, 8 g of von in D29 were significantly reduced compared to that in the vehicle ip group.
  • the rate of withdrawal response to 4 g of von Frey stimulation in D22 was significantly reduced compared to that in the vehicle intraperitoneal administration group.
  • the SNCV in the 0.1 mmol / kg L-serine intraperitoneal administration group and the 0.1 mmol / kg L-serine oral administration group were compared with the vehicle intraperitoneal administration group.
  • the decline of was improved.
  • SNCV at D15, D22, D29, and D36 in the 0.1 mmol / kg L-serine intraperitoneal administration group and 0.1 mmol / kg L-serine oral administration group was compared with SNCV in the vehicle intraperitoneal administration group. was significantly increased.
  • intraperitoneal administration group of STZ 50 mg / kg of STZ was injected intraperitoneally into D1, and in the intraperitoneal administration group of STZ and L-serine, intraperitoneal injection of 50 mg / kg of STZ into D1, 0.1 mmol / kg L-serine was injected intraperitoneally.
  • 50 mg / kg STZ was intraperitoneally injected into D1, and then 0.1 mmol / kg L-serine was orally administered every day from D1.
  • Streptozotocin is generally used when preparing a diabetes model because it has the property of being taken up by ⁇ cells of the pancreas and causing a radical reaction there to destroy ⁇ cells.
  • Rats of the above four groups non-administration group, STZ intraperitoneal administration group, STZ and L-serine intraperitoneal administration group, STZ intraperitoneal administration and L-serine oral administration group) were subjected to the method described in Example 2 above.
  • the results of the von Frey test performed at D29 and the sensory nerve conduction velocity test at D29 are shown in the upper panel of FIG. As can be seen from the results in the upper panel of FIG.
  • the sensory nerve conduction velocity was significantly reduced in the STZ intraperitoneal administration group (STZ) compared to the non-administration group, and STZ and L-serine intraperitoneal administration group (STZ + 0.1S (ip))
  • STZ and L-serine intraperitoneal administration group STZ + 0.1S (ip)
  • STZ intraperitoneal administration and L-serine oral administration group STZ + 0.1S (po) significantly increased compared with the STZ intraperitoneal administration group (STZ).
  • STZ intraperitoneal administration group STZ and L-serine intraperitoneal administration group, STZ intraperitoneal administration and L-serine oral administration group (STZ + 0.1S (po))
  • BS blood glucose level
  • the present invention can be particularly suitably used in the field of prevention / treatment of peripheral neuropathy and the field of screening methods for prophylactic / therapeutic agents of peripheral neuropathy.

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Abstract

L'invention porte sur : un agent prophylactique/thérapeutique utile sur le plan pratique pour une neuropathie périphérique ; et sur un procédé pour le dépistage d'un agent prophylactique/thérapeutique utile sur le plan pratique pour une neuropathie périphérique. L'agent prophylactique/thérapeutique et le procédé sont caractérisés par l'utilisation de la L-sérine.
PCT/JP2011/001303 2010-03-10 2011-03-04 Agent prophylactique/thérapeutique pour neuropathie périphérique comprenant de la l-sérine WO2011111355A1 (fr)

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JP2014172846A (ja) * 2013-03-07 2014-09-22 Kyushu Univ がん化学療法剤に起因する末梢神経障害予防剤及び／又は治療剤
WO2015163316A1 (fr) * 2014-04-22 2015-10-29 味の素株式会社 Composition pour prévenir ou améliorer la neuropathie périphérique

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WO1999004781A1 (fr) * 1997-07-28 1999-02-04 The Institute Of Physical And Chemical Research Agent promoteur de protection et de survie des cellules du systeme nerveux central

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WO1999004781A1 (fr) * 1997-07-28 1999-02-04 The Institute Of Physical And Chemical Research Agent promoteur de protection et de survie des cellules du systeme nerveux central

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014172846A (ja) * 2013-03-07 2014-09-22 Kyushu Univ がん化学療法剤に起因する末梢神経障害予防剤及び／又は治療剤
WO2015163316A1 (fr) * 2014-04-22 2015-10-29 味の素株式会社 Composition pour prévenir ou améliorer la neuropathie périphérique
JPWO2015163316A1 (ja) * 2014-04-22 2017-04-20 味の素株式会社 末梢神経障害の予防又は改善用組成物
JP2019182881A (ja) * 2014-04-22 2019-10-24 味の素株式会社 末梢神経障害の予防又は改善用組成物
US10653655B2 (en) 2014-04-22 2020-05-19 Ajinomoto Co., Inc. Composition for preventing or improving peripheral neuropathy

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