JP5099620B2 - Amyotrophic lateral sclerosis drug - Google Patents

Description

  The present invention relates to a therapeutic agent for amyotrophic lateral sclerosis designated as a specific intractable disease.

  Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with a poor prognosis that develops after middle age and causes progressive paralysis of skeletal muscles. Yes. About 90% of ALS are sporadic and the cause is unknown. The remaining 10% are familial, and the newly acquired cytotoxicity of mutant SOD1 causes death of motor neurons due to point mutations in the Cu / Zn superoxide dismutase (SOD1) gene (gain-of-toxic function). The theory is powerful (Non-Patent Document 1).

Currently, only riluzole (Riltech ^™ , Aventis), which is an antagonist of glutamate receptor and has glutamate inhibitory action, is sold as an ALS therapeutic drug (Patent Document 1).

Neurotrophic factors, caspase inhibitors, copper chelators, etc. are being investigated as ALS therapeutic agents. Among these, D-penicillamine (Non-Patent Document 2), trientine (Non-Patent Document 3) and diethyldithiocarbamate (Patent Document 2) have been studied for copper chelating agents. However, these copper chelators increased survival in ALS model animals by 7.8% with D-penicillamine 100 mg / kg and 8.0% with trientine 800 mg / kg, and in vivo (in In vivo) has not been studied.
Japanese Patent No. 2713384 Special Table 2000-515491 Bruijn, LI, et al., 2004. Annu. Rev. Neurosci. 27, 723-749. Fottinger, AF, et al., 1997. Eur. J. Neurosci. 9, 1548-1551. Andreassen, OA, et al., 2001. Exp. Neurol. 168, 419-424.

  Accordingly, an object of the present invention is to provide an ALS therapeutic agent having an excellent therapeutic effect against ALS.

  Therefore, when the present inventors searched for a new therapeutic agent using an ALS model animal, tetrathiomolybdic acid had a remarkable 23.6% increase in survival rate at a dose as low as 5 mg / kg. Thus, the ALS therapeutic effect of tetrathiomolybdate was found to be 60 times as strong as D-penicillamine and 640 times that of trientine in consideration of the dose, and the present invention was completed.

  That is, the present invention provides an ALS therapeutic agent containing tetrathiomolybdic acid or a salt thereof as an active ingredient.

The therapeutic effect of tetrathiomolybdic acid or its salt on ALS is 60 times stronger than that of D-penicillamine, which has been known as a copper chelator, and 640 times that of trientine. Considering what is known to have is totally unexpected.
Tetrathiomolybdic acid has the effect of improving motor dysfunction, which is the main symptom of ALS. Furthermore, tetrathiomolybdate significantly corrects the oxidative stress and hyperapoptotic state that existed in the background of ALS-induced motor neuron death. In addition to ALS death, motor dysfunction represented by progressive paralysis of skeletal muscle It is also effective in preventing and improving the above.

  Tetrathiomolybdic acid or a salt thereof, which is an active ingredient of the ALS therapeutic agent of the present invention, is known as a copper chelator, but it has not been known at all whether it has an ALS therapeutic action. Examples of the salt of tetrathiomolybdic acid include ammonium tetrathiomolybdate.

  As shown in the examples below, tetrathiomolybdic acid has a significant survival rate of 23.6% in ALS model animals at doses as low as 5 mg / kg compared to the doses of D-penicillamine and trientine. Has an increasing effect. Tetrathiomolybdate significantly improves motor dysfunction typified by progressive paralysis of skeletal muscle, which is the main symptom of ALS. Furthermore, tetrathiomolybdate suppresses the production of lipid peroxides, which are said to be involved in the onset and progression of ALS, and significantly suppresses the hyperapoptotic state induced thereby (various caspases). Suppression and caspase inhibitory factor survivin activity). Furthermore, it is known that tetrathiomolybdic acid is highly safe (D-penicillamine is a drug with emergency safety information) and can be administered orally. Therefore, the ALS therapeutic agent of the present invention is an excellent therapeutic agent capable of preventing or improving the progression of motor paralysis, which is the main symptom of ALS, as well as the life-prolonging effect of ALS patients.

  The administration route of the medicament of the present invention is not particularly limited, and can be administered orally or parenterally. Examples of parenteral administration include intrathecal, intravenous, intraarterial, intramuscular, subcutaneous or intradermal injection, inhalation, rectal, intranasal administration, and nasal drop, ear drop, eye drop, and external administration. . Among them, the medicament of the present invention is preferably administered orally.

  As the medicament of the present invention, tetrathiomolybdic acid or a salt thereof which is an active ingredient may be administered to a patient as it is, but preferably a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier. Should be administered as a formulation. Examples of pharmaceutically acceptable carriers include excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers, solubilizers, isotonic agents. Agents, pH adjusters, stabilizers and the like can be used.

  Examples of preparations for oral administration include, for example, tablets, capsules, powders, fine granules, granules, solutions, syrups, etc. Examples of preparations for parenteral administration include injection Agents, drops, suppositories, inhalants, nasal drops, ear drops, eye drops, or external preparations (including patches, ointments, creams, gels, lotions, sprays, etc.).

Preparations for oral administration include, for example, excipients such as glucose, lactose, D-mannitol, starch, or crystalline cellulose; disintegrants such as carboxymethylcellulose, starch, or carboxymethylcellulose calcium; hydroxypropylcellulose, hydroxypropylmethylcellulose A binder such as magnesium stearate or talc; a coating agent such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol or titanium oxide; petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, Bases such as glycerin, purified water, or hard fat can be used. For injection, instillation, nasal drop, ear drop or eye drop, aqueous solution such as distilled water for injection, physiological saline, propylene glycol or the like, or a solubilizing agent that can be dissolved at the time of use; glucose, sodium chloride , D-mannitol, glycerin and other isotonic agents; and pharmaceutical additives such as pH regulators such as inorganic acids, organic acids, inorganic bases and organic bases. For the suppository, for example, bases such as polyethylene glycol, lanolin, cocoa butter, fatty acid triglyceride, and additives such as surfactants such as nonionic surfactants can be used as necessary.
In the ointment, bases, stabilizers, wetting agents, preservatives and the like that are usually used are used as necessary. Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, paraffin and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, and propyl paraoxybenzoate.

  Examples of the patch include those obtained by applying the ointment, cream, gel, paste and the like to a normal support by a conventional method. As the support, films such as cotton, woven fabric made of chemical fiber, non-woven fabric, soft vinyl chloride, polyethylene, and polyurethane are suitable.

  The dosage of the medicament of the present invention can be appropriately selected according to various conditions such as the progress of ALS or the degree of symptoms, the age and weight of the patient. It is preferable to administer 0.5 mg / kg to 50 mg / kg of adults as tetrathiomolybdic acid for ALS patients in 1 to 4 divided doses per day.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Example 1
A. Method (1) A G93A SOD1 transgenic mouse (Tg), one of ALS model animals, was used. This model has the mutated SOD1 gene found in familial ALS and exhibits motor paralysis starting in the lower extremities after 14-16 weeks of age.
Twenty-four mice at 4 weeks of age were divided into a tetrathiomolybdate (TTM) group and a control group (13 mice in each group). TTM (5 mg / kg) was administered to the TTM group and phosphate buffered saline (PBS) was administered to the control group every day until the individual died. The motor function of mice was evaluated by Rota-Rod, and the survival rate was evaluated by Kaplan-Meier method. At the age of 8 weeks (4 weeks after the start of TTM administration), a part of each group (n = 3) was killed, and the spinal copper (Cu) concentration, lipid peroxide ( The amount of LPO) produced and the behavior of apoptosis-related factors were examined by inductively coupled plasma mass spectrometry, thiobarbituric acid method, and Western blot method, respectively.

(2) Measurement of motor function Motor function was evaluated using Rota-Rod (Columbus Instruments, Columbus, OH, USA). In order to familiarize the mice with Rota-Rod, the mice were trained one week before the measurement start date. Eight-week-old mice were housed in the opposite direction of rotation in a separator disk box with a rotating rod. The rotation speed of Rota-Rod was 15 rpm. Measurements were made once a week and up to 3 attempts. The time (seconds) until the mouse fell from the Rota-Rod was measured. If the mouse continued to try for 300 seconds or dropped, the best record of the three attempts was scored that week.

(3) Measurement of Cu concentration The spinal cord was wet ashed with concentrated nitric acid (65%) at room temperature overnight. The spinal cord was completely dissolved by heating in an 80 ° C water bath for 1 hour. An appropriate amount of this solution was diluted with ultrapure water. The Cu concentration was measured using inductively coupled plasma mass spectrometry (Agilent 7500, Yokogawa Analytical Systems) and determined as the Cu concentration per spinal tissue wet weight (μg / g wet tissue).

(4) Measurement of lipid peroxide (LPO) production The lipid peroxide concentration was measured by the thiobarbituric acid (TBA) method (Ono et al, 1998. Radiat. Res. 150, 52-57). The spinal cord was homogenized with ice-cold PBS, and the protein was removed with sulfuric acid and phosphotungstic acid. TBA reagent was added under acidic conditions and boiled at 100 ° C for 1 hour. After running water treatment, lipid peroxide was extracted with n-butanol. The n-butanol layer was determined as the malondialdehyde concentration (nmol / g wet tissue) with respect to the standard solution (1,1,3,3-tetraethoxypropane) at an excitation wavelength of 515 nm and a fluorescence wavelength of 553 nm.

(5) Behavior of apoptosis-related factors The behavior of caspase-3 and survivin as apoptosis-related factors was examined by Western blotting as apoptosis-related factors. The protein mass of the spinal cord tissue was quantified by the pyrogallol red method. The protein was separated by 15% sodium dodecylsulfate-polyacryl amide gel (SDS-PAGE). After transferring the separated protein to the polyvinelydene difluoride membrane, mouse monoclonal anti caspase-3 antidbody or mouse monoclonal anti survivin antibody was reacted. Protein detection was visualized by enhanced chemiluminescence after labeling the primary antibody with mouse monoclonal horseradish conjugated anti IgG antibody. Band intensity was quantified by NIH image (National Institutes of Health, Bethesda, MD, USA), and was determined as the relative protein expression level for β-tubulin.
B. Results (1) Motor function of ALS model mouse Figure 1 shows the effect of TTM on the motor function of Tg. As shown in Figure 1, the progression of motor function decline due to ALS was significantly suppressed in the TTM group compared to the PBS group (control group) (p = 1.89 × 10 ^-8 , repeated-measures ANOVA).

(2) Increase in survival rate As shown in Fig. 2, the survival period of ALS model mice was extended by 23.6% by TTM for about 30 days (p = 4.01 × 10 ^-5 , log-rank test).

(3) Spinal Cu Concentration and LPO Production As shown in Figure 3, the spinal Cu concentration and LPO production were significantly lower in the TTM administration group than in the PBS group.

(4) Effect on the state of hyperapoptosis As shown in Fig. 4, in the TTM administration group, the expression level of caspase-3, which is an apoptosis execution factor, was significantly reduced, and conversely, the expression level of survivin, an apoptosis inhibitor, was significantly Increased.

It is a figure which shows the effect | action which acts on the motor function (Rota-Rod Time) of TTM. It is a figure which shows the effect | action which acts on the survival time of the ALS model mouse of TTM. It is a figure which shows the change of Cu density | concentration in spinal cord and the amount of LPO production | generation by TTM administration. It is a figure which shows the change of the amount of caspase 3 and survivin by TTM administration.

Claims (1)

A therapeutic agent for amyotrophic lateral sclerosis comprising tetrathiomolybdic acid or a salt thereof as an active ingredient, which is orally administered as tetrathiomolybdic acid per adult per day from 0.5 mg / kg to 50 mg / kg. medicine.