Use of carbamate compound for prevention, alleviation, or treatment of demyelinating disease

Abstract

The present invention relates to a use for preventing, alleviating or treating a demyelinating disease by administering a pharmaceutical composition containing a carbamate compound of chemical formula 1.

Claims

1. A method for alleviating or treating demyelinating diseases in a subject, comprising administering to the subject a therapeutically effective amount of a carbamate compound of the following Formula 1, or a pharmaceutically acceptable salt, solvate or hydrate thereof: ##STR00005## wherein, R.sub.1 and R.sub.2 are each independently selected from the group consisting of hydrogen, halogen, C.sub.1-C.sub.8 alkyl, halo-C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 thioalkoxy and C.sub.1-C.sub.8 alkoxy; and one of A.sub.1 and A.sub.2 is CH, and the other is N, wherein the demyelinating diseases are one or more selected from the group consisting of multiple sclerosis, Devic's disease, inflammatory demyelinating diseases, central pontine myelinolysis, tabes dorsalis, leukoencephalopathies, leukodystrophies, optic neuritis, transverse myelitis, Guillain-Barré syndrome, and Charcot-Marie-Tooth disease.

2. The method according to claim 1, wherein R.sub.1 and R.sub.2 are each independently selected from the group consisting of hydrogen, halogen and C.sub.1-C.sub.8 alkyl.

3. The method according to claim 1, wherein the carbamate compound of Formula 1 is carbamic acid (R)-1-(2-chlorophenyl)-2-tetrazol-2-yl)ethyl ester of the following Formula 2: ##STR00006##

4. The method according to claim 1, wherein the method is for the alleviation or treatment of multiple sclerosis.

5. The method according to claim 4, wherein the multiple sclerosis is one or more selected from the group consisting of Clinically isolated syndrome (CIS), Radiologically isolated syndrome (RIS), Clinically definite multiple sclerosis (CDMS), Relapsing remitting multiple sclerosis (RR-MS), Secondary progressive multiple sclerosis (SP-MS), Primary progressive multiple sclerosis (PP-MS) and Progressive relapsing multiple sclerosis (PR-MS).

6. The method according to claim 1, wherein the method is for the alleviation or treatment of optic neuritis.

7. The method according to claim 1, wherein the subject is a mammal.

8. The method according to claim 7, wherein the mammal is a human.

9. The method according to claim 1, wherein the therapeutically effective amount of the carbamate compound of Formula 1 is 50 mg to 500 mg based on the free form.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing the results of a systematic motor symptom alleviation experiment in an Experimental Autoimmune Encephalomyelitis (EAE) model.

(2) FIG. 2 is a graph showing the results of measuring pattern retinal conduction, which is one of the indicators of functional vision, in an Experimental Autoimmune Encephalomyelitis (EAE) model.

(3) FIGS. 3A and 3B show the results of measuring the number of retinal ganglion cells per unit area in an Experimental Autoimmune Encephalomyelitis (EAE) model, using Brn3a (FIG. 3A) and RBPMS (FIG. 3B) as retinal ganglion cell markers to examine the degree of protection of the optic nerve.

DETAILED DESCRIPTION

(4) Hereinafter, the present invention will be explained in more detail through working examples. However, the following working examples are only intended to illustrate one or more embodiments and are not intended to limit the scope of the invention.

Preparation Example: Synthesis of carbamic acid (R)-1-(2-chlorophenyl)-2-tetrazol-2-yl)ethyl ester

(5) Carbamic acid (R)-1-(2-chlorophenyl)-2-tetrazol-2-yl)ethyl ester (the compound of Formula 2, hereinafter referred to as “the test compound”) was prepared according to the method described in Preparation Example 50 of PCT Publication No. WO 2010/150946.

Example 1: Systemic Motor Symptom Alleviation Effect in an Experimental Autoimmune Encephalomyelitis (EAE) Model

(6) Experimental Animals

(7) For this experiment, male C57BL/6 mice were purchased from Orient Bio, Inc. of Korea. The mice were placed in a wire mesh cage under conditions of ambient temperature of 20 to 24° C., relative humidity of 55 to 75%, an automatically controlled light-and-darkness cycle of 12 hours and free access to feed (purchased from Agri Brands Purina Korea, Inc.) and water. The mice were housed and maintained in accordance with the Laboratory Animal Care Standards of the Institutional Animal Care and Use Committee (IACUC). After about one week of stabilization, mice weighing 18 to 23 g were used in the experiment.

(8) Preparation of EAE-Inducing Samples

(9) To 100 mg of Mycobacterium tuberculosis H37RA (killed and desiccated; Difco, 231141, USA) which had been well triturated in mortar, was mixed 10 ml of Incomplete Freund's adjuvant (IFA; Difco, 263910, USA) to prepare 10 mg/ml of Complete Freund's adjuvant (CFA) storage solution, followed by refrigeration at 4° C. Myelin oligodendrocyte glycoprotein (MOG35-55; Peptron, Korea) was diluted with PBS (phosphate-buffered saline; Gibco, 10010, USA) to a final concentration of 2 mg/ml and stored at −20° C. (MOG35-55 storage solution).

(10) In order to prepare EAE-inducing samples for administration to mice, 1 ml of CFA storage solution diluted to 2 mg/ml with IFA solution and 1 ml of MOG.sub.35-55 storage solution were each put into different syringes, and a 3-way stopcock was used to mix them well at a ratio of 1:1 for over 10 minutes such that air bubbles did not form inside to obtain an emulsion solution having a final concentration of 1 mg/ml.

(11) 50 μg of PTX (pertussis toxin, PTX; Sigma, P7208, USA) was dissolved in 500 μl of PBS to prepare 100 μg/ml of pertussis toxin (PTX) storage solution, followed by registration at 4° C. In the experiment, the PTX storage solution was diluted 50 times with PBS and used.

(12) EAE-Inducing Samples/Drug Administration and Clinical Symptom Observation

(13) On the first day of the experiment (Day-1), the EAE-inducing sample solution was administered to mice by subcutaneous injection at 200 μl per mouse. On the same day (Day-1) and two days later (Day-3), 200 μl of PTX solution was intraperitoneally administered to mice twice. In addition, the mice were observed once every day to check whether the EAE-related clinical symptoms were exhibited, and evaluated by EAE clinical score.

(14) The EAE clinical score is as follows.

(15) 0.0: normal gait

(16) 0.5: normal gait, partially limp tail, tip of tail droops

(17) 1.0: normal gait, paralyzed limp tail, tail droops

(18) 1.5: uncoordinated gait, limp tail, hind limb paresis

(19) 2.0: one hind limb dragging, one hind limb paralyzed

(20) 2.5: both hind limb dragging, both hind limbs paralyzed

(21) 3.0: forelimb weakness, forelimb reflex after pinching

(22) 3.5: one forelimb paralyzed, loss of movement

(23) 4.0: both forelimb paralyzed, loss of movement

(24) 4.5: moribund, altered breathing

(25) 5.0: death

(26) From Day-11, on which the EAE clinical score was observed to be 0.5 or more, the test compound dissolved in 30% polyethylene glycol 400 (Sigma, USA) was administered to mice at a dose of 10 ml/kg, in a volume of 10 ml per body weight of a mouse, orally once a day, and changes in the EAE clinical score were observed.

(27) Statistical Analysis

(28) All data were expressed as mean±SEM. The EAE clinical scores in the group treated with the test compound were compared to those in the negative control vehicle group by date. Statistical analysis was performed using GraphPad Prism (ver. 4.0) program, and the differences between the two groups were analyzed by Student's t-test.

(29) Experiment Results

(30) The EAE clinical score changes by day in the vehicle group as a negative control group and the test compound treated group are shown in Table 1 and FIG. 1. The EAE-related clinical symptoms began to be observed on the 9th day after treatment with EAE-inducing sample (Day-10), and the vehicle and the test compound were administered from Day-11 once daily. As a result, the vehicle group showed clinical symptoms of the highest level of 2.6±0.8 on Day-17, and the test compound treated group showed clinical symptoms in the degree of 2.1±0.6 on Day-17. The test compound treated group showed significant inhibition of EAE clinical symptoms compared to the vehicle group on Day-19 to Day-22, indicating that the test compound exhibited a significant efficacy in the EAE model, a representative animal model of multiple sclerosis and demyelinating diseases.

(31) TABLE-US-00001 TABLE 1 EAE clinical score change table Experiment Test Statistical significance day Vehicle compound (vs. vehicle) Day-1~Day-9 0.0 ± 0.0 0.0 ± 0.0 None Day-10 0.4 ± 1.0 0.5 ± 0.8 None Day-11 1.0 ± 1.1 0.8 ± 1.0 None Day-12 1.9 ± 1.3 1.2 ± 1.1 None Day-13 2.3 ± 0.8 1.3 ± 0.7 None Day-14 2.3 ± 0.8 2.0 ± 0.6 None Day-15 2.5 ± 0.7 1.9 ± 0.5 None Day-16 2.6 ± 0.7 1.9 ± 0.6 None Day-17 2.6 ± 0.8 2.1 ± 0.6 None Day-18 2.3 ± 0.9 1.8 ± 0.5 None Day-19 2.4 ± 0.8 1.7 ± 0.6 p < 0.05 Day-20 2.3 ± 0.9 1.4 ± 0.5 p < 0.05 Day-21 2.2 ± 0.7 1.3 ± 0.4 p < 0.01 Day-22 2.2 ± 0.6 1.1 ± 0.5 p < 0.01

Example 2: Optic Nerve Damage Alleviation Effect in an Experimental Autoimmune Encephalomyelitis (EAE) Model

(32) Experimental Animals

(33) For this experiment, female C57BL/6 mice were purchased from Janvier Labs of France and were maintained under conditions of temperature (22±1° C.), a light-and-darkness cycle of 12 hours and free access to water and food according to the protocol approved and investigated by the Animal Experiment Committee of Finland. All animals were kept isolated for a week after purchase and had time to adapt to the kennel. 11-week-old animals were used in the experiment. According to the criteria of the Animal Experiment Committee of Finland, in the case of a weight loss of 20% or more compared to the starting point, a degree of disease progression (clinical score) of 4 or more, or a failure in the righting reflex test in clinical score of 3, the experiment was stopped and animals were sacrificed for humanitarian purposes.

(34) EAE Induction

(35) To induce EAE, an inoculum was purchased from Hooke Laboratories of the United States: the inoculum contains 100 μg of MOG35-55 peptide and 200 μg of heat-inactivated Mycobacterium tuberculosis, both dissolved in 100 μl of mineral oil. 100 μl of the inoculum was injected subcutaneously into both high and low parts of the back of mice. 100 μl pertussis toxin was injected into the peritoneum of the mice twice, two hours after inoculation and after 24 hours.

(36) Observation of Clinical Symptoms by EAE

(37) EAE-induced mice were observed daily, and the progression of the disease was measured using the following criteria (B Gran, G-X Zhang, S Yu, J Li, X H Chen, E S Ventura, M Kamoun, A Rostami, J. Immunol., 2002, 169, 7104-7110).

(38) 0=No disease

(39) 0.5=Partial tail weakness

(40) 1.0=Complete tail paralysis

(41) 1.5=Flaccid tail and abnormal gait (waddling gait)

(42) 2.0=Flaccid tail and clear weakness of hind limbs

(43) 2.5=Partial paralysis in one hind limb

(44) 3.0=Complete paralysis in both hind limbs

(45) 4.0=Complete paralysis in both hind limbs and weakness in forelimbs (requires euthanasia)

(46) 5.0=Complete paralysis in all limbs, moribund, death (requires euthanasia)

(47) Preparation and Administration of Compound

(48) The test compound was dissolved in distilled water containing 30% polyethylene glycol 400 (PEG 400) and treated with an ultrasonic sonicator for 30 minutes. The dissolved compound (20 mg/kg or 30 mg/kg) or vehicle was injected into the peritoneum once daily from the 12th day after EAE induction.

(49) Measurement of Pattern Retinal Conduction (pERG)

(50) Pattern retinal conduction was measured two days before and 28 days after EAE induction. A drop of oxybuprocaine was placed on the cornea for partial anesthesia, and the mice were placed on a rodent test bench that maintained the body temperature at approximately 37° C. After performing the test, the results were analyzed using Matlab software (MathWorks, Natick, Mass.).

(51) Animal Sacrifice and Tissue Collecting

(52) When the experiment was terminated or when the time of sacrifice for humanitarian purposes was reached, the mice were sacrificed by cardiac perfusion using a tissue fixative, and the eye and optic nerve were removed for further analysis.

(53) Morphological Assessment of Retinal Whole Mount

(54) The retinal whole mount samples were extracted from both eyes and stained with antibodies of RBPMS (RNA binding protein with multiple splicing) and Brn3a (brain-specific homeobox/POU domain protein 3A) which are retinal ganglion cell markers, followed by analyzation in a stereological manner (Stereo Investigator, MicroBrightfield, VT, USA).

(55) Statistical Analysis

(56) Quantitative data were graphed (plotted) and analyzed using Prism software version 7 (GraphPad Software Inc. La Jolla, Calif.), the data were expressed as mean±standard deviation or standard error of mean. Comparisons with the vehicle group were analyzed using a t-test, and when the significance (p value) was less than 0.05, it was judged as a statistically significant difference.

(57) Experiment Results

(58) 1) Pattern Retinal Conduction Amplitude (pERG Amplitude)

(59) The measurement results of the pattern retinal conduction in the vehicle group as a negative control group and in the compound treatment group are shown in Table 2 and FIG. 2. The amplitude of pattern retinal conduction was reduced by about 40% in the vehicle group compared to the baseline measured two days before EAE-inducing sample treatment (t-test, ***p<0.001). In contrast, in the treatment group treated with 20 mg/kg or 30 mg/kg of the test compound, no significant difference was found compared to the baseline. This suggests that the test compound has a tendency to protect the functional vision of the EAE animal model.

(60) TABLE-US-00002 TABLE 2 Pattern retinal conduction amplitude (pERG amplitude) measured values Test Test Treatment compound compound group Baseline Vehicle 20 mg/kg 30 mg/kg Mean ± SEM 9.34 ± 5.435 ± 7.803 ± 7.378 ± 0.5866 0.7171 1.268 1.324 Statistical ***p < 0.001 None None significance (vs. Baseline)

(61) 2) Number of Retinal Ganglion Cells

(62) The measurement results of the number of retinal ganglion cells per unit area in the vehicle group as the negative control group and in the compound treatment group are shown in Table 3 and FIG. 3. A larger number of retinal ganglion cells per unit area were observed in the group treated with 20 mg/kg of the test compound compared to the vehicle group in EAE-induced animals. This means that the test compound showed a significant efficacy on the protection of the retinal ganglion cells of the EAE animal model.

(63) TABLE-US-00003 TABLE 3 Number of retinal ganglion cells per unit area Brn3a RBPMS Test Test Treatment compound compound group Vehicle 20 mg/kg Vehicle 20 mg/kg 1689.82111 1389.43278 2194.48371 2291.69805 1205.24606 1918.969766 1851.58626 2567.37062 1472.22518 2675.372857 1851.12316 3005.11983 606.640405 1750.912472 1614.33127 2250.60258 1801.89007 1442.818497 2431.17226 1777.88991 1895.57712 2138.596922 1937.38139 2976.21405 747.713011 719.985283 1484.84161 1780.68542 1345.61213 2220.629888 2218.24485 2296.67886 1785.76248 1858.996916 2269.22449 2479.9735 871.845571 2488.569838 1517.47713 2722.31576 1006.1851 1673.504971 1707.99971 2362.36326 2035.384604 2485.7761 2328.305062 2784.6808 Mean ± SEM 1312 ± 1895 ± 1916 ± 2445 ± 137.8 144.3 97.56 106.9 Statistical **p < 0.01 **p < 0.01 significance (vs. vehicle)