Application of a composition containing riluzole and (+)-2-borneol to the preparation of a cerebrovascular drug

Abstract

A composition used for preparing a drug for cerebrovascular disease, particularly ischemic cerebrovascular disease, comprising 2-amino-6-trifluoromethoxybenzothiazole or a pharma- ceutical acceptable salt thereof and borneol. The results of non-clinical cell tests and animal efficacy tests show that 2-amino-6-trifluoromethoxybenzothiazole and (+)-2-borneol, when used in combination, have the function of synergistically increasing the efficacy against cerebrovascular disease.

Description

[Technical Field]

[0001] (cross reference) This application claims priority based on a Chinese patent application filed with the State Intellectual Property Office of China on February 2, 2021, bearing application number 202110141522.3 and entitled "Application of a composition containing riluzole and borneol to the preparation of a cerebrovascular drug," the entire contents of which are incorporated herein by reference.

[0002] The present invention belongs to the field of pharmacy and relates to the application of a composition of 2-amino-6-trifluoromethoxybenzothiazole and borneol or (+)-2-borneol in the preparation of a medicament for treating cerebrovascular diseases, especially ischemic cerebrovascular diseases. [Background technology]

[0003] Cerebrovascular disease (CVD) refers to brain lesions caused by various cerebrovascular diseases and can be divided into acute cerebrovascular disease (stroke) and chronic cerebrovascular disease depending on the pathogenesis. Acute cerebrovascular diseases include transient ischemic attack, cerebral thrombosis, cerebral embolism, hypertensive encephalopathy, cerebral hemorrhage, and subarachnoid hemorrhage. Chronic cerebrovascular diseases include cerebral arteriosclerosis, vascular dementia, cerebral steal syndrome, and Parkinson's disease. Ischemic stroke is a general term for necrosis of brain tissue caused by stenosis or occlusion of the arteries supplying blood to the brain (carotid arteries and vertebral arteries) or by insufficient blood supply to the brain. Cerebral ischemia includes four types: transient ischemic attack (TIA), reversible ischemic neurological deficit (RIND), progressive stroke (SIE), and complete stroke (CS). No cerebral infarction was observed in TIA, but cerebral infarction of varying degrees was observed in RIND, SIE, and CS.

[0004] 2-Amino-6-trifluoromethoxybenzothiazole (riluzole), a member of the benzothiazole family of compounds, was first developed in the 1950s as a centrally acting muscle relaxant and was approved by the US FDA in 1995 for the treatment of amyotrophic lateral sclerosis (ALS). Riluzole inhibits glutamate release from cultured neurons, brain slices, and in vivo cerebral cortical neurons. This effect is thought to be due to the inactivation of voltage-gated sodium channels in glutamatergic nerve terminals and the activation of pertussis toxin (PTX)-sensitive G protein-dependent signaling processes. Riluzole also inhibits N-methyl-D-aspartate (NMDA) receptors and kainic acid receptors (IC receptors). 50Riluzole may also block some of the postsynaptic effects of glutamate by noncompetitively blocking glutamate-dependent neurotransmission (167 μM). In vivo, riluzole has neuroprotective, anticonvulsant, and sedative effects. In a rodent model of transient global cerebral ischemia, riluzole can completely inhibit the ischemia-induced surge in glutamate release. In vitro, riluzole can protect cultured neurons from hypoxic damage, toxic damage caused by glutamate uptake inhibitors, and damage caused by toxic factors in the cerebrospinal fluid of patients with amyotrophic lateral sclerosis (Neurology, 1996, 47(6 Suppl 4), S233S-41). Since then, preclinical animal studies and human clinical trials have demonstrated that riluzole has certain therapeutic effects on spinal cord neuroprotection, neuropathic pain, epilepsy, anxiety, and depression (Chinese Journal of Pharmacology, 2015, 50 (14): 1165-1168; Chemical and Bioengineering, 2017, 34 (2): 6-9). Furthermore, a systematic review and meta-analysis of riluzole in the treatment of neurodegenerative movement disorders, including Parkinson's disease (PD), atypical Parkinson's disease (AD), Huntington's disease (HD), and hereditary ataxia, showed that riluzole may be useful for improving symptoms in patients with hereditary ataxia, but further clinical validation studies are needed (Drug Delivery, 2017, 25 (1), 43-48). Furthermore, in a rat model of medium-sized arterial occlusion (MCAO), a single intravenous injection of 4 mg / kg or 8 mg / kg of riluzole 30 minutes after ischemia significantly reduced the neurological deficit score and cerebral infarction area (CNS Drug Reviews, 1997, 3(1), 83-101).

[0005] The structural formula of riluzole is:

[0006] [ka]

[0007] Natural borneol (borneolum) is extracted, processed, and crystallized from the fresh branches and leaves of the Lauraceae plant (Cinnamomum camphora). Its main component is (+)-2-borneol. "The 2015 edition of the Chinese Pharmacopoeia stipulates that the (+)-2-borneol content in natural borneol must be 96.0% or more." Ice-flavored borneol is extracted, processed, and crystallized from the fresh leaves of the Asteraceae plant (Blumea balsamifera). Its main component is (-)-2-borneol. "The 2015 edition of the Chinese Pharmacopoeia stipulates that the (-)-2-borneol content in natural borneol must be 85.0% or more." Synthetic borneol (borneolum syntheticum) is a chemically synthesized compound composed primarily of (+)-2-borneol and (-)-2-borneol. (+)-2-borneol exhibits various biological activities, including anti-inflammatory, antioxidant, and γ-aminobutyric acid (GABA) receptor enhancement (Euro J Pharmacol, 2017 811, 1-11). Furthermore, (+)-2-borneol can provide drug delivery to the central nervous system by temporarily and reversibly increasing the permeability of the blood-brain barrier (BBB) ​​under physiological conditions, while maintaining BBB integrity and protecting brain tissue under pathological conditions (Drug Deliv 2017, 24:1037-1044; Drug Deliv 2018, 25:1617-1633; Biomed Pharmacother 2018, 102:874-883). Furthermore, (+)-2-borneol inhibited the low-concentration GABA-induced increase in recombinant human GABA in Xenopus oocytes. A R(α1β2γ 2L ) function can be increased by more than 10 times, and its EC 50The value was 248 μM (Biochemical Pharmacology, 2005, 69(7), 1101-1111). Natural borneol has the effect of protecting neuronal damage induced by glutamate (Nanjing Medical University Journal (Natural Sciences) 2013, 33(5), 630-635). (+)-2-Borneol has already been used as a raw material pharmaceutical in the first-class new drug, Edaravone (+)-2-Borneol Infusion, and is used to treat ischemic stroke (CDE Accession No. CXHS1800031).

[0008] The chemical structure of (+)-2-borneol is as follows:

[0009] [ka]

[0010] Therefore, providing a composition containing riluzole and borneol for use in treating cerebrovascular diseases is of great practical significance. Summary of the Invention [Problem to be solved by the invention]

[0011] In view of the above, the present invention provides use of a composition containing riluzole and borneol in the preparation of a cerebrovascular therapeutic agent. The composition contains 2-amino-6-trifluoromethoxybenzothiazole or a pharmaceutically acceptable salt thereof, and borneol or (+)-2-borneol. The composition further contains 2-amino-6-trifluoromethoxybenzothiazole or a pharmaceutically acceptable salt thereof, and (+)-2-borneol. [Means for solving the problem]

[0012] In order to achieve the object of the present invention, the present invention provides the following technical solutions:

[0013] According to a first aspect, the composition provided by the present invention comprises the following components: Component (I) is 2-amino-6-trifluoromethoxybenzothiazole, a derivative thereof, a pharmaceutically acceptable salt thereof, or a prodrug molecule thereof; and (+)-2-borneol, borneol, or ingredient (II) which is a drug using (+)-2-borneol as an active ingredient.

[0014] In some specific embodiments of the present invention, the weight ratio of the component (I) to the component (II) is 30:1 to 1.5:1 or 27:1 to 1:27.

[0015] In some specific embodiments of the present invention, the weight ratio of the component (I) to the component (II) is 15:1 to 1.5:1.

[0016] In some specific embodiments of the present invention, the weight ratio of the component (I) to the component (II) is 15:1 to 7.5:1.

[0017] In some specific embodiments of the present invention, the weight ratio of component (I) to component (II) is 1:1, 1:3, 3:1, 9:1, 27:1, 1:9, 1:27, 20:1, 5:1, 15:1, and / or 8:1.

[0018] In some specific embodiments of the present invention, the borneol is one or more of synthetic borneol, (-)-2-borneol, and natural borneol.

[0019] In addition, according to a second aspect, the present invention provides a pharmaceutical agent comprising the above composition and a pharmaceutically acceptable additive.

[0020] Furthermore, according to a third aspect, the present invention also provides the use of the composition or the medicament in the preparation of a medicament for the prevention and / or treatment of cerebrovascular disease.

[0021] In some specific embodiments of the present invention, the cerebrovascular disease is an ischemic cerebrovascular disease.

[0022] In some specific embodiments of the invention, said ischemic cerebrovascular disease is ischemic stroke.

[0023] The borneol in the composition is natural borneol, (-)-2-borneol, or synthetic borneol.

[0024] The pharmaceutical combination according to the present invention is used to prepare a drug for cerebrovascular disease, in which the cerebrovascular disease is preferably ischemic cerebrovascular disease, more preferably ischemic stroke. [Effects of the Invention]

[0025] In the present invention, the results of non-clinical cell tests and animal efficacy tests have shown that when 2-amino-6-trifluoromethoxybenzothiazole and (+)-2-borneol are used in combination, they have the advantageous effect of synergistically increasing the efficacy against cerebrovascular diseases. [Brief explanation of the drawings]

[0026] [Figure 1] Figure 1 shows the effects of riluzole and (+)-2-borneol on NMDA-induced neuronal damage, where A shows the effect of riluzole on NMDA-induced neuronal damage, and B shows the effect of (+)-2-borneol on NMDA-induced neuronal damage. [Figure 2] FIG. 2 shows the inhibitory rates of riluzole and (+)-2-borneol against NMDA-induced neuronal damage. [Figure 3] FIG. 3 shows the effects of riluzole, (+)-2-borneol and the two compositions on NMDA-induced primary neuronal excitability damage. [Figure 4]4 shows the effects of riluzole, (+)-2-borneol, and composition (20:1) on NMDA-induced neuronal excitability damage, where A shows the effects of riluzole, (+)-2-borneol, and composition (20:1) on NMDA-induced neuronal excitability damage, B shows the effect of (+)-2-borneol on NMDA-induced neuronal excitability damage, and C shows the effect of composition (20:1) of riluzole and (+)-2-borneol on NMDA-induced neuronal excitability damage. [Figure 5] Figure 5 shows the dose-effect curve (A), median-effect plot (B), and combination coefficient plot (C) of riluzole and (+)-2-borneol (20:1) as calculated by CompuSyn software. [Figure 6] 6 shows the effects of riluzole, (+)-2-borneol, and the composition (5:1) on NMDA-induced neuronal excitability damage, where A shows the effect of riluzole on NMDA-induced neuronal excitability damage, B shows the effect of (+)-2-borneol on NMDA-induced neuronal excitability damage, and C shows the effect of the composition (5:1) of riluzole and (+)-2-borneol on NMDA-induced neuronal excitability damage. [Figure 7] 7 shows the effects of riluzole, (+)-2-borneol, and the composition (1:1) on NMDA-induced neuronal excitability damage, where A shows the effect of riluzole on NMDA-induced neuronal excitability damage, B shows the effect of (+)-2-borneol on NMDA-induced neuronal excitability damage, and C shows the effect of the composition (1:1) of riluzole and (+)-2-borneol on NMDA-induced neuronal excitability damage. [Figure 8] FIG. 8 shows the effect of a composition of riluzole and (+)-2-borneol on neurological deficit symptoms in MCAO rats. [Figure 9]FIG. 9 shows the effect of a composition of riluzole and (+)-2-borneol on the area of ​​cerebral infarction. [Figure 10] Figure 10 shows the dose-effect curve (A), median-effect plot (B), and combination coefficient (Fa-CI) plot (C) of the effect of riluzole and (+)-2-borneol (mass ratios of 8:1 and 15:1) on reducing cerebral infarction area in MCAO rats, as calculated using CompuSyn software. [Figure 11] 11 shows the effect of a composition of riluzole, (+)-2-borneol, (-)-2-borneol, and synthetic borneol on the neurological deficit score and cerebral infarction area in MCAO rats, where A shows the effect of a composition of riluzole, (+)-2-borneol, (-)-2-borneol, and synthetic borneol on the neurological deficit score in MCAO rats, and B shows the effect of a composition of riluzole, (+)-2-borneol, (-)-2-borneol, and synthetic borneol on the cerebral infarction area in MCAO rats. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention discloses the application of a composition containing riluzole and borneol in the preparation of a cerebrovascular drug. Those skilled in the art can realize the present invention by appropriately modifying the process parameters based on the contents of this specification. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. Although the method and application of the present invention have been described using preferred embodiments, it is clear that those skilled in the art can modify or make appropriate changes and combinations of the method and application described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0028] All of the raw materials and reagents used in the application of the composition containing riluzole and borneol provided by the present invention to the preparation of a cerebrovascular drug can be purchased from the market.

[0029] The present invention will now be further described with reference to the following examples. [Example]

[0030] Example 1 Effects of Riluzole and (+)-2-Borneol on NMDA-Induced Excitotoxicity Damage in Primary Cortical Neurons

[0031] 1. Materials and Methods 1.1 Animals Pregnant SD rats, Shanghai Slack Experimental Animal Co., Ltd. (Production permit number: SCXK (Shanghai) 2017-0005)

[0032] [ka]

[0033] 1.3 Generation of primary cortical neurons Pregnant SD rats on day 18 were sacrificed by cervical dislocation, and E18 fetal rat brains were removed from the uterus. The cerebral cortical tissues of the fetal rats were isolated and placed in ice-cold DMEM. The meninges and blood vessels on the cortical tissue were removed under a dissecting microscope, and the cortical tissues were transferred to ice-cold DMEM and cut into small pieces (approximately 1 mm). 3 The cells were cut into small pieces and digested with trypsin at 37°C for 10 minutes. The digestion was then stopped with FBS, gently blown with a Pasteur pipette, and filtered through a 200-mesh sieve. The filtered cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cell mass at the bottom of the tube was gently blown off with complete medium (Neurobasal + B27 + GlutaMax + 1% P / S) preheated to 37°C. The cells were then counted using a hemocytometer. Then, 5 × 10 cells were collected in complete medium. 5The cells were diluted to 100 cells / mL and seeded into PDL-coated 96-well plates (100 μL / well). Half of the medium was replaced with complete medium every other day, and the cells were cultured in vitro until the neurons differentiated and matured on day 11. The cells were then used for the OGD (oxygen glucose deprivation) assay.

[0034] 1.4 Testing NMDA-induced excitotoxicity damage in primary cortical neurons Culture media for in vitro-matured primary neurons was replaced with various concentrations of riluzole (R) or (+)-2-borneol (+)-2-borneol (100, 33.3, 11.1, 3.7, 1.23, and 0.41 μM) in Locke's buffer (NaCl 154 mM, KCl 5.6 mM, NaHCO3 3.6 mM, CaCl2 2.3 mM, D-glucose 5.6 mM, HEPES 5 mM, pH 7.4) and incubated at 37°C for 10 min. Excitatory inducers (NMDA, final concentration 100 μM; glycine, final concentration 10 μM) were added and induced for 30 min. After discarding the induction buffer, the cells were washed once with Locke's buffer containing 1 mM MgCl2, and replaced with complete medium (100 μL / well) for 4 h.

[0035] 1.5 Measurement of neuronal cell viability Primary neuron cell viability was detected by a luminescent cell viability assay. 100 μL of reagent was added per well according to the protocol, and the plate was shaken for 10 minutes. The luminescent absorbance (LUM) was then read using a SpectraMaxi3X (Molecule Device) multimode microplate reader, and the relative viability of the neuron cells was calculated.

[0036] Calculation formula: relative neuronal viability V (%) = (LUM - LUM) ブランク ) / (LUM 正常対照群 -LUM ブランク群 )×100%. LUM ブランク is the blank reading of the complete medium wells without cells to which the luminescent cell viability assay reagent was added.

[0037] 1.6 Neuroprotective effects of compounds The neuroprotective effect of the compounds against excitotoxic injury was expressed as the compound's inhibition of neuronal excitotoxic injury. Relative inhibition rate = 100% × (V 化合物 -V NMDAモデル ) / (V 正常対照群 -V NMDAモデル ) where V is the relative survival rate of neurons.

[0038] Compound EC 50 Fitting: Using Prism8 (GraphPad), log (inhibitor) vs. response—variable slope (four parameters) curves were fitted with log [compound concentration] as the horizontal axis and relative inhibition as the vertical axis, and compound IC 50 obtained.

[0039] 1.7 Data Statistics Experimental data are expressed as mean ± standard deviation (Mean ± SD) (n = 3). One-way ANOVA was performed using Prism8 (GraphPad), followed by uncorrected Fisher's LSD to analyze differences between two groups. P < 0.05 indicates a significant difference. ###p < 0.001 compared to the control group. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the NMDA model group.

[0040] 2. Experimental Results 2.1 Effects of riluzole and (+)-2-borneol on NMDA-induced neuronal damage Riluzole concentration-dependently increased NMDA-induced neuronal cell viability at concentrations ranging from 0.41 to 100 μM, and significantly increased neuronal cell viability at concentrations of 33.3 and 100 μM (Figure 1A). (+)-2-Borneol concentration-dependently increased neuronal cell viability after excitotoxic injury at concentrations ranging from 0.41 to 3.7 μM, but further increases in concentration (10 to 100 μM) led to a significant decrease in neuronal cell viability (Figure 1B).

[0041] According to the compound concentration-inhibition rate curves, the EC values ​​of the neuroprotective effects of riluzole and (+)-2-borneol were 50 are approximately 40 μM (E max ~60%) and 2 μM (E max ~15%) (Figure 2).

[0042] Example 2 Effect of a Composition of Riluzole and (+)-2-Borneol on NMDA-Induced Primary Cortical Neuron Excitability Damage

[0043] 1. Materials and Methods 1.1 Animals were the same as in Example 1.

[0044] 1.2 Reagents and consumables were the same as in Example 1.

[0045] 1.3 Primary cortical neurons were prepared in the same manner as in Example 1.

[0046] 1.4 Testing NMDA-induced excitotoxicity damage in primary cortical neurons Cultures of in vitro-matured primary neurons were incubated in Locke's buffer (NaCl 154 mM, KCl 5.6 mM, NaHCO3 3.6 mM, CaCl2 2.3 mM, d-glucose 5.6 mM, HEPES 5 mM, pH 7.4) containing various concentrations of riluzole (R), (+)-2-borneol (B), or a combination of riluzole and (+)-2-borneol (RB) (see Table 1) at 37°C for 10 minutes. After incubation, excitatory inducers (NMDA, final concentration 100 μM, glycine, final concentration 10 μM) were added and induced for 30 minutes. After discarding the induction buffer, the cells were washed once with Locke's buffer containing 1 mM MgCl2 and replaced with complete medium (100 μL / well) for an additional 4 hours.

[0047] [Table 1]

[0048] Note: R is riluzole, B is (+)-2-borneol, RX B Y The X / Y data 1-6 represent the composition of riluzole and (+)-2-borneol, and the X / Y data 1-6 represent the concentrations of each compound, 100, 33.3, 11.1, 3.7, 1.23, and 0.41 μM, respectively. Five replicate wells (n=5) were set up for each drug group. Each compound was dissolved in DMSO so that the final concentration of DMSO in the cell culture medium was 0.2%.

[0049] 1.5 Neuronal cell viability was measured as in Example 1.

[0050] 1.6 Data statistics were the same as in Example 1.

[0051] Experimental data are expressed as mean ± standard deviation (Mean ± SD) (n = 3). One-way ANOVA was performed using Prism8 (GraphPad), followed by uncorrected Fisher's LSD to analyze differences between two groups. P < 0.05 indicates a significant difference. ###p < 0.001 compared to the control group. *p < 0.05, **p < 0.01 compared to the NMDA model group.

[0052] 2. Experimental Results 2.1 Effect of a Composition of Riluzole and (+)-2-Borneol on Primary Neuronal Excitatory Injury As shown in Figure 3, when administered alone in the concentration range of 0.41 to 100 μM, riluzole significantly reduced the cell viability of neurons with NMDA-induced excitotoxicity at the R2 concentration (33.3 μM), whereas (+)-2-borneol significantly improved the cell viability of neurons with NMDA-induced excitotoxicity only at the R4 concentration (3.7 μM). The compositions of riluzole and (+)-2-borneol R1B1 (1:1), R2B1 (1:3), R2B2 (1:1), R2B3 (3:1), R2B4 (9:1), R2B5 (27:1), R3B1 (1:9), R3B2 (1:3), R3B3 (1:1), R3B4 (3:1), R3B5 (9:1), R3B6 (1:27), R4B1 (1:27), R4B2 (1:9), R4B3 (1:3), R4B4 (1:1), R4B5 (3:1), R4B6 (9:1) could significantly improve NMDA-induced neuronal cell viability. This indicates that combining riluzole and (+)-2-borneol in a ratio of 27:1 to 1:27 can produce a synergistic effect on neuroprotection.

[0053] Example 3: Study on the synergism of the composition of riluzole and (+)-2-borneol (20:1) in protecting primary neuronal excitatory injury

[0054] 1. Materials and Methods 1.1 Animals were the same as in Example 1.

[0055] 1.2 Reagents and consumables were the same as in Example 1.

[0056] 1.3 Primary cortical neurons were prepared in the same manner as in Example 1.

[0057] 1.4 Testing NMDA-induced excitotoxicity damage in primary cortical neurons Cultures of in vitro-matured primary neurons were incubated with various concentrations of riluzole (R), (+)-2-borneol (B), or a 20:1 mixture of riluzole and (+)-2-borneol in Locke's buffer (see Table 2) at 37°C for 10 minutes. Then, excitatory inducers (NMDA, final concentration 100 μM; glycine, final concentration 10 μM) were added and induced for 30 minutes. After discarding the induction buffer, the cells were washed once with Locke's buffer containing 1 mM MgCl2, replaced with complete medium (100 μL / well), and re-incubated for 4 hours.

[0058] [Table 2]

[0059] 1.5 Measurement of neuronal cell viability was as in Example 1.

[0060] 1.6 The neuroprotective effect of the compounds was examined in the same manner as in Example 1.

[0061] 1.7 Analysis of composition synergy Synergy analysis was performed using CompuSyn software (ComboSyn, Inc.) on the neuroprotective effect of a fixed ratio combination of riluzole and (+)-2-borneol.

[0062] 1.8 Data Statistics Experimental data are expressed as mean ± standard deviation (Mean ± SD) (n = 3–6). One-way ANOVA was performed using Prism8 (GraphPad), followed by uncorrected Fisher's LSD to analyze differences between two groups. P < 0.05 indicates a significant difference. ###p < 0.001 compared to the control group. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the NMDA model group.

[0063] 2. Experimental Results 2.1 Effects of Riluzole, (+)-2-Borneol, and Composition (20:1) on Primary Neuronal Excitatory Injury Based on the results of Example 1, the compound concentrations were designed to be 1 / 4×ED50, 1 / 2×ED50, 1×ED50, 2×ED50, and 4×ED50, the riluzole concentrations were 10, 20, 40, 80, and 160 μM, the (+)-2-borneol concentrations were 0.5, 1, 2, 4, and 8 μM, and the molar ratio of riluzole to (+)-2-borneol was 20:1. Riluzole, (+)-2-borneol, and the composition of both can increase the cell viability of NMDA-induced injured neurons in a concentration-dependent manner, and the effect of the composition is superior to that of riluzole or (+)-2-borneol (FIG. 3).

[0064] 2.2 Synergistic analysis of neuroprotective effects of a composition of riluzole and (+)-2-borneol According to the principle of the Chou-Talalay equation, the combination coefficient (CI) of the fixed proportion composition was calculated using CompuSyn software (see Table 3 and Figure 5). The CI of the composition of riluzole and (+)-2-borneol (the molar ratio of riluzole to (+)-2-borneol was 20:1) was <1, indicating that riluzole and (+)-2-borneol have a synergistic effect on protecting against neuronal excitatory injury.

[0065] [Table 3]

[0066] Example 4: Study on the synergism of the composition of riluzole and (+)-2-borneol (5:1) in protecting primary neuronal excitatory injury

[0067] 1. Materials and Methods 1.1 Animals were the same as in Example 1.

[0068] 1.2 Reagents and consumables were the same as in Example 1.

[0069] 1.3 The generation of primary cortical neurons was the same as in Example 1.

[0070] 1.4 Testing NMDA-induced excitotoxicity damage in primary cortical neurons Cultures of in vitro matured primary neurons were incubated with various concentrations of riluzole (R), (+)-2-borneol (B), or a 5:1 mixture of riluzole and (+)-2-borneol in Locke's buffer (see Table 2) at 37°C for 10 minutes. Then, excitatory inducers (NMDA, final concentration 100 μM; glycine, final concentration 10 μM) were added and induced for 30 minutes. After discarding the induction buffer, the cells were washed once with Locke's buffer containing 1 mM MgCl2, replaced with complete medium (100 μL / well), and re-incubated for 4 hours.

[0071] [Table 4]

[0072] 1.5 Neuronal cell viability was measured as in Example 1.

[0073] 1.6 The neuroprotective effect of the compounds was examined in the same manner as in Example 1.

[0074] 1.7 Analysis of composition synergy Synergy analysis was performed using CompuSyn software (ComboSyn, Inc.) on the neuroprotective effects of a fixed ratio combination of riluzole and (+)-2-borneol.

[0075] 1.8 Data statistics were performed as in Example 3.

[0076] 2. Experimental Results 2.1 Effects of Riluzole, (+)-2-Borneol, and Composition (5:1) on Primary Neuronal Excitatory Injury The concentrations of riluzole were 2.5, 5, 10, 20, and 40 μM, and the concentrations of (+)-2-borneol were 0.5, 1, 2, 4, and 8 μM. The molar ratio of riluzole to (+)-2-borneol was 5:1. Riluzole, (+)-2-borneol, and both compositions were able to increase the cell viability of NMDA-induced injured neurons in a concentration-dependent manner, and the effect of the composition (5:1) was superior to that of riluzole or (+)-2-borneol (Figure 6).

[0077] 2.2 Synergistic analysis of neuroprotective effects of a composition of riluzole and (+)-2-borneol According to the principle of the Chou-Talalay equation, the combination coefficient (CI) of the fixed proportion composition was calculated using CompuSyn software (see Table 5). The CI of the composition of riluzole and (+)-2-borneol (the molar ratio of riluzole to (+)-2-borneol was 5:1) was <1, indicating that riluzole and (+)-2-borneol have a synergistic effect on protecting against neuronal excitatory injury.

[0078] [Table 5]

[0079] Example 5: Study on the synergism of the composition of riluzole and (+)-2-borneol (1:1) in protecting primary neuronal excitatory injury

[0080] 1. Materials and Methods 1.1 Animals were the same as in Example 1.

[0081] 1.2 Reagents and consumables were the same as in Example 1.

[0082] 1.3 Primary cortical neurons were prepared in the same manner as in Example 1.

[0083] Testing 1.4N MDA-induced excitotoxic damage in primary cortical neurons Cultures of in vitro-matured primary neurons were incubated with various concentrations of riluzole (R), (+)-2-borneol (B), or a 1:1 mixture of riluzole and (+)-2-borneol in Locke's buffer (see Table 2) at 37°C for 10 minutes. The neurons were then induced for 30 minutes by adding excitatory inducers (NMDA, final concentration 100 μM; glycine, final concentration 10 μM). After discarding the induction buffer, the cells were washed once with Locke's buffer containing 1 mM MgCl2 and replaced with complete medium (100 μL / well) for 4 hours.

[0084] [Table 6]

[0085] 1.5 Neuronal cell viability was measured as in Example 1.

[0086] 1.6 The neuroprotective effect of the compounds was examined in the same manner as in Example 1.

[0087] 1.7 Analysis of composition synergy Synergy analysis was performed using CompuSyn software (ComboSyn, Inc.) on the neuroprotective effects of a fixed ratio combination of riluzole and (+)-2-borneol.

[0088] 1.8 Data statistics were the same as in Example 3.

[0089] 2. Experimental Results 2.1 Effects of Riluzole, (+)-2-Borneol, and Composition (1:1) on Primary Neuronal Excitatory Injury The concentrations of riluzole were 0.5, 1, 2, 4, and 8 μM, and the concentrations of (+)-2-borneol were 0.5, 1, 2, 4, and 8 μM. The molar ratio of riluzole to (+)-2-borneol was 1:1. Riluzole's protective effect was somewhat concentration-dependent within this concentration range. Both (+)-2-borneol and the composition (1:1) were able to increase the cell viability of NMDA-induced damaged neurons in a concentration-dependent manner, and the effect of the composition (1:1) was superior to that of riluzole or (+)-2-borneol (Figure 7).

[0090] 2.2 Synergistic analysis of neuroprotective effects of a composition of riluzole and (+)-2-borneol According to the principle of the Chou-Talalay equation, the combination coefficient (CI) of the fixed proportion composition was calculated using CompuSyn software (see Table 7). The CI of the composition of riluzole and (+)-2-borneol (the molar ratio of riluzole to (+)-2-borneol was 1:1) was <1, indicating that riluzole and (+)-2-borneol have a synergistic effect on protecting against neuronal excitatory injury.

[0091] [Table 7]

[0092] Example 6 Study on the protective effect of a composition of riluzole and (+)-2-borneol against focal cerebral ischemia-reperfusion injury

[0093] 1. Materials and Methods 1.1 Animals Sprague-Dawley (SD) rats, male, SPF grade, weighing 250-280g.

[0094] 1.2 Test Drug Riluzole and (+)-2-borneol were the same as in Example 1.

[0095] 1.3 Experimental Method 1.3.1 Creation of a focal cerebral ischemia-reperfusion model A rat model of focal cerebral ischemia-reperfusion was created using the intracarotid artery suture method. The limbs and head of an anesthetized rat were tied with rubber bands (the hind limbs were tied above the knee joints, and the forelimbs were tied above the wrist joints). The animal was then placed supine on the operating table. The hair from the head to the chest was shaved with an animal shaver, and the skin was disinfected with alcohol. A midline neck incision was made, and the subcutaneous tissue was bluntly dissected. A thin fascia on the surface of the anterior cervical triangle was dissected, and the inferior end of the cliohyoid muscle was exposed. The arterial shell was opened, exposing the right carotid bifurcation. The right common carotid artery, external carotid artery, and internal carotid artery were then dissected. The vagus nerve was gently dissected, and the external carotid artery was ligated and transected. The proximal end of the common carotid artery was occluded, and an incision was made distal to the external carotid artery ligature. An embolic line was inserted through the common carotid artery bifurcation and into the internal carotid artery. It was then slowly advanced until resistance was felt (approximately 20 mm from the bifurcation), completely blocking the blood supply to the middle cerebral artery. The embolic line was slightly secured below the incision in the external carotid artery with a thread, and the occluding thread at the proximal end of the common carotid artery was loosened. The wound was covered with gauze soaked in sterile saline, and the rat was placed on a heating pad to maintain warmth. After 2.0 hours of right cerebral ischemia, the embolic line was slowly withdrawn to restore blood supply and allow reperfusion. The external carotid artery was ligated with the secured embolic line thread, and the skin was sutured and disinfected. The rats were placed on clean feed and monitored for normal behavior and breathing until they recovered from anesthesia. They were then provided with food and water and housed in the usual manner.

[0096] 1.3.2 Animal Grouping and Dosing The test animals were divided into nine groups: riluzole group (6 mg / kg and 12 mg / kg, iv), (+)-2-borneol group (0.4 mg / kg and 0.8 mg / kg, iv), riluzole and (+)-2-borneol composition group (6.4 mg / kg and 12.8 mg / kg, iv, riluzole:(+)-2-borneol = 15:1), and model group. After the cerebral ischemia model was established, animals were assigned to each group at equal probability using a single-blind method. The animals received a single intravenous dose of the drug immediately after reperfusion, while animals in the model group received an equal volume of saline. Neurological deficits were evaluated 24 hours after cerebral ischemia, after which the animals were sacrificed. The brains were removed, stained, and photographed to measure the area of ​​cerebral infarction.

[0097] 1.3.3 Neurological deficit score and measurement of cerebral infarction area Neurological deficits were assessed using a modified Bederson 5-point scale. Neurological deficits in rats after cerebral ischemia were assessed using a single-blind method. The test designer assigned the animals to different groups, and the testers scoring the neurological deficits were blinded to the group assignments. After scoring was completed, the testers handed over the scores for the various marks to the designer, who then assessed the scores for each animal in each test group.

[0098] [Table 8]

[0099] The extent of cerebral infarction was measured using TTC staining. After evaluating the animals for neurological deficits, they were sacrificed with CO2 and decapitated. The brains were removed, and the olfactory bulbs, cerebellum, and lower brainstem were removed. The brains were washed with saline to remove blood from the surface, and the remaining water on the surface was absorbed. The brains were then placed at -20°C for 20 min. Immediately after removal, coronal sections were cut at 2 mm intervals in a cross-section perpendicular to the viewing plane and stained in 1% TTC solution (37°C, 30 min). Normal brain tissue stained dark red, while ischemic brain tissue stained lightly. After washing with saline, the brain sections were quickly arranged in a line from front to back, the remaining water on the surface was absorbed, the surfaces were dried, and then photographed.

[0100] Calculation of cerebral infarct area: The photographs were processed using Image J software, and the corresponding area in the left brain and the area of ​​the non-infarcted region in the right brain were calculated using the following formula, and the percentage of the infarct area was calculated.

[0101] How to calculate cerebral infarct volume: V=t(A1+A2+A3+……+An) where t is the thickness of the slice and A is the infarct area.

[0102] %I = 100% × (V C -V L ) / V C where %I is the percentage of infarct volume, VC is the brain volume on the target side (left hemisphere), and VL is the volume of the non-infarcted region on the infarcted side (right hemisphere).

[0103] 1.4 Analysis of composition synergy Synergy analysis is performed using CompuSyn software (ComboSyn, Inc.) on the neuroprotective effects of a non-fixed ratio combination of riluzole and (+)-2-borneol.

[0104] 1.5 Data Statistics The experimental data are expressed as mean ± standard deviation (Mean ± SD) (n = 10). One-way ANOVA (one-way analysis of variance) was performed, followed by Fisher's LSD to analyze the differences between two groups. P < 0.05 indicates a significant difference. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the MCAO model. ns indicates no statistical difference between the groups shown in the figure.

[0105] 2. Experimental Results 2.1 Effect of a Composition of Riluzole and (+)-2-Borneol on Neurological Deficit Symptoms As shown in Figure 8, riluzole, (+)-2-borneol, and the composition all dose-dependently reduced the neurological deficit scores in MCAO rats. Furthermore, compared with the MCAO model group, the neurological deficit scores in MCAO rats were significantly reduced in the 8 mg / kg riluzole group, the 1 mg / kg (+)-2-borneol group, the 4.5 mg / kg and 9 mg / kg composition groups (riluzole to (+)-2-borneol mass ratio of 8:1), and the 6.4 mg / kg and 12.8 mg / kg composition groups (riluzole to (+)-2-borneol mass ratio of 15:1). Furthermore, the composition group tended to have a lower neurological deficit score than the riluzole or (+)-2-borneol groups.

[0106] 2.2 Effect of a composition of riluzole and (+)-2-borneol on the size of cerebral infarction As shown in Figure 8, compared with the MCAO model group, the riluzole 4 and 8 mg / kg groups, the (+)-2-borneol 0.5 and 1 mg / kg groups, the composition (riluzole to (+)-2-borneol mass ratio 8:1) 4.5 mg / kg and 9 mg / kg (8:1), and the composition (riluzole to (+)-2-borneol mass ratio 15:1) 6.4 mg / kg and 12.8 mg / kg significantly reduced the area of ​​cerebral infarction in a dose-dependent manner. Furthermore, compared with the riluzole or (+)-2-borneol groups, the composition group showed a smaller area of ​​cerebral infarction in the animals.

[0107] 2.3 Synergistic analysis of the composition of riluzole and (+)-2-borneol in reducing the size of cerebral infarction The combination index (CI) of non-fixed proportion compositions was calculated using CompuSyn software according to the principle of the Chou-Talalay equation (see Table 9 and Figure 10). The CI of the composition of riluzole and (+)-2-borneol (the mass ratio of riluzole to (+)-2-borneol was 8:1) was <1, indicating that riluzole and (+)-2-borneol have a synergistic effect in reducing the area of ​​cerebral infarction in MCAO rats.

[0108] [Table 9]

[0109] Example 7 Study on the protective effect of compositions of riluzole with (+)-2-borneol, (-)-2-borneol, and synthetic borneol against focal cerebral ischemia-reperfusion injury

[0110] 1. Materials and Methods 1.1 The test animals were the same as in Example 6.

[0111] 1.2 Test Drug Riluzole and (+)-2-borneol were the same as in Example 1. (-)-2-Borneol and synthetic borneol were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0112] 1.3 Experimental Method 1.3.1 A focal cerebral ischemia-reperfusion model was prepared in the same manner as in Example 6.

[0113] 1.3.2 Animal Grouping and Dosing The test animals were divided into four groups: a riluzole and (+)-2-borneol composition group (8 mg / kg riluzole and 1 mg / kg (+)-2-borneol, iv), a riluzole and (-)-2-borneol composition group (8 mg / kg riluzole and 1 mg / kg (-)-2-borneol), a riluzole-synthetic-borneol composition group (8 mg / kg riluzole and 1 mg / kg synthetic borneol), and a model group. After creating the cerebral ischemia model, animals were assigned to each group at equal probability using a single-blind method. The animals received a single intravenous dose of the drug immediately after reperfusion, while animals in the model group received an equal volume of saline. Neurological deficits were evaluated 24 hours after cerebral ischemia, after which the animals were sacrificed. The brains were removed, stained, and photographed to measure the area of ​​cerebral infarction.

[0114] 1.3.3 The scores of neurological deficit symptoms and the area of ​​cerebral infarction were measured in the same manner as in Example 6.

[0115] 1.4 Data Statistics The experimental data are expressed as mean ± standard deviation (Mean ± SD) (n = 10). One-way ANOVA (one-way analysis of variance) was performed, followed by Fisher's LSD to analyze the differences between two groups. P < 0.05 indicates a significant difference. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the MCAO model. ns indicates no statistical difference between the groups shown in the figure.

[0116] 2. Experimental results 2.1 Effect of the composition on neurological deficit symptoms and cerebral infarction size in MCAO rats As shown in Figure 11, the composition of riluzole (8 mg / kg) and (+)-2-borneol (1 mg / kg), the composition of riluzole (8 mg / kg) and (-)-2-borneol (1 mg / kg), and the composition of riluzole (8 mg / kg) and synthetic borneol (1 mg / kg) all significantly reduced the neurological deficit score and cerebral infarction area in MCAO rats. Furthermore, there was no difference in the neurological deficit score and cerebral infarction area among these three compositions.

[0117] The application of the composition containing riluzole and borneol provided by the present invention to the preparation of a cerebrovascular drug has been described in detail above. The principles and embodiments of the present invention are explained in this specification using specific examples, and the explanations in the above examples are only used to understand the method and gist of the present invention. It should be noted that those skilled in the art may make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications are also within the patentable scope of the present invention. <Additional Notes> The present invention includes the following aspects. <Section 1> Component (I) is 2-amino-6-trifluoromethoxybenzothiazole, a derivative thereof, a pharmaceutically acceptable salt thereof or a prodrug molecule thereof; A composition comprising (+)-2-borneol, borneol, or a drug containing (+)-2-borneol as an active ingredient (II). <Section 2> Item 2. The composition according to item 1, wherein the weight ratio of component (I) to component (II) is 30:1 to 1.5:1. <Section 3> Item 2. The composition according to item 1, wherein the weight ratio of component (I) to component (II) is 15:1 to 1.5:1. <Section 4> Item 2. The composition according to item 1, wherein the weight ratio of component (I) to component (II) is 15:1 to 7.5:1. <Section 5> Item 2. The composition according to item 1, wherein the weight ratio of component (I) to component (II) is 1:1, 1:3, 3:1, 9:1, 27:1, 1:9, 1:27, 20:1, 5:1, 15:1, and / or 8:1. <Section 6> Item 6. The composition according to any one of Items 1 to 5, wherein the borneol is one or more selected from the group consisting of synthetic borneol, (-)-2-borneol, and natural borneol. <Section 7> A pharmaceutical agent comprising the composition according to any one of items 1 to 6 and a pharmaceutically acceptable additive. <Section 8> Use of the composition according to any one of Items 1 to 6 or the agent according to Item 7 in the preparation of a pharmaceutical for preventing and / or treating cerebrovascular disease. <Section 9> Item 9. The use according to Item 8, wherein the cerebrovascular disease is an ischemic cerebrovascular disease. <Section 10> Item 10. The use according to Item 9, wherein the ischemic cerebrovascular disease is ischemic stroke.

Claims

1. Component (I) is 2-amino-6-trifluoromethoxybenzothiazole or a pharmaceutically acceptable salt thereof; and a component (II) which is (+)-2-borneol, wherein the weight ratio of said component (I) to said component (II) is 20:1 to 1:

1.

2. 2. The pharmaceutical composition according to claim 1, wherein the weight ratio of component (I) to component (II) is 1:1, 3:1, 9:1, 20:1, 5:1, 15:1 or 8:

1.

3. A drug comprising the pharmaceutical composition of claim 1 or 2 and a pharmaceutically acceptable additive.

4. Use of a pharmaceutical composition according to claim 1 or claim 2 or an agent according to claim 3 in the preparation of a medicament for the prevention and / or treatment of cerebrovascular diseases.

5. The use according to claim 4, characterized in that the cerebrovascular disease is an ischemic cerebrovascular disease.

6. The use according to claim 5, characterized in that the ischemic cerebrovascular disease is ischemic stroke.

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