CN112569217B - Application of valeric acid derivatives in treating hereditary cerebellar ataxia - Google Patents
Application of valeric acid derivatives in treating hereditary cerebellar ataxia Download PDFInfo
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Abstract
The invention discloses application of a compound shown in a formula I in preparation of medicines for preventing, treating or improving hereditary cerebellar ataxia, in particular to autosomal recessive hereditary spinocerebellar ataxia 20(SCAR 20). The compound of the invention can obviously improve the motor ataxia of a model mouse and slow down the death of Purkinje cells, thereby providing clinical support for the treatment of hereditary cerebellar ataxia, especially SCAR 20.
Description
Technical Field
The present invention relates to the field of pharmacy. In particular, the invention relates to the use of pentanoic acid derivatives for the treatment, prevention and amelioration of hereditary cerebellar ataxia, in particular autosomal recessive hereditary spinocerebellar ataxia 20(SCAR 20).
Background
Hereditary ataxia is a highly heterogeneous disease, which is clinically manifested by functional deterioration of motor balance, unstable gait, dyskinesia of eyeball and dysarthria, etc., with a morbidity of about 8.9/10 ten thousand. According to the genetic pattern, the genetic patterns can be divided into autosomal dominant, autosomal recessive, X-linked inheritance and the like. Among them, the incidence of autosomal recessive ataxia is about 3/10 ten thousand, and patients show early onset motor balance and coordination disorders, often accompanied by cerebellar atrophy or hypoplasia. An Autosomal recessive spinocerebellar ataxia 20(SCAR20) is a serious ataxia disease caused by functional deletion mutation of sortingNexin 14(SNX14), and is firstly reported in 2014, and subsequent studies show that the SCAR20 accounts for the highest proportion (9.9%) in the population suffering from early cerebellar atrophy and ataxia. The disease is manifested by early-onset cerebellar atrophy, severe ataxia, moderate-severe intellectual impairment, rough complexion, etc. At present, the mechanism by which SNX14 mutations cause scarr 20 is not clear, and effective drug therapy is less likely to be mentioned.
Therefore, there is an urgent need in the art for a technological means capable of treating, preventing and ameliorating hereditary ataxia, particularly SCAR 20.
Disclosure of Invention
The present invention aims to provide compounds for the treatment, prevention and amelioration of hereditary cerebellar ataxia, in particular, autosomal recessive hereditary spinocerebellar ataxia 20.
In a first aspect, the invention provides the use of a compound of formula I, various crystalline forms, hydrates or solvates thereof in the preparation of a medicament for the prevention, treatment or amelioration of hereditary cerebellar ataxia,
in the formula (I), the compound is shown in the specification,
r is selected from: H. metal ion, substituted or unsubstituted C1-6Alkyl (preferably substituted or unsubstituted C)1-3Alkyl groups);
R1、R2、R3and R4Each independently selected from: H. substituted or unsubstituted C1-6Alkyl, substituted or unsubstituted C1-6Alkoxy, substituted or unsubstituted C3-6Cycloalkyl, halogen, nitro, amino, hydroxy.
In a preferred embodiment, the "substitution" is with C1-3Alkyl, halogen, nitro, amino and hydroxyl.
In particular toIn the embodiment (1), R2、R3And R4Is H.
In a specific embodiment, R1Is substituted or unsubstituted C1-6An alkyl group; more preferably substituted or unsubstituted C1-3An alkyl group; most preferred is propyl.
In a preferred embodiment, R is a metal ion, including but not limited to sodium, magnesium, potassium.
In a specific embodiment, R is a metal ion selected from the group consisting of: sodium ions, magnesium ions.
In a specific embodiment, the compounds of formula I are as follows:
in a specific embodiment, the hereditary cerebellar ataxia is autosomal recessive hereditary spinocerebellar ataxia 20(SCAR 20).
In a second aspect, the present invention provides a compound of formula I, various crystalline forms, hydrates or solvates thereof,
in the formula (I), the compound is shown in the specification,
r is selected from: H. metal ion, substituted or unsubstituted C1-6Alkyl (preferably substituted or unsubstituted C)1-3Alkyl groups);
R1、R2、R3and R4Each independently selected from: H. substituted or unsubstituted C1-6Alkyl, substituted or unsubstituted C1-6Alkoxy, substituted or unsubstituted C3-6Cycloalkyl, halogen, nitro, amino, hydroxy;
can be used for preventing, treating or improving hereditary cerebellar ataxia.
In a preferred embodiment, the "substitution" is with C1-3Alkyl, haloSubstitution of element, nitryl, amino and hydroxyl.
In a preferred embodiment, R2、R3And R4Is H.
In a preferred embodiment, R1Is substituted or unsubstituted C1-6An alkyl group; more preferably substituted or unsubstituted C1-3An alkyl group; most preferred is propyl.
In a preferred embodiment, R is a metal ion selected from the group consisting of: sodium ions, magnesium ions, potassium ions; sodium ions or magnesium ions are preferred.
In a preferred embodiment, the compounds of formula I are as follows:
in a specific embodiment, the hereditary cerebellar ataxia is autosomal recessive hereditary spinocerebellar ataxia 20(SCAR 20).
In a third aspect, the invention provides a medicament for preventing, treating or ameliorating hereditary cerebellar ataxia comprising a compound of formula I, various crystalline forms, hydrates or solvates thereof,
in the formula (I), the compound is shown in the specification,
r is selected from: H. metal ion, substituted or unsubstituted C1-6Alkyl (preferably substituted or unsubstituted C)1-3Alkyl groups);
R1、R2、R3and R4Each independently selected from: H. substituted or unsubstituted C1-6Alkyl, substituted or unsubstituted C1-6Alkoxy, substituted or unsubstituted C3-6Cycloalkyl, halogen, nitro, amino, hydroxy.
In a preferred embodiment, the "substitution" is with C1-3Alkyl, halogen, nitro, amino, hydroxyAnd (4) carrying out substitution.
In a preferred embodiment, R2、R3And R4Is H.
In a preferred embodiment, R1Is substituted or unsubstituted C1-6An alkyl group; more preferably substituted or unsubstituted C1-3An alkyl group; most preferred is propyl.
In a preferred embodiment, R is a metal ion selected from the group consisting of: sodium ions, magnesium ions, potassium ions; sodium ions or magnesium ions are preferred.
In a preferred embodiment, the compounds of formula I are as follows:
in a preferred embodiment, the prevention, treatment, or amelioration of hereditary cerebellar ataxia refers to the treatment, prevention, and amelioration of autosomal recessive hereditary spinocerebellar ataxia 20(SCAR 20).
In a fourth aspect, the present invention provides a method for preventing, treating or ameliorating hereditary cerebellar ataxia, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, or various crystalline forms, hydrates or solvates thereof,
in the formula (I), the compound is shown in the specification,
r is selected from: H. metal ion, substituted or unsubstituted C1-6Alkyl (preferably substituted or unsubstituted C)1-3Alkyl groups);
R1、R2、R3and R4Each independently selected from: H. substituted or unsubstituted C1-6Alkyl, substituted or unsubstituted C1-6Alkoxy, substituted or unsubstituted C3-6Cycloalkyl, halogen, nitro, amino, hydroxy.
In a preferred embodiment, theBy "substituted" is meant with C1-3Alkyl, halogen, nitro, amino and hydroxyl.
In a preferred embodiment, R2、R3And R4Is H.
In a preferred embodiment, R1Is substituted or unsubstituted C1-6An alkyl group; more preferably substituted or unsubstituted C1-3An alkyl group; most preferred is propyl.
In a preferred embodiment, R is a metal ion selected from the group consisting of: sodium ions, magnesium ions, potassium ions; sodium ion and magnesium ion are preferred.
In a preferred embodiment, the compounds of formula I are as follows:
in a preferred embodiment, the hereditary cerebellar ataxia is autosomal recessive hereditary spinocerebellar ataxia 20(SCAR 20).
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. For reasons of space, they will not be described in detail.
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FIG. 1 is a graph of the strategy for constructing conditional knockout mice in the Snx14 brain and the results of identifying knockout mice by PCR and immunoblotting. In the figure, Ctx represents cortex, Hip represents hippocampus, Cb represents cerebellum, and Sp represents spinal cord.
Figure 2 is a timeline of Sodium Valproate (VPA) treatment trials (including dosing regimens and follow-up trials) and a line graph of mouse body weight change during VPA treatment. P25 represents postnatal day 25, P40, P50 and P60 and so on.
FIG. 3 is a graph of the assessment of VPA treatment versus knockout mice (Snx14) using the behavioral paradigm of Balance beamf/f(ii) a Nestin-cre) influence of motor coordination abilityThe Time for the mice to pass through the balance bar (Time to cross the beam) and the Number of hind limb glides (Number of slides) were recorded and plotted statistically.
FIG. 4 is a graph of VPA treatment versus Snx14f/f(ii) a Influence of the number of Nestin-cre mouse cerebellar Purkinje cells (Purkinje cell, PKJ). Shown in the figure are representative images of immunohistochemical staining of PKJ cell marker protein Calbindin and PKJ quantitative statistical images.
FIG. 5 is a graph evaluating the effect of VPA treatment on the expression of the cerebellum PKJ marker protein Calbinin. The immunoblot and gray scale analysis of Calbinin and internal reference beta-actin are shown.
FIG. 6 is a graph of VPA treatment versus Snx14f/f(ii) a Influence of the number of cerebellar microglia in Nestin-cre mice. Representative immunofluorescent staining and PKJ quantitative statistical plots for the microglia marker protein IBA1 are shown.
Detailed Description
The inventor constructs a conditional knockout mouse (Snx14) in the brain of Sorting Nexin 14(Snx14)f/f(ii) a Nestin-cre) mimics an Autosomal recessive spinocerebellar ataxia 20(SCAR20) disease, followed by VPA vs. Snx14f/f(ii) a Nestin-cre for treatment. The results show that VPA administration significantly improved motor ataxia and slowed purkinje cell death in model mice. Thus, the present invention provides clinical support for the treatment of hereditary cerebellar ataxia, in particular SCAR 20. The present invention has been completed based on this finding.
Definition of terms
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. For the purpose of understanding the present invention, the following definitions will be made for terms related to the present invention, but the scope of the present invention is not limited to these specific definitions.
Herein, "alkyl" refers to a straight or branched chain saturated group consisting of carbon atoms and hydrogen atoms. For example, "C1-C6Alkyl "refers to a saturated branched or straight chain alkyl group with a carbon chain length of 1 to 6 carbon atoms,alkyl groups of 1 to 3 carbon atoms are preferred. Examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, heptyl, pentyl, and the like.
Herein, "alkoxy" refers to an oxy group substituted with an alkyl group. In a particular embodiment, alkoxy as used herein is an alkoxy group of 1 to 6 carbon atoms in length, more preferably 1 to 4 carbon atoms in length. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, and the like. In further embodiments, the alkoxy group may be a substituted alkoxy group, for example, a halogen substituted alkoxy group. In particular embodiments, halogen substituted C is preferred1-3An alkoxy group.
"cycloalkyl" as used herein refers to saturated cyclic alkyl groups, such as saturated cyclic alkyl groups having a carbon chain length of 3 to 6 carbon atoms, including but not limited to those containing cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
Herein, "halogen" refers to fluorine, chlorine, bromine and iodine. In a preferred embodiment, the halogen is chlorine or fluorine.
As used herein, "halo" refers to fluoro, chloro, bromo, and iodo.
As used herein, "substituted or unsubstituted" or "optionally substituted" means that the substituent modified by the term may be optionally substituted with 1 to 5 (e.g., 1, 2, 3, 4, or 5) substituents selected from the group consisting of: halogen, C1-4Aldehyde group, C1-6Straight or branched chain alkyl, halogen substituted C1-6Straight or branched chain alkyl (e.g. trifluoromethyl), C1-6Alkoxy, halogen substituted C1-6Alkoxy (e.g. trifluoromethoxy), cyano, nitro, amino, hydroxy, hydroxymethyl, carboxy, ethoxyformyl, -N (CH)3) And C1-4An acyl group.
Compounds of the invention
The invention provides a compound capable of effectively treating hereditary cerebellar ataxia, in particular to autosomal recessive hereditary spinocerebellar ataxia 20.
As used herein, the "compound of the present invention", "compound of formula I" or "compound of formula I" have the same meaning. The compounds herein have the structure shown in formula I:
in the formula (I), the compound is shown in the specification,
R、R1、R2、R3and R4As described above.
Based on the teachings of the present invention, those skilled in the art know that the specific choice of R corresponds to the acid, salt, ester forms, respectively, of the compound of formula I. Thus, in formula I, R may be selected from H, a metal ion, or a substituted or unsubstituted C1-6An alkyl group. In particular embodiments, R may be a metal ion, including but not limited to: sodium ions, magnesium ions, potassium ions. In a preferred embodiment, the metal ion is a sodium ion or a magnesium ion.
Based on the teachings of the present invention and the general knowledge in the art, one skilled in the art will appreciate that various groups in the compounds of the present invention can be further substituted to provide derivatives that have the same or similar activity as the specifically disclosed compounds of the present invention. Each group in the compounds of the present invention may be substituted with various substituents which are conventional in the art, as long as such substitution does not violate the rules of chemical synthesis or the rules of valency.
The term "substituted" as used herein means that one or more hydrogen atoms on a particular group are replaced with a particular substituent. The specific substituents may be those described above in correspondence with the description, or may be specific substituents appearing in each example or substituents conventional in the art. Therefore, in the present invention, the substituents in the general formula may also each independently be the corresponding group in the specific compounds in the examples; that is, the present invention includes both combinations of the respective substituents in the above general formulae and combinations of partial substituents shown in the general formulae with other specific substituents appearing in the examples. Preparing compounds having such combinations of substituents and testing the resulting compounds for activity is readily accomplished by those skilled in the art based on routine skill in the art.
Unless otherwise specified, the structural formulae depicted herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers and geometric isomers (or conformers): e.g., the R, S configuration containing an asymmetric center, (Z), (E) isomers of double bonds, etc. accordingly, a single stereochemical isomer of a compound of the present invention or a mixture of enantiomers, diastereomers or geometric isomers (or conformers) thereof is within the scope of the present invention.
As used herein, the term "tautomer" means that structural isomers having different energies may exceed the low energy barrier and thus be converted to each other. For example, proton tautomers (i.e., proton transmutations) include interconversion by proton shift, such as 1H-indazoles and 2H-indazoles. Valence tautomers include interconversion by recombination of some of the bonding electrons.
As used herein, the term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specified ratio.
As used herein, the term "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.
In particular embodiments, the compounds of the present invention include sodium valproate, magnesium valproate, and other valproate salts. In a most preferred embodiment, the compounds of the invention are as follows:
the compound is Sodium Valproate (VPA), CAS number is 1069-66-5, molecular weight is 166.193, and molecular formula is C8H15NaO2. Sodium valproate is a first-line drug clinically used for treating epilepsy and manic-depressive illness, is rapidly and completely absorbed by oral administration, can rapidly permeate blood brain barrier, and has good safety. In addition, a series of researches prove that VPA can also increase the expression of neurotrophic factors and growth related proteins, promote the growth of neuron axons and dendrites, play a role in neuroprotection and can be used for treating Alzheimer's diseaseThe medicament has good treatment effect on neurodegenerative diseases such as the sea-organ disease, the Parkinson disease, the amyotrophic lateral sclerosis and the like. However, no study of the application of VPA to scarr 20, a rare cerebellar degenerative disease, is currently available.
Pharmaceutical compositions and methods of administration
Because the compound can effectively treat hereditary cerebellar ataxia, in particular autosomal recessive hereditary spinocerebellar ataxia 20, the compound, various crystal forms, hydrates or solvates thereof and the pharmaceutical composition containing the compound as a main active ingredient can be used for effectively treating hereditary cerebellar ataxia, in particular autosomal recessive hereditary spinocerebellar ataxia 20.
The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention in combination with a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 10-250mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.
"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), and the like
) Wetting agents (such as sodium lauryl sulfate), colorants,Flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.
The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous). Therefore, the dosage form of the medicine can be any clinically acceptable oral administration dosage form or injection administration dosage form.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.
In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.
The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
When administered in combination, the pharmaceutical composition further comprises one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds. One or more of the other pharmaceutically acceptable compounds may be administered simultaneously, separately or sequentially with a compound of the invention.
When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, the daily dosage of the compound is 15mg per body weight/(kg body weight) or 600-1200 mg per day, and the maximum daily dosage is not more than 30mg per body weight/(kg body weight) or 1.8-2.4 g per day. The common dosage of the children is the same as that of adults according to the weight, and can be 20-30 mg per day/(kg body weight) for 2-3 times or 15mg per day/(kg body weight) and is increased by 5-10 mg per kg body weight every other week as required until the children are effective or can not tolerate the diseases. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The main advantages of the invention are:
1. the invention firstly constructs a specific knockout mouse of Snx14 in brain (Snx14)f/f(ii) a Nestin-cre) which can well reproduce the phenotype of clinical hereditary cerebellar ataxia disease, especially SCAR20, and is expressed by severe ataxia and a large amount of cerebellar Purkinje cells deletion;
2. the invention evaluates the continuous VPA administration pair Snx14 by means of classical ethological paradigm, immunostaining and the likef/f(ii) a The influence of the mobility coordination ability and purkinje cell survival of Nestin-cre mice showed that VPA could well improve the motor ataxia of the mice and slow down the death of purkinje cells.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
Example 1 construction and characterization of conditional knockout mice in Snx14 brain
The animal used in the experiment is a knockout mouse of Snx14 brain condition constructed by using a LoxP-Cre system, namely Snx14f/f(ii) a Nestin-cre mice. Involved transgene Snx14flox/+(Snx14f/+) Mice were constructed by the company B6.Cg-Tg (Nes-cre)1Kln/J (Nestin-cre) and purchased from Jackson lab, USA. Snx14 is firstlyf/+Mating with Nestin-cre to obtain Snx14f/+(ii) a Nestin-cre mice, which were in turn reacted with Snx14f/fObtaining a conditional knockout mouse Snx14 in the brain by matingf/f(ii) a Nestin-cre (FIG. 1A), in addition toObtaining Snx14f/+、Snx14f/fAnd Snx14f/+(ii) a Nestin-cre mice. By genotyping the progeny mice, four genotyped mice could be identified (fig. 1B). Verified by immunoblotting, Snx14f/f(ii) a The Nestin-cre mice did not express SNX14 in the brain region of the central nervous system (FIG. 1C) and expressed peripheral organs (FIG. 1D), indicating that the Snx14 gene was specifically knocked out only in the central nervous system.
Example 2 Experimental protocol for VPA administration
The drug VPA was purchased from sigma-Aldrich (P4543) and prepared at 20mg/kg with physiological saline (NS) for use. The experiments were divided into three groups: snx14f/+(ii) a Nestin-cre + NS group, Snx14f/f(ii) a Nestin-cre + NS group and Snx14f/f(ii) a Nestin-cre + VPA group. Mice in the administration group received an i.p. injection of VPA (250mg/kg body weight/day) starting at day P25 for 35 days, while mice in the control group received the same volume of NS (fig. 2A). The behavioral tests were started 3 days after the end of the administration (P63), and after the tests were completed, the mice were sacrificed and the brains were removed for subsequent experiments.
Example 3 VPA treatment had no significant effect on mouse body weight (body weight).
Referring to fig. 2B and 2C, during treatment (P25, P40, P50 and P60), Snx14f/f(ii) a Body weight and Snx14 of Male mice in the Nestin-cre + VPA groupf/f(ii) a There was no significant difference in the Nestin-cre + NS group comparisons, as was the case in the female. Suggesting that long-term VPA treatment had no significant adverse effects on the physical condition of male and female mice. Data are expressed as Mean ± standard error (Mean ± SEM) and statistically analyzed using Two-way ANOVA, with 8-11 mice per group. NS represents no significant difference.
Example 4 VPA treatment can reverse Snx14f/f(ii) a Atomotic ataxia in Nestin-cre mice
We used a classical behavioral paradigm-balance beam (balance beam) to examine the motor balance ability of mice. In brief, both ends of a log stick having a diameter of 10cm and a length of 1m were supported by an iron stand, the starting end was 40cm from the ground, the final end was 50cm from the ground, and a safety box (safe box) was fixed to the final end to attract a mouse. A training stage: placing the test mouseAt the beginning of the stick, it was guided to move towards the end and finally to the safety box, and each mouse was trained 2 times with 1min interval. And (3) a testing stage: and marking a mark at the starting end and the terminal end respectively as a test starting point and a test terminal point, wherein the distance between the two points is 60 cm. Mice were placed at the start, allowed to move autonomously to the end, and the Time taken for the mice to travel from the start to the end (Time to cross the beam) and the Number of hind limbs slipped during this period (Number of slips) were recorded. The test result shows that Snx14f/f(ii) a The time for male mice in the Nestin-cre + NS group to pass through the balance beam and the number of hind limb slides are remarkably higher than those of Snx14f/+(ii) a Nestin-cre + NS group male mice (P)<0.001 and P<0.001) (FIG. 3A), and VPA treatment significantly reversed these defects (P)<0.001 and P<0.001) (fig. 3A). Snx14f/f(ii) a Nestin-cre + NS females also had similar motor coordination impairment, manifested by a significantly higher time to passage through the balance and hind limb shedding than Snx14f/+(ii) a Nestin-cre + NS group female mice (P)<0.001 and P<0.001) (FIG. 3B), whereas VPA administration significantly reduced Snx14f/f(ii) a The hind limb number of Nestin-cre females slipped (P0.03), and had no significant effect on their passage through the balance bar (P0.35). Data are expressed as Mean ± standard error (Mean ± SEM) and statistically analyzed using One-way ANOVA, with 15-20 male mice per group and 14-15 female mice per group. Represents P<0.001 represents P<0.05, NS represents no significant difference.
Example 5 partial reversal of Snx14 by VPA treatmentf/f(ii) a Loss of Nestin-cre mouse cerebellar purkinje cells
After the behavioral testing was completed, mice were anesthetized with isoflurane, perfused with pre-cooled PBS and 4% Paraformaldehyde (PFA), brain tissue was rapidly removed, fixed with 4% PFA, dehydrated with 25% and 30% sucrose in sequence, and then OCT-embedded and cryosectioned. To observe changes in cerebellar Purkinje cells (Purkinje cells, PKJ), we immunostained brain sections with an anti-calbinin antibody (CST, 13176S) to label PKJ cells. The results show that at 2 months of age, Snx14f /f(ii) a A massive deletion of Nestin-cre + NS mouse cerebellum PKJ cells (FIGS. 4A and 4B, P)<0.001) and VPA treatment may be performed to some extentPreventing deletion of PKJ cells (FIGS. 4A and 4B, P)<0.01), most cells survived. Immunoblotting also confirmed this: snx14f/f(ii) a The protein level of Nestin-cre + NS mouse cerebellum is higher than that of Snx14f/+(ii) a Nestin-cre + NS significantly decreased (FIGS. 5A and 5B, P)<0.01), whereas VPA treatment significantly increased the level of calbinin (FIGS. 5A and 5B, P)<0.05). The immunoblot grey analysis was performed using Image J software, data were expressed as Mean. + -. standard error (Mean. + -. SEM), statistical analysis was performed using One-way ANOVA, 4-5 mice per group were immunohistochemical, and 5 mice per group were immunoblot experiments. Represents P<0.001, represents P<0.01, represents P<0.05。
Example 6 VPA treatment to reduce Snx14f/f(ii) a Activation of microglia in the cerebellum of Nestin-cre mice
Microglia are an important immune cell in the central nervous system, secrete inflammatory factors, mediate central inflammatory response, and have the functions of proliferation, migration and phagocytosis. Microglia are considered to play an important role in neurodegenerative diseases (alzheimer disease, parkinson disease, and the like). We used anti-IBA-1 antibody (Wako, 019-19-and 19741) to immunostain brain sections to label microglia, and the results showed: compare Snx14f/+(ii) a Nestin-cre + NS mice, microglia cells in Snx14f/f(ii) a Nestin-cre + NS mice had significantly larger soma and significantly increased numbers in the cerebellum (FIGS. 6A and 6B, P)<0.001); VPA treatment can attenuate Snx14f/f(ii) a Activation of microglia in the cerebellum of Nestin-cre mice was manifested by a decrease in soma and a significant decrease in cell number (FIGS. 6A and 6B, P)<0.01). Data are expressed as Mean ± standard error (Mean ± SEM), statistical analysis using One-way ANOVA, 3 mice per group used for immunofluorescence, and P<0.001, represents P<0.01, NS represents no significant difference.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (8)
1. The application of the compound shown in the formula I in the preparation of the medicine for preventing, treating or improving autosomal recessive spinocerebellar ataxia 20,
in the formula (I), the compound is shown in the specification,
r is selected from: H. a metal ion;
R1selected from: c1-6An alkyl group;
R2、R3and R4Each independently selected from: H. c1-6An alkyl group.
2. The use according to claim 1, wherein R is2、R3And R4Is H.
3. The use according to claim 1, wherein R is1Is C1-3An alkyl group.
4. Use according to claim 3, wherein R is1Is propyl.
5. The use of claim 1, wherein R is a metal ion.
6. Use according to claim 5, wherein the metal ions are selected from: sodium ions, magnesium ions, potassium ions.
7. The use according to claim 6, wherein the metal ion is selected from the group consisting of: sodium ions, magnesium ions.
8. The use according to claim 1, wherein the compound of formula I is as follows:
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| CA2524551A1 (en) * | 2005-10-14 | 2007-04-14 | Alltech, Inc. | Use of selenium yeasts in the treatment of alzheimer's disease |
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