Use of n-phenylpiperazine derivatives for the preparation of a medicament for the prevention and / or treatment of cognitive dysfunction caused by PPT1 gene deletion

Abstract

The application belongs to the technical field of medicine, and particularly relates to the use of N-phenylpiperazine derivatives in the preparation of drugs for preventing and / or treating cognitive dysfunction caused by PPT1 gene deletion. Behavioral evaluation shows that the N-phenylpiperazine derivative can significantly improve the cognitive defects of PPT1 gene knockout mice. In the new object recognition and Y maze experiments, the recognition index and spontaneous alternation rate of the mice in the treatment group are significantly improved compared with the model group, and are restored to a level close to the wild type, which confirms that the N-phenylpiperazine derivative can effectively improve non-spatial recognition memory and spatial working memory. At the same time, the open field experiment proves that the drug has strong action specificity, and excludes the interference of motor toxicity and anxiety. In addition, the compound provided by the application has a flexible administration route, and can be used for preparing drugs for treating cognitive impairment related to neurodegenerative diseases such as neuronal ceroid lipofuscinosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of N-phenylpiperazine derivatives in the preparation of drugs for the prevention and / or treatment of cognitive impairment caused by PPT1 gene deletion. Background Technology

[0002] Infantile neuronal ceroid lipofuscin deposition disease (INCL, also known as CLN1 disease) is an autosomal recessive neurodegenerative disorder caused by mutations in the PPT1 (palmitoyl-protein thioesterase 1) gene. PPT1 is a lysosomal enzyme responsible for catalyzing the depalmitoylation of S-palmitoylated proteins. Its loss of function leads to the abnormal accumulation of ceroid lipofuscin in neurons, causing progressive central nervous system dysfunction. Clinical manifestations include cognitive and motor impairment, vision loss, seizures, and early death.

[0003] Current treatment strategies for INCL mainly include enzyme replacement therapy, gene therapy, and small molecule drugs. Enzyme replacement therapy has immunogenicity issues, and the PPT1 enzyme has difficulty effectively crossing the blood-brain barrier; the long-term safety and immunogenicity of gene therapy need to be addressed; existing small molecule drugs (such as antioxidants and autophagy modulators) have not shown significant effects in improving cognitive impairment and lack targeted mechanisms against neuroinflammation. Summary of the Invention

[0004] Dopamine receptors contain palmitoylation modification sites, and abnormal palmitoylation function can lead to abnormal dopamine function. Loss of PPT1 function can affect the normal palmitoylation / depalmitoylation cycle of dopamine receptors, thereby causing abnormal dopamine signaling, which is closely related to the pathological process of neurological diseases.

[0005] Dopamine D3 receptors (D3Rs) participate in neural network regulation and are distributed in key brain regions involved in motivation and cognition, such as the nucleus accumbens, olfactory tubercle, hippocampus, and cortex. Dopamine itself has a 30-fold affinity for D3Rs compared to D2 receptors. Studies have shown that D3R ligands hold promise for reducing adverse reactions while maintaining therapeutic efficacy, demonstrating significant therapeutic potential in treating central nervous system disorders such as schizophrenia, Parkinson's disease, and drug addiction.

[0006] The inventors previously designed and synthesized a novel N-phenylpiperazine derivative, HS-81, based on the core structure of the dopamine D3 receptor. In vitro binding experiments showed that it exhibits high affinity and subtype selectivity for D2 / D3 receptors. Therefore, this invention attempts to use this N-phenylpiperazine derivative, HS-81, for the treatment of INCL.

[0007] PPT1 knockout (PPT1KO) mice are a commonly used animal model for studying intracranial leukemia (INCL), exhibiting pathological and clinical characteristics similar to human INCL. Previous studies have shown that PPT1KO mice exhibit cognitive impairment, astrocyte activation, and increased inflammatory mediators. Behavioral tests have revealed reduced spontaneous alternation rates in the Y-maze, prolonged latency for exploring novel arms, and decreased new object recognition index in PPT1KO mice, indicating spatial learning and memory impairment and recognition memory deficits.

[0008] This invention selects PPT1KO mice to verify the therapeutic effect of N-phenylpiperazine derivative HS-81 on cognitive impairment caused by PPT1 gene deletion.

[0009] In view of this, the present invention provides the use of N-phenylpiperazine derivative HS-81 in the treatment of INCL.

[0010] In one aspect, the present invention provides the use of N-phenylpiperazine derivatives or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention and / or treatment of cognitive impairment caused by PPT1 gene deletion, the chemical structural formula of said N-phenylpiperazine derivatives being shown below: .

[0011] In some embodiments, the cognitive impairment caused by PPT1 gene deletion includes infantile neuronal ceroid lipofuscin deposition disease (INCL).

[0012] In some embodiments, the pharmaceutically acceptable salt includes, but is not limited to: hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, or trifluoroacetate.

[0013] In some embodiments, the drug is used to improve cognitive impairment.

[0014] In some implementations, the cognitive impairment includes a decline in spatial working memory and / or a decline in recognition memory.

[0015] In some embodiments, the drug is used to improve one or more of the following pathological manifestations: (1) Reduce or inhibit inflammation of the central nervous system; (2) Reduce the abnormal accumulation of lipofuscin in neurons; (3) Improve synaptic plasticity; (4) Protect neurons from damage caused by lipid metabolism disorders.

[0016] In some embodiments, the N-phenylpiperazine derivative or a pharmaceutically acceptable salt thereof can cross the blood-brain barrier.

[0017] In some embodiments, the drug also includes pharmaceutically acceptable excipients or excipients.

[0018] As an optional implementation, the excipient or excipient is selected from one or more of the following: solvent, diluent, disintegrant, precipitation inhibitor, surfactant, flow aid, adhesive, lubricant, dispersant, suspending agent, isotonic agent, thickener, emulsifier, preservative, stabilizer, hydrating agent, emulsification accelerator, buffer, absorbent, colorant, flavoring agent, sweetener, ion exchanger, release agent, coating agent, flavoring agent, or antioxidant.

[0019] In some embodiments, the drug is configured as a dosage form capable of crossing the blood-brain barrier.

[0020] In some preferred embodiments, the dosage form of the drug is an oral preparation, an injectable preparation, a nasal preparation, a transdermal patch, or a sustained-release preparation.

[0021] As an optional implementation, the oral formulation is selected from tablets, capsules, granules, oral liquids, suspensions, or microcapsules.

[0022] As an optional implementation, the injectable formulation is selected from injectable solutions, lyophilized powder injections, or liposome injections.

[0023] In some embodiments, the drug is administered to a subject, preferably a mammal, more preferably a human being.

[0024] In some embodiments, the route of administration of the drug includes oral administration, injection administration, nasal administration, or transdermal administration.

[0025] In some embodiments, the N-phenylpiperazine derivative or a pharmaceutically acceptable salt thereof is used as the sole active ingredient, or in combination with other drugs for treating cognitive impairment caused by PPT1 gene deletion.

[0026] In some preferred embodiments, the other therapeutic agents include enzyme replacement therapy agents, gene therapy vectors, antioxidants, or autophagy regulators.

[0027] This invention provides the use of N-phenylpiperazine derivative HS-81 or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of cognitive impairment caused by PPT1 gene deletion. The invention validated the effect of N-phenylpiperazine derivative HS-81 on cognitive impairment caused by PPT1 gene deletion in PPT1KO mice. The results showed that HS-81 significantly alleviated cognitive impairment caused by PPT1 gene deletion, and significantly improved spatial working memory and recognition memory abilities.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Significant improvement in cognitive function: In the Y maze test, the spontaneous alternation rate of the HS-81 treatment group increased from 33.91% in the model group to 56.79%, which is close to the normal WT level (59.57%). The latency to enter the new arm was significantly shorter than that of the model group, and there was no significant difference in the total number of arm entries among the groups, thus excluding the interference of motor ability differences on the cognitive evaluation results. In the new object recognition test, the discrimination index was significantly improved compared with the model group.

[0029] (2) High specificity of action: The open field test showed no significant difference in the total movement distance and central area activity time among the mice in each group, which ruled out the possibility that HS-81 indirectly improves cognition by affecting spontaneous activity or anxiety level. This indicates that HS-81 did not show obvious motor toxicity or anxiety-like behavior and other mental adverse reactions at the therapeutic dose, suggesting that the compound has good tolerability and safety characteristics.

[0030] (3) Drug advantages: HS-81 is a small molecule compound with mature preparation process, flexible administration route (can be injected intraperitoneally) and low production cost.

[0031] (4) It fills a technological gap: For the first time, N-phenylpiperazine derivatives are applied to a neurodegenerative disease model caused by PPT1 deficiency, expanding the application scope of this class of drugs. Attached Figure Description

[0032] Figure 1 The effect of HS-81 treatment on spatial memory in PPT1KO mice is shown. A through D are representative movement trajectories of mice in the Y maze for the WT normal control group (A), PPT1KO model group (B), solvent control group (PPT1+SO) (C), and HS-81 treatment group (PPT1+CC) (D), respectively. E is a statistical graph of the spontaneous alteration rate for each group; F is a statistical graph of the latency to enter the novel arms for each group; and G is a statistical graph of the total number of arm entries for each group.

[0033] Figure 2 The effect of HS-81 treatment on recognition memory in PPT1KO mice is shown. A is a schematic diagram of the Novel Object Recognition test; B is a statistical graph of the Discrimination Index (DI) for each group of mice.

[0034] Figure 3 The effects of HS-81 treatment on PPT1KO mice are shown. A through D represent the representative movement trajectories of mice in an open field for the WT normal control group (A), PPT1KO model group (B), solvent control group (PPT1+SO) (C), and HS-81 treatment group (PPT1+CC) (D), respectively. E shows the total distance of movement for each group; F shows the time spent in the center zone for each group; and G shows the number of center zone entries for each group. Detailed Implementation

[0035] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0036] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0037] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0038] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0039] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0040] As used herein, the term "comprising" or "including" means "including, but not limited to." This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "substantially consisting of." In one embodiment, the term "comprising" as used throughout the application, particularly in the claims, may be replaced by the term "consisting of."

[0041] As used herein, the terms “optional,” “any,” “arbitrary,” or “any one” mean that the event or situation subsequently described may, but does not have to, occur, including the circumstances in which the event or situation occurs or does not occur. As used herein, “an” and “a” refer to one or more grammatical objects.

[0042] The term “and / or” as used herein should be understood to mean any one of the options or any combination of two or more of the options.

[0043] Experimental materials and methods: The method for synthesizing the N-phenylpiperazine derivative HS-81 of the present invention is described in the inventor's prior application CN201810040879.0.

[0044] Methods for constructing the PPT1KO mouse model: PPT1 knockout (PPT1KO) mice are constructed using gene targeting technology: a targeted vector is designed to disrupt the protein-coding sequence of the mouse PPT1 gene; chimeric mice are obtained through homologous recombination in embryonic stem cells and injection into blastocysts; and heterozygous mice are obtained by backcrossing with C57BL / 6 mice. + / - Heterozygous crossbreeding yields homozygous knockout (PPT1) - / - The mice used were PPT1KO mice and littermate wild-type (WT) controls. Mouse genotypes were confirmed by PCR identification of tail genomic DNA. Homozygous knockout mice exhibited a lack of PPT1 enzyme activity in their brains, displaying pathological and behavioral characteristics similar to human INCL, including ceramide lipofuscin accumulation, astrocyte activation, and progressive cognitive impairment. The mice were housed in SPF-grade animal facilities with free access to food and water, and underwent 12-hour / 12-hour light / dark cycles. All animal experimental procedures were reviewed and approved by the laboratory animal ethics committee.

[0045] Example 1: Effects of N-phenylpiperazine derivative HS-81 on spatial memory in PPT1KO mice Male PPT1KO mice aged 5-6 months were randomly divided into a model group, a solvent control group, and an HS-81 treatment group (HS-81 was dissolved in sesame oil, 1 mg / kg, intraperitoneally injected daily for 5 consecutive days; the solvent control group was injected intraperitoneally with an equal volume of sesame oil). Age-matched WT mice were used as normal controls. The Y-maze test was performed after the drug administration was completed.

[0046] Experimental method of the Y-maze experiment: The Y-maze experiment uses a Y-maze apparatus consisting of three arms of equal length, with the angle between the three arms being 120°. The experiment is conducted in a quiet, softly lit behavioral laboratory, with a camera above the maze connected to a behavioral analysis system to record the movement trajectory of the mice. The experiment consisted of two parts: a spontaneous alternation experiment and a novel arm exploration experiment. (1) Spontaneous alternation experiment: The mice were placed in the central area of ​​the maze and allowed to explore the three arms freely for 8 minutes. The order in which each mouse entered the arm and the total number of times it entered the arm were recorded. Entering three different arms consecutively (e.g., A→B→C or A→C→B) was recorded as one spontaneous alternation. The spontaneous alternation rate (%) = number of spontaneous alternations / (total number of arm entries - 2) × 100%. (2) Novel arm exploration experiment: The experiment was divided into a learning period and a testing period. During the learning period, one arm (novel arm) was randomly closed. The mice were placed into the maze to explore freely from the starting arm. After a 1-hour interval, the testing period was conducted. All three arms were opened. The mice were placed into the maze to explore freely from the original starting arm for 5 minutes. The latency to enter the novel arm and the total number of arm entries were recorded. After each mouse was tested, the maze was wiped with 75% alcohol to eliminate odor interference.

[0047] The results are as follows Figure 1 As shown, in the Y-maze experiment, the spontaneous alternation rate in the HS-81 treatment group was (56.79±5.492)%, which was significantly higher than that in the PPT1KO model group (33.91±8.379)%. P <0.05, close to the WT group level (59.57±1.381)%.

[0048] The Y-maze spontaneous alternation experiment is a classic behavioral paradigm for assessing spatial working memory in rodents.

[0049] Normal rodents possess an innate exploratory instinct, tending to enter three different arms consecutively in a Y-maze (e.g., A→B→C or A→C→B), a phenomenon known as "spontaneous alternation." To complete the correct alternation, the animal must remember which arm it just entered and actively choose a new arm. This process is highly dependent on spatial working memory. In the Y-maze experiment of this example, the spontaneous alternation rate of WT mice was at the normal baseline level, indicating intact working memory and a strong tendency for alternating exploration. The spontaneous alternation rate of the model group mice was only 33.91%, close to the level of random selection, indicating severe working memory impairment. The spontaneous alternation rate of the treatment group mice was as high as 59.57%, indicating significant recovery of working memory, approaching normal levels (no significant difference from the WT group).

[0050] In addition to the spontaneous alternation rate, this embodiment further analyzed two indicators: the latency to enter the novel arm and the total number of arm entries. The latency to enter the novel arm reflects the animal's ability to recognize and remember stimuli in a novel space: the latency to enter the novel arm was significantly longer in the model group mice than in the WT group ( P <0.05, indicating impaired spatial memory; the latency in the HS-81 treatment group was significantly shorter than that in the model group ( P <0.01, recovering to levels close to the WT group; while there was no significant difference between the solvent control group and the model group ( P >0.05), indicating that the improvement in latency was indeed due to the pharmacological effects of HS-81.

[0051] The total number of arm infeeds reflects the animal's level of spontaneous activity and willingness to explore: there was no significant difference in the total number of arm infeeds among the groups of mice. P The result >0.05 indicates that neither PPT1 gene knockout nor HS-81 administration affected the basic motor ability of mice. This result excludes the interference of motor ability differences on the two cognitive indicators of spontaneous alternation rate and novel arm latency, confirming that the above inter-group differences specifically reflect changes in spatial working memory, that is, the effect of HS-81 on improving spatial cognitive function in PPT1KO mice is real and specific.

[0052] Example 2: Effects of N-phenylpiperazine derivative HS-81 on recognition and memory in PPT1KO mice Male PPT1KO mice aged 5-6 months were randomly divided into a model group, a solvent control group, and an HS-81 treatment group (HS-81 was dissolved in sesame oil, 1 mg / kg, intraperitoneally injected daily for 5 consecutive days; the solvent control group was injected intraperitoneally with an equal volume of sesame oil). Age-matched WT mice were used as normal controls. After the drug administration was completed, a new object recognition experiment was performed.

[0053] Experimental methodology for the Novel Object Recognition test: The new object recognition experiment was conducted in an open field chamber and consisted of three phases: adaptation, learning, and testing. Adaptation: Before the formal experiment, mice were placed in an empty chamber and allowed to explore freely for 10 minutes to acclimatize to the environment. Learning: Two identical objects were placed in fixed positions within the chamber. Mice were placed with their backs to the objects and allowed to explore freely for 5 minutes, recording their exploration of the two identical objects. Testing: One hour later, one of the old objects was replaced with a new object of different shape and color. The mice were again placed in the chamber and allowed to explore freely for 5 minutes. The video analysis system recorded the exploration time for the new and old objects (exploration was defined as the mouse's nose facing the object and the distance between the mouse and the object not exceeding 2 cm). Discrimination Index (DI) = (New object exploration time - Old object exploration time) / (New object exploration time + Old object exploration time). After each mouse's test, the chamber and objects were wiped with 75% alcohol to eliminate odor interference.

[0054] The results are as follows Figure 2 As shown, in the new object recognition experiment, the discrimination index of the model group mice was close to zero, significantly lower than that of the WT group ( P <0.05 indicates that PPT1 gene deletion causes mice to be unable to effectively distinguish between new and old objects, resulting in severe impairment of recognition memory; the discrimination index of mice in the HS-81 treatment group was significantly improved compared with that in the model group ( P <0.05, recovering to levels close to the WT group, while there was no significant difference between the solvent control group and the model group ( P >0.05). The above results indicate that HS-81 can significantly improve non-spatial recognition memory impairment caused by PPT1 gene deletion, and this improvement is due to the pharmacological activity of the compound itself, rather than the effects of solvent or administration.

[0055] In the Novel Object Recognition (NOR) experiment, the Discrimination Index (DI) is a core direct indicator for measuring recognition memory in animals. Rodents naturally exhibit a "novelty preference." After being exposed to two identical objects during the training period, mice in the control group spent more time exploring the new object during the testing period because they remembered the old object. However, mice in the model group could not remember the old object, and there was no significant difference in exploration time between the old and new objects, resulting in an extremely low recognition index, indicating memory deficit. The treatment group showed a significant increase in the recognition index, indicating that the mice, after drug administration, regained a strong preference for novel objects. This directly demonstrates that the drug effectively rescued or improved the non-spatial recognition memory ability of mice.

[0056] Example 3: Evaluation of the specificity of N-phenylpiperazine derivative HS-81 Male PPT1KO mice aged 5-6 months were randomly divided into a model group, a solvent control group, and an HS-81 treatment group (HS-81 was dissolved in sesame oil, 1 mg / kg, intraperitoneally injected daily for 5 consecutive days; the solvent control group was injected intraperitoneally with an equal volume of sesame oil). Age-matched WT mice were used as normal controls. Open field tests were performed after the drug administration was completed.

[0057] Experimental method of open field experiment: The open field experiment was conducted in a square open field chamber. Mice were gently placed in the central area of ​​the open field and allowed to explore freely for 10 minutes. A camera above the open field recorded the mice's movement trajectories. The behavioral analysis system automatically divided the open field into a central area (occupying 25% of the total area) and a peripheral area, and quantitatively analyzed each mouse's total distance, time spent in the central zone, and number of entries into the central zone. After each mouse's test, the open field chamber was wiped with 75% alcohol to eliminate odor interference.

[0058] The results are as follows Figure 3 As shown, there were no significant differences among the groups in the open field test, and no significant differences were found in the total movement distance, central area activity time, and number of times mice entered the central area among the groups. This ruled out the possibility that HS-81 indirectly improves cognition by affecting spontaneous activity or anxiety levels, and also indicated that HS-81 did not exhibit significant motor toxicity or anxiety-like behaviors or other psychiatric adverse reactions at therapeutic doses, suggesting that this compound has good tolerability and safety characteristics.

[0059] Specifically, there was no significant difference in total distance, demonstrating that the drug did not alter the mice's basic motor abilities and activity levels, while most cognitive function tests (such as the Y maze and new object recognition) are highly dependent on the animals' motor abilities.

[0060] Rodents are naturally thigmotaxis-like, exhibiting an instinctive fear of open, bright central areas. Therefore, time spent in the center zone is a classic indicator for assessing anxiety-like behavior in animals. The longer the animal spends in the center zone, the lower its anxiety level (an anti-anxiety effect). The lack of significant difference in center zone activity time demonstrates that the drug did not alter the mice's baseline mood or anxiety state.

[0061] The number of times a rodent enters the center zone is an important indicator for assessing anxiety-like behavior and exploration intentions.

[0062] In summary, a comprehensive analysis combining the time spent in the central area and the total distance traveled can eliminate the interference of drugs on basic motor abilities and specifically reflect the effects of drugs on the animal's emotional state and exploration motivation.

[0063] It should be noted that, since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of the present invention have been described, those skilled in the art, once they understand the inventive concept of the present invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of the present invention.

[0064] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. The use of N-phenylpiperazine derivatives or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention and / or treatment of cognitive impairment caused by PPT1 gene deletion, characterized in that, The chemical structural formula of the N-phenylpiperazine derivative is shown below: 。 2. The use according to claim 1, characterized in that, The cognitive impairment caused by PPT1 gene deletion includes infantile neuronal ceroid lipofuscin deposition disease.

3. The use according to claim 2, characterized in that, The drug is used to improve cognitive impairment.

4. The use according to any one of claims 1 to 3, characterized in that, The cognitive impairments include a decline in spatial working memory and / or a decline in recognition memory.

5. The use according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients or excipients.

6. The use according to claim 5, characterized in that, The excipients or excipients are selected from one or more of the following: solvents, diluents, disintegrants, precipitation inhibitors, surfactants, flow aids, adhesives, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavorings, sweeteners, ion exchangers, release agents, coating agents, flavoring agents, or antioxidants.

7. The use according to claim 5 or 6, characterized in that, The dosage form of the drug is an oral preparation, an injectable preparation, a nasal preparation, a transdermal patch, or a sustained-release preparation.

8. The use according to claim 7, characterized in that, The oral preparation is selected from tablets, capsules, granules, oral liquids, suspensions, or microcapsules.

9. The use according to claim 8, characterized in that, The injectable formulation is selected from injectable solutions, lyophilized powder injections, or liposome injections.

10. The use according to claim 9, characterized in that, The drug can be administered orally, by injection, by nasal administration, or by transdermal administration.

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