Medicine for relieving conditional position preference behaviors of morphine-dependent mice and application

Abstract

The invention discloses a medicine for relieving conditional position preference behaviors of morphine-dependent mice and application of the medicine. The medicine comprises polypeptide and siRNA, and the amino acid sequence of the polypeptide is SCSRRLLAAALLPWCCAAW; the siRNA is a double strand, the sequence of a sense strand of the siRNA is UGUCAAAGUCAACAUGGUGdTdT, and the sequence of an antisense strand of the siRNA is CACCAUGUUGACUGdTdT. According to the application disclosed by the invention, by discovering the specific association between the Ppt1 gene in the anterior clasp cortex and morphine dependence and developing a Ppt1-targeted polypeptide (Ppt1-4-22) and siRNA intervention tool, not only is key theoretical inspiration provided for clinical research on an opioid drug-dependent molecular mechanism, but also an accurate theoretical basis and an effective molecular target can be provided for drug research and development.

Description

Technical Field

[0001] This invention relates to a drug and its application for alleviating conditional positional preference behavior in morphine-dependent mice, belonging to the field of pharmaceutical technology. Background Technology

[0002] Drug dependence is characterized by persistent pathological changes in specific brain regions induced by addictive drugs, leading to compulsive drug cravings and consumption behaviors. Its core feature is uncontrolled drug use regardless of negative consequences, and it has become a major challenge in global public health. Research on its pathogenesis and intervention strategies continues to advance among scholars worldwide. In my country, opioids are the primary type of abuse. While morphine, as the core alkaloid of opioids, has significant analgesic effects on both acute and chronic pain, its clinical application is limited by tolerance developed after short-term repeated use. Gradual increases in dosage are required to maintain the original analgesic effect, and it easily induces drug dependence, severely restricting the full realization of its clinical value.

[0003] The anterior cingulate cortex (ACC) is a key brain region in the central nervous system regulating reward mechanisms (Baker, et al. 2017; Elman, et al. 2013), pain perception (Elman, et al. 2013; Wang, et al. 2024; Wang, et al. 2021; Yang and Hua 2015), emotion regulation, and cognitive function (Elman, et al. 2013; Hu, et al. 2021; Wang, et al. 2024). Abnormalities in its function are closely related to the development of drug dependence. Functional magnetic resonance imaging studies have confirmed that ACC activity is significantly reduced in drug-dependent states (Camchong et al., 2017; Lu, et al. 2023; Wang et al., 2013); while artificial activation of the ACC can effectively improve self-control in drug-dependent individuals and reduce negative emotions and excessive stress responses (Tervo et al., 2021). Longitudinal studies on cannabis abuse among adolescents have shown that resting-state functional connectivity in the ACC brain region was significantly reduced in the abuse group after 18 months, while it showed an increasing trend in the control group (Bush et al., 2000; Tolomeo and Yu, 2022). Other studies have shown that opioids can directly inhibit glutamatergic synaptic transmission from the medial thalamus to the ACC (Jaeckel, et al. 2023). These studies all suggest that reduced activity in the ACC brain region or abnormal functional connectivity with surrounding brain regions directly weakens its core role in reward regulation and cognitive executive function, ultimately leading to pathological characteristics such as uncontrolled drug cravings, exacerbated negative emotions, and excessive stress responses in drug-dependent individuals.

[0004] The above studies have clarified that ACC dysfunction is a key pathological feature of opioid dependence, but there are still core gaps in current technology: no key molecular targets have been found that can specifically regulate the ACC brain region and are directly related to opioid dependence, which makes it impossible to develop targeted intervention drugs that can precisely act on ACC and balance efficacy and safety. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention provides a drug and its application for alleviating conditional position preference behavior in morphine-dependent mice.

[0006] To achieve the above objectives, the present invention employs a polypeptide that alleviates conditional position preference behavior in morphine-dependent mice. The amino acid sequence of the polypeptide is SCSRRLLAAALLPWCCAAW, and the polypeptide is located in the linear domain region where Ppt1 protein interacts with other molecules. The polypeptide interferes with the role of Ppt1 in morphine-dependent signaling pathways by competitively binding to interacting molecules of Ppt1 protein.

[0007] In a second aspect, the present invention also provides an siRNA for alleviating conditional positional preference behavior in morphine-dependent mice. The siRNA is double-stranded, with the sense strand sequence being UGUCCAAGUCAACAUGGUGdTdT and the antisense strand sequence being CACCAUGUUGACUUGGACAdTdT. The siRNA inhibits the expression of Ppt1 in the anterior cingulate cortex of morphine-dependent mice by specifically degrading the mRNA of the Ppt1 gene.

[0008] A third aspect of the present invention also provides the use of the described polypeptide or the described siRNA in the preparation of a medicament for alleviating conditional position preference behavior in morphine-dependent mice.

[0009] As an improvement, the drug is an injectable drug, which is stereotactically injected into the anterior cingulate cortex of morphine-dependent mice. The polypeptide interferes with the role of Ppt1 in morphine-dependent signaling pathways by competitively binding to interacting molecules of the Ppt1 protein; the siRNA reduces Ppt1 protein levels by inhibiting Ppt1 mRNA expression.

[0010] As an improvement, the injection concentration of the polypeptide is 2 μg / μL, and the solvent is physiological saline containing 10% acetic acid. The injection concentration of siRNA was 10 μg / μL, and the solvent was siRNA-Mate transfection reagent.

[0011] In a fourth aspect, the present invention also provides a medicament for alleviating conditional positional preference behavior in morphine-dependent mice, the active ingredient of the medicament comprising the polypeptide or the siRNA described above; and further comprising a pharmaceutically acceptable carrier.

[0012] The mechanism of action of this invention is: In morphine-dependent states, the Ppt1 gene is abnormally highly expressed in the anterior cingulate cortex (ACC) of mice. The polypeptide (Ppt1-4-22) of this invention is located in the linear domain region where the Ppt1 protein interacts with other molecules. By competitively binding to the interacting molecules of the Ppt1 protein, it blocks the activation of the above-mentioned abnormal signaling pathway. The siRNA (siRNA-Ppt1) of this invention inhibits the abnormal expression of the Ppt1 protein in the ACC region of morphine-dependent mice by specifically degrading the mRNA of the Ppt1 gene. Both can ultimately alleviate the conditional position preference behavior of morphine-dependent mice.

[0013] Among them, the localization verification of the polypeptide (Ppt1-4-22) of the present invention on the Ppt1 protein: the bioinformatics website (https: / / www.uniprot.org / uniprotkb / O88531 / entry#ptm_processing) shows that this peptide is located in the 1-27 amino acid signaling region of Ppt1, which is easy to interact with other molecules; the bioinformatics website (https: / / mobidb.org / O88531) shows that this peptide is located in the linear domain of Ppt1, which is the region that interacts with other signaling molecules.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. Filling a target gap with outstanding innovation. Existing technologies have not discovered morphine-dependent specific molecular targets in the ACC region. This invention is the first to demonstrate that Ppt1 expression in the ACC region of morphine-dependent mice is associated with conditional position preference behavior. Furthermore, a peptide (Ppt1-4-22) and siRNA (siRNA-Ppt1) targeting Ppt1 were developed. By precisely regulating Ppt1 function, the gap in morphine-dependent targets in the ACC region is filled.

[0015] 2. Precise mechanism, safe and efficient. The two core reagents have clearly defined targets: the peptide binds only to the Ppt1 interaction domain without interfering with its normal physiological function; the siRNA specifically degrades Ppt1 mRNA, with no off-target risk.

[0016] 3. The solution is feasible and supports clinical translation. This invention clearly defines the sequence, concentration, and solvent of the core reagents, and verifies their effectiveness through qPCR, transcriptomics, and behavioral experiments; the carrier in the drug composition is adapted to stereotactic injection requirements, and the technical parameters are clear and reproducible, providing a reliable basis for subsequent preparation, verification, and preclinical translation, and overcoming the shortcomings of existing technologies that only propose hypotheses without implementation plans.

[0017] 4. Expanding the application of Ppt1 and providing new research directions. Existing Ppt1 research focuses on neurogenetic diseases and oncology. This invention is the first to discover its morphine dependence regulatory function, expanding it into a target for morphine dependence intervention. This not only provides new treatment options but also broadens the research field of Ppt1, laying the foundation for research on other opioid dependence.

[0018] 5. Empowering Clinical Treatment with Significant Social Value. There is a lack of effective clinical treatments for morphine dependence. This invention, by targeting Ppt1 to alleviate conditioned positional behavior in mice, provides core support for the development of adjuvant or specific treatments that combine morphine analgesia with reduced dependence risk. This is expected to reduce the health and social harms of morphine dependence and has significant clinical value. Attached Figure Description

[0019] Figure 1 A schematic diagram illustrating the preparation of a mouse model of acute morphine addiction using a 5-day incremental dose addiction method; Figure 2 This diagram shows the expression and signaling pathway association analysis of the Ppt1 gene in the anterior cingulate cortex (ACC) of morphine-dependent mice. In the diagram, A is the qPCR validation result of the Ppt1 gene mRNA expression level; B is the KEGG signaling pathway association diagram from transcriptome bioinformatics analysis; and C is the Reactome signaling pathway association diagram from transcriptome bioinformatics analysis. Figure 3 The diagram shows the effect of the reagent of the present invention on the conditional position preference (CPP) behavior of morphine-dependent mice; A is the result of CPP behavioral testing after intervention with peptide (Ppt1-4-22); B is the result of CPP behavioral testing after intervention with siRNA (siRNA-Ppt1). Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specified in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer. Example

[0021] 1. Reagents and detection methods used in this invention (1) Peptide synthesis: The polypeptide Ppt1-4-22 of this invention, amino acid sequence: SCSRRLLAAALLPWCCAAW (SEQ ID NO.1), was synthesized by Shanghai Botai Biotechnology Co., Ltd.; (2) Synthesis of siRNA and primers: double-stranded siRNA targeting Ppt1 (siRNA-Ppt1). Justice Chain: UGUCCAAGUCAACAUGGUGdTdT (SEQ ID NO.2); Antonym: CACCAUGUUGACUUGGACAdTdT (SEQ ID NO.3); The primers used for qPCR detection were all synthesized by Gemma Gene Technology Co., Ltd.; among them, siRNA-Ppt1 was designed to target the functional domain of the Ppt1 protein to ensure efficient interference with Ppt1 gene expression. (3) qPCR related reagents: The kit (Code No. 9109-036-820) used for total RNA extraction, reverse transcription and quantitative PCR (qPCR) was purchased from TaKaRa. It contains RNA extraction buffer, reverse transcriptase, Taq enzyme and fluorescent dye to ensure experimental repeatability and data reliability. (4) Transcriptomics detection: Transcriptome sequencing and bioinformatics analysis (including differential gene screening, KEGG and Reactome signaling pathway enrichment analysis) of the anterior cingulate cortex (ACC) of morphine-dependent mice and normal control group were completed by Shanghai Aqu Biotechnology Co., Ltd.

[0022] 2. Experimental animals: Male C57 mice, clean grade, weighing 20-25g, provided by the Experimental Animal Center of Xuzhou Medical University.

[0023] 3. Preparation of morphine-dependent mouse models like Figure 1 As shown, an acute morphine dependence model in mice was established using a 5-day incremental dose subcutaneous injection method. The specific steps are as follows: (1) Route of administration: Subcutaneous injection in the back of the neck of mice (avoiding blood vessels and skin ruptures to ensure uniform drug absorption); (2) Dosage frequency and time: once a day, at 8:00 am, for 5 consecutive days; (3) Dosage: The morphine dose was 10 mg / kg on day 1, 20 mg / kg on day 2, 30 mg / kg on day 3, 40 mg / kg on day 4, and 50 mg / kg on day 5. The control group mice were injected with the same volume of physiological saline (0.9% sodium chloride solution) at the same time point and the same site to eliminate the interference of the injection operation itself on the experimental results.

[0024] 4. Morphine-induced conditioned position preference (CPP) behavioral testing methods A complete behavioral model of conditional position preference (CPP) includes the following four consecutive phases. The experiment used a three-chamber CPP test chamber (the middle chamber connects to the two side chambers, and the two side chambers have horizontal and vertical stripes to distinguish the environment): (1) Exploration and Adaptation Period (Day 1) Mice in the morphine group received a subcutaneous injection of morphine (10 mg / kg) via the back of the neck at 8:00 AM; the control group received the same volume of saline at the corresponding time point. Adaptation training was conducted twice that day, at 9:00 AM and 3:00 PM, with each session lasting 30 minutes. During training, the striped box and the vertical striped box were connected, allowing mice to freely move between the two boxes and the intermediate box, fully familiarizing themselves with and adapting to the experimental environment (including light, temperature, humidity, and odor).

[0025] (2) Base value testing period (or conditionalization pre-test, Day 2) The morphine group received a subcutaneous injection of morphine (20 mg / kg) into the back of the neck at 8:00 AM; the control group received the same volume of saline at the corresponding time point. Mice that had completed the exploration and adaptation period were placed from the intermediate box into the experimental box, the isolation plate was removed, and the video acquisition system was activated 1 minute later to record 15 minutes (900 seconds) of free movement video of the mice (i.e., Pretest). After all animal video information was collected, the data was analyzed: mice that spent more than 630 seconds (≥70% of the total test time) in any box were defined as exhibiting obvious natural preference behavior and were excluded; the remaining mice that met the requirements were included in the next stage of the experiment, and the total activity time of each mouse in each box was recorded to determine the matching boxes with relatively shorter and longer activity times. When receiving conditioned stimuli (morphine injection), the mice were placed in the box with shorter activity times; when receiving saline injection, they were placed in the box with longer activity times.

[0026] (3) CPP behavioral conditioning period (Day 3-5, 3-day cycle) During this period, the horizontal and vertical striped boxes were kept separate. The morphine group was treated as follows: Morphine (30 mg / kg, 40 mg / kg, and 50 mg / kg doses, respectively) was subcutaneously injected into the neck and back at 8:00 AM on Day 3 (morning), Day 4 (afternoon), and Day 5 (morning); the same volume of saline was injected subcutaneously into the neck and back at the afternoon of Day 3, Day 4 (morning), and Day 5 (afternoon). After each injection, the mice were placed in the corresponding matching box (i.e., after morphine injection, they were placed in the box with a relatively shorter activity time during the baseline testing period; after saline injection, they were placed in the box with a relatively longer activity time during the baseline testing period), allowing them to move freely for 45 minutes. The control group received subcutaneous injections of saline into the neck and back at the corresponding time periods, before being placed in the matching or non-matching boxes. After each experiment, the mice were returned to their cages; after the morning and afternoon experiments, the experimental boxes were thoroughly cleaned to eliminate the influence of individual animal odor on subsequent experiments.

[0027] (4) CPP Behavioral Testing Period (Day 6-8, 3-day cycle) The morphine group received a subcutaneous injection of morphine (50 mg / kg) into the back of the neck at 8:00 AM; the control group received the same volume of saline at the corresponding time point. The key objective at this stage was to obtain the CPP score after completing the morphine-conditioned induction training to determine whether the mice exhibited CPP behavior. During the experiment, the partition was removed, connecting the horizontal and vertical striped boxes. Mice that had completed the previous experimental phase were placed directly into the experimental box from the middle box. One minute later, the video acquisition system was activated, capturing 15 minutes (900 seconds) of video footage of the mice moving freely within the experimental box. The activity time of the mice in the morphine-matched box (i.e., the box with the shorter activity time during the baseline testing period) was used as their CPP behavioral score. This score was used to determine whether the mice's CPP behavior was induced by the conditioned stimulus (morphine and a specific box).

[0028] 5. qPCR analysis The mice were decapitated, and brain tissue was rapidly dissected on ice. Following stereotactic brain localization standards, a region of the cerebral cortex measuring 2.34 mm anterior to, 0.22 mm posterior to, and 0.6 mm lateral to the anterior fontanelle was harvested; this region is the anterior cingulate cortex (ACC region) of interest in the experiment. Subsequently, following the instructions of the TaKaRa RNA extraction + RT + qPCR kit (model: 9109-036-820), total RNA extraction, reverse transcription, and PCR amplification were performed to detect the mRNA expression level of the Ppt1 gene in the ACC region.

[0029] 6. Stereoscopic injection of two drugs Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg, ip) and then fixed on a mouse-specific electronic stereotaxic instrument. Care was taken to adjust the position of the ear rods, ensuring the depth of fixation on both sides was just enough to produce a crisp click sound. Simultaneously, the height of the ear rods was adjusted to be on the same plane as the height of the two incisors. The mouse head was then treated: the fur was clipped, the scalp was disinfected with iodine, and an incision was made 0.5 cm posterior to the midline connecting the eyes. The incision was repeatedly wiped with a cotton swab soaked in 3% H2O2 to disrupt the fascia and fully expose the skull. The anterior fontanelle was located as the origin for stereotaxic positioning.

[0030] Adjust the injection needle position to 1.11 mm anterior to the anterior fontanelle and 0.25 mm lateral to it. Slowly inject 0.5 μL of the prepared reagent to a depth of 2.10 mm below the skull surface (injection duration 30 seconds). Leave the needle in place for 10 minutes after injection, then slowly withdraw the needle. The reagents used include: ①Ppt1-4-22 polypeptide: Dissolved in physiological saline containing 10% acetic acid, with a solution concentration of 2μg / μL; ②siRNA-Ppt1: Dissolved using siRNA-Mate transfection reagent at a concentration of 10 μg / μL; ③siRNA-Scramble (negative control): Dissolved using siRNA-Mate transfection reagent at a concentration of 10 μg / μL.

[0031] After the injection, the skin was sutured, disinfected with povidone-iodine, and antibiotic solution was applied to the incision to prevent infection. Throughout the entire process from anesthesia to the mice's awakening, the ambient temperature was maintained at approximately 26°C.

[0032] Figure 2 The results showed that, compared with the normal control group, the mRNA expression level of the Ppt1 gene in the anterior cingulate cortex (ACC) of morphine-dependent mice was significantly increased; and transcriptomic bioinformatics analysis showed that the high expression of this gene was significantly associated with the KEGG signaling pathway and the Reactome signaling pathway related to morphine dependence, confirming that Ppt1 is involved in the regulation of pathological molecular pathways of morphine dependence.

[0033] Figure 3 The results showed that the Ppt1-4-22 peptide and siRNA-Ppt1 of the present invention could significantly shorten the time spent in the morphine-associated box in morphine-dependent mice by targeting and regulating Ppt1 function, clearly demonstrating the negative association between the two and the conditional position preference (CPP) behavior in morphine-dependent mice, and confirming the alleviating effect of the two reagents on morphine-dependent CPP behavior.

[0034] This invention, by discovering the specific association between the Ppt1 gene in the anterior cingulate cortex (ACC) and morphine dependence, and developing a peptide (Ppt1-4-22) and siRNA intervention tool targeting Ppt1, not only provides key theoretical inspiration for the study of the molecular mechanisms of clinical opioid (such as morphine) dependence, but also provides a precise theoretical basis and effective molecular targets for drug development. It provides translatable target basis and technical direction for developing adjunctive therapies that take into account the analgesic effect of morphine and reduce the risk of dependence, as well as for developing specific intervention drugs for opioid dependence. Furthermore, those skilled in the art will understand that although some embodiments described herein include certain features found in other embodiments but not others, combinations of features from different embodiments are also within the scope of protection of this invention and form different embodiments. For example, in the embodiments described above, those skilled in the art can use them in combination based on known technical solutions and the technical problems to be solved by this application.

Claims

1. A polypeptide for alleviating conditioned position preference behavior in morphine-dependent mice, characterized in that, The amino acid sequence of the polypeptide is SCSRRLLAAALLPWCCAAW, and the polypeptide is located in the linear domain region where Ppt1 protein interacts with other molecules. The polypeptide interferes with the role of Ppt1 in morphine-dependent signaling pathways by competitively binding to interacting molecules of Ppt1 protein.

2. A siRNA for alleviating conditional position preference behavior in morphine-dependent mice, characterized in that, The siRNA is double-stranded. The sense strand sequence of the siRNA is UGUCCAAGUCAACAUGGUGdTdT, and the antisense strand sequence is CACCAUGUUGACUUGGACAdTdT. The siRNA inhibits the expression of Ppt1 in the anterior cingulate cortex of morphine-dependent mice by specifically degrading the mRNA of the Ppt1 gene.

3. The use of the polypeptide of claim 1 or the siRNA of claim 2 in the preparation of a drug for alleviating conditioned position preference behavior in morphine-dependent mice.

4. The application according to claim 3, characterized in that, The drug is an injectable drug, which is stereotactically injected into the anterior cingulate cortex of morphine-dependent mice. The polypeptide interferes with the role of Ppt1 in morphine-dependent signaling pathways by competitively binding to interacting molecules of the Ppt1 protein; the siRNA reduces Ppt1 protein levels by inhibiting Ppt1 mRNA expression.

5. The application according to claim 4, characterized in that, The injection concentration of the polypeptide is 2 μg / μL, and the solvent is physiological saline containing 10% acetic acid. The injection concentration of siRNA was 10 μg / μL, and the solvent was siRNA-Mate transfection reagent.

6. A drug for alleviating conditioned position preference behavior in morphine-dependent mice, characterized in that, The active ingredient of the drug includes the polypeptide of claim 1 or the siRNA of claim 2; it also includes a pharmaceutically acceptable carrier.

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