Oxytocin receptor-selective cyclic peptide molecules and their use in the preparation of medicaments against neuropathic pain

Abstract

The present application relates to oxytocin receptor selective cyclic peptide molecules and their use in the preparation of anti neuropathic pain drugs. The present application provides an oxytocin receptor (OXTR) selective cyclic peptide molecule, the OXTR selective cyclic peptide molecule comprises an amino acid sequence as shown in formula I and salts thereof: formula I: [CYIX1NC]X2X3G-NH2. The OXTR selective polypeptide provided by the present application, by establishing a bioluminescence resonance energy transfer high-throughput screening system for OXTR, detecting the G alpha q protein decoupling triggered after the polypeptide ligand activates OXTR, in order to screen out candidate molecules that can selectively agonize OXTR and play an anti neuropathic pain, and is expected to realize the discovery of anti neuropathic pain new drugs.

Description

Technical Field

[0001] This invention relates to an oxytocin receptor (OXTR) selective cyclic peptide molecule, a selective cyclic peptide agonist of the oxytocin receptor comprising these molecules, a pharmaceutical composition, and the use of said pharmaceutical composition in the preparation of a medicament for treating neuropathic pain. Background Technology

[0002] Oxytocin receptor (OXTR) is a G protein-coupled receptor widely distributed in the central nervous system and peripheral tissues, possessing broad physiological functions and significant targeting value in clinical diagnosis and treatment. In the central nervous system, OXTR regulates emotional homeostasis and social memory in brain regions such as the amygdala and hypothalamus. Abnormalities in its mediated anti-anxiety signaling pathways can easily induce anxiety disorders. Furthermore, deficiencies in OXTR phosphorylation in the medial amygdala, leading to impaired social memory regulation, significantly increase the risk of autism spectrum disorder (ASD). At the peripheral level, OXTR is expressed in root ganglion neurons of the peripheral nervous system. Studies have shown that in paclitaxel-induced peripheral neuropathy, OXTR expression increases compensatorily, and activating OXTR can significantly enhance the paclitaxel-induced reduction in the mechanical withdrawal reflex threshold in mice. In addition, decreased OXTR expression in uterine smooth muscle can lead to dystocia, while abnormal OXTR activity in mammary myoepithelial cells can induce lactation disorders. OXTR presence or absence is associated with the occurrence and maintenance of various neuropathic pain conditions, making it an important therapeutic target.

[0003] The endogenous ligand of OXTR is oxytocin (OT), a cyclic 9-peptide with the sequence [CYIQNC]PLG. Cys1 and Cys6 form an intramolecular disulfide bond, followed by three amino acids: Pro, Leu, and Gly. This polypeptide sequence is highly similar to arginine vasopressin, the ligand of the vasopressin receptor family, differing only in the third and eighth amino acids. Therefore, oxytocin is associated with the V1 receptor of the vasopressin family. A R, V1 B Both R and V2R exhibit some agonistic activity. Peptide modifications targeting oxytocin have been reported, among which [Thr4, Gly7]OT is a peptide derived from the OT sequence, with the sequence [CYITNC]GLG. [Thr4, Gly7]OT shows approximately 40-fold decreased OXTR agonistic activity compared to OT, and also exhibits activity against V2R and V1R. B R still possesses some agonistic activity. Currently, there is a lack of selective agonists with high agonistic activity against OXTR to serve as molecular probes for in-depth analysis of disease mechanisms or to develop therapeutic drugs for neuropathic pain with OXTR as a key target. Summary of the Invention

[0004] The present invention aims to provide an OXTR-selective cyclic peptide molecule, characterized in that the OXTR-selective cyclic peptide molecule comprises an amino acid sequence as shown in Formula I and its salts: Formula I: [CYIX1NC]X2X3G-NH2, i.e. SEQ ID NO: 6; X1 is (2S,3R)-2-amino-3-methoxybutyric acid, serine, (2S)-2-amino-3-methoxypropionic acid, threonine or (2S,3R)-2-amino-3-methoxybutyric acid. X2 is glycine or 2-(methylamino)acetic acid; X3 is leucine or (2S)-2-amino-5-methylhexanoic acid; The first cysteine ​​and the sixth cysteine ​​form an intramolecular disulfide bond.

[0005] As a preferred embodiment, the oxytocin receptor-selective cyclic peptide molecule is selected from the group consisting of the following amino acid sequences: SEQ ID NO: 1: [CYI-((2S,3R)-2-amino-3-methoxybutyric acid)NC]GLG-NH2; SEQ ID NO: 2: [CYISNC]GLG-NH2; SEQ ID NO: 3: [CYI-((2S)-2-amino-3-methoxypropionic acid)NC]GLG-NH2; SEQ ID NO: 4: [CYITNC]G-((2S)-2-amino-5-methylhexanoic acid)G-NH2; and SEQ ID NO: 5: [CYI-((2S,3R)-2-amino-3-methoxybutyric acid)NC]-(2-(methylamino)acetic acid)LG-NH2.

[0006] Another object of the present invention is to provide a selective cyclic peptide agonist of OXTR, the selective cyclic peptide agonist of OXTR comprising the selective cyclic peptide molecule of OXTR or a salt thereof or a mixture thereof.

[0007] Another object of the present invention is to provide use in the preparation of a pharmaceutical composition for the prevention or treatment of neuropathic pain, characterized in that the pharmaceutical composition comprises a selective cyclic peptide agonist of the OXTR and a pharmaceutically acceptable carrier.

[0008] As a preferred embodiment, the neuropathic pain is anxiety disorder or autism spectrum disorder.

[0009] The OXTR selective cyclic peptide molecule of the present invention was synthesized using a solid-phase synthesis method known to those skilled in the art, and purified using high-performance liquid chromatography.

[0010] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neuropathic pain, characterized in that the pharmaceutical composition contains a selective cyclic peptide agonist of the OXTR and a pharmaceutically acceptable carrier.

[0011] The pharmaceutical compositions of the present invention can be formulated into various conventional dosage forms known in the art, including but not limited to tablets (including various coated tablets, sustained-release or controlled-release tablets), lozenges, capsules (including soft capsules and hard capsules), granules, dispersible powders, aqueous or oily suspensions, emulsions, elixirs or syrups, etc., suitable for oral administration; creams, ointments, gels, aqueous or oily solutions or suspensions, etc., suitable for topical use; powders or liquid aerosols suitable for inhalation; sterile aqueous or oily intravenous, subcutaneous or intramuscular injections, suppositories, etc., suitable for parenteral administration.

[0012] Those skilled in the art will recognize that the appropriate amounts of the OXTR selective cyclic peptide molecule for anti-neuropathic pain and the pharmaceutically acceptable carrier, which are the active ingredients in the pharmaceutical compositions of the present invention, can be determined according to conventional methods in the art. Those skilled in the art will also know how to prepare pharmaceutical compositions containing the anti-diabetes insipidus polypeptide compounds of the present invention.

[0013] The advantages of this invention are as follows: This invention relates to an OXTR selective cyclic peptide molecule or its salt, an OXTR selective cyclic peptide agonist, and their applications for screening candidate new drugs for neuropathic pain. This invention provides a novel OXTR selective cyclic peptide molecule or its salt, which can be used as an OXTR selective cyclic peptide agonist for screening candidate new drugs for neuropathic pain. The sequence of this OXTR selective cyclic peptide molecule is derived from the reported OXTR selective agonist, [Thr4, Gly7]OT, with the sequence [CYITNC]GLG. This application explores and optimizes some amino acids in the peptide sequence, introducing non-standard amino acids to improve the activity of the peptide ligand, while also considering the peptide molecule's effect on V2R and V1. A R and V1 B The selectivity of different R isoforms ultimately yielded the polypeptide of this invention. The OXTR-selective cyclic peptide molecule of this invention, as an OXTR-selective peptide agonist, targets V2R and V1... A R and V1 B R has weak or even no activation of V2R or V1. A R and V1 BR, but with high affinity for OXTR and strong agonistic activity, and can significantly improve the reduction of the mechanical withdrawal reflex threshold induced by paclitaxel in mice, can be considered as a potential candidate for anti-neuropathic pain drugs. Attached Figure Description

[0014] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 The results of the bioluminescence resonance energy transfer (BRET) assay show that the downstream Gαq dissociation caused by the activation of OXTR after SEQ ID NO: 1 to SEQ ID NO: 5 was detected. Figure 2 The results of the BRET test show the selective test results of SEQ ID NO: 5 on OXTR. Figure 3 The BRET test showed that SEQ ID NO: 5 was positive for V1. A Results of R selectivity test experiments; Figure 4 The BRET test showed that SEQ ID NO: 5 was positive for V1. B Results of R selectivity test experiments; Figure 5 The results of the BRET test show the V2R selectivity test results of SEQ ID NO: 5; Figure 6 The results, obtained by the Von Frey method, show the threshold of the mechanical withdrawal reflex in mice induced by paclitaxel induced by SEQ ID NO: 5. Detailed Implementation

[0015] The present invention will be further illustrated below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0016] Example 1: Solid-phase synthesis, cleavage, purification, and identification of cyclic peptide molecules 1.1 Solid-phase synthesis of SEQ ID NO: 1 This invention utilizes a peptide synthesizer to synthesize SEQ ID NO: 1: [CYI-((2S,3R)-2-amino-3-methoxybutyric acid)NC]GLG-NH2 via solid-phase synthesis. Using N,N-dimethylformamide (DMF) as a solvent, various amino acid solutions (0.25 mol / L) with α-amino groups protected by fluorenebenzyloxycarbonyl (Fmoc), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) solution, 1-hydroxybenzotriazole (HOBt) solution (0.33 mol / L), piperidine solution (200 mL / L), diisopropylcarbodiimide (DIC) solution (0.5 mol / L), and 1-hydroxyphenyl-4,5-diphenylimidazole (Oxyma) solution (1 mol / L) were prepared.

[0017] 1 g of Fmoc-protected butyramide resin was placed in the reactor. The corresponding amino acid raw materials for the polypeptide chain were weighed and prepared into a 0.2 mol / L solution with DMF. The prepared 20% piperidine solution, activator DIC, and activator base Oxyma solution were placed in their respective reagent bottles on the polypeptide solid-phase synthesizer. Following the instrument software's preset program, after the resin swelled for 5 min, 20% piperidine was used for deprotection, followed by sequential condensation, deprotection, and condensation of the amino acid sequence of SEQ ID NO: 1. After synthesis was complete, the resin was washed with DMF for 2-3 minutes, filtered, and the filtrate was discarded to obtain resin containing the crude polypeptide.

[0018] 1.2 Cutting of Excess Groups and Resin The dried resin was placed in a 25 mL round-bottom flask, and 15 mL of resin cutting solution (containing 95% trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% water) was slowly added dropwise under an ice-water bath. The mixture was heated to room temperature and shaken for 2 to 3 hours. After the reaction was complete, the reaction solution and resin were transferred to a vacuum filtration device to remove the resin. The collected filtrate was placed in a round-bottom flask, allowed to settle in ice-cold ether, and then heated at 4°C and 7000× g Centrifuge at 15 minutes, discard the supernatant, repeat the operation 3 times, and finally dry in a vacuum drying oven at 50°C for 48 hours to obtain about 0.5g of crude peptide.

[0019] 1.3 Purification and identification of linearized SEQ ID NO: 1 The crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). A C18 preparative column was used as the stationary phase, and the mobile phase consisted of an aqueous phase and an organic phase. The aqueous phase was deionized water (containing 0.1% trifluoroacetic acid by mass); the organic phase was an aqueous solution containing 80% acetonitrile (containing 0.1% trifluoroacetic acid by mass). The flow rate was 20.00 mL / min; the elution gradient was: aqueous phase (90%→10%), organic phase (10%→90%). The eluent at the highest peak in the HPLC chromatogram was collected, and acetonitrile was removed from the eluent using a rotary evaporator at 50℃. The remaining aqueous solution was rapidly frozen and then freeze-dried to remove moisture, yielding a white, fluffy powder. HPLC-MS (High-Performance Liquid Chromatography-Mass Spectrometry) verified that the solid purity was greater than 98%, ESI: 954.5 [M+H]. + .

[0020] 1.4 Linearization of SEQ ID NO: 1 disulfide cyclization The collected first white, fluffy powdery solid was dissolved in 200 mL of pure water. The pH was adjusted to approximately 8 with ammonia, and 5 mL of 1% hydrogen peroxide was added. The mixture was stirred and reacted for 30 minutes. After a negative result was obtained by testing with the 5,5'-dithio-bis-[2-nitrobenzoic acid] thiol assay reagent, the pH was adjusted to acidic with acetic acid. The mixture was then rapidly frozen and the moisture was removed using a freeze dryer to obtain the second white, fluffy powdery solid. HPLC-MS (High Performance Liquid Chromatography-Mass Spectrometry) verified that the solid purity was greater than 98%, ESI: 523.8 [M+2H]. 2+ .

[0021] Example 2: SEQ ID NO: 2: [CYISNC]GLG-NH2 was prepared using the same method as in Example 1. The solid purity was greater than 98%, and the ESI was 926.5 [M+H]. + ; Example 3: SEQ ID NO: 3: [CYI-((2S)-2-amino-3-methoxypropionic acid)NC]GLG-NH2 was prepared using the same method as in Example 1. The solid purity was greater than 98%, and the ESI was 940.5 [M+H]. + ; Example 4: SEQ ID NO: 4: [CYITNC]G-((2S)-2-amino-5-methylhexanoic acid)G-NH2 was prepared using the same method as in Example 1. The solid purity was greater than 98%, and the ESI was 954.5 [M+H]. + ; Example 5: SEQ ID NO: 5: [CYI-((2S,3R)-2-amino-3-methoxybutyric acid)NC]-(2-(methylamino)acetic acid)LG-NH2 was prepared using the same method as in Example 1. The solid purity was greater than 98%, ESI: 968.4 [M+H] + ; Example 6: Detection of in vitro activity of SEQ ID NO: 1 to SEQ ID NO: 5 on downstream Gαq decoupling after OXTR activation using a bioluminescence resonance energy transfer (BRET) method. (1) The wild-type OXTR plasmid was purchased from the SnapGene gene library. This plasmid contains the codon-optimized full-length OXTR gene. Primers were designed and the target fragment was amplified by PCR. The full-length OXTR gene was inserted between the BamHI and EcoRI restriction sites of the pcDNA3.1 vector. The HiBiT tag, Igl signal peptide tag, and Kozak start sequence were connected to the N-terminus of the OXTR gene using a flexible linker. The plasmid was verified by first-generation sequencing after construction before it could be used.

[0022] (2) HEK293T cells were injected at a concentration of 1.2 × 10⁻⁶. 6 The cells were seeded at a density of 10 cells per 6 cm cell culture dish, and 5 ml of DMEM medium was added. The cells were then incubated at 37°C for 24 hours. (3) Replace with fresh complete culture medium 2 hours before transfection. Prepare transfection solution by adding 250µL of Opti-MEM containing the following components to a 6cm culture dish. The components of the transfection system are shown in Table 1.

[0023] Table 1: Components of the G protein dissociation detection transfection system (BRET assay)

[0024] (4) 24 hours after transfection, discard the original culture medium, digest the cells with trypsin, add 500 μL of digestion solution to each dish, incubate at 37°C for 2 minutes, transfer the cells to centrifuge tubes, and centrifuge at 200g for 5 minutes. Discard the supernatant, resuspend in DMEM medium, mix well by pipetting, and count the cells. Spread the cell suspension at 3 × 10⁻⁶ cells / mL. 4 100 µL of cells were seeded into 96-well pure white cell culture plates. After mixing, the plates were incubated at 37°C.

[0025] (5) After 24 hours of incubation, the culture medium in the 96-well plate was aspirated, and 80 µL of BRET assay buffer was added to each well for equilibration. 20 µL of 5× analyte compound (diluted with assay buffer) was added to each well, and the plate was incubated for 3 to 5 minutes. Then, 20 µL of diluted coelenterate 400a (working concentration 5 µM) was added to each well, and the fluorescence value was measured using the chemiluminescence module of the microplate reader. The microplate reader was set to switch filters for each well to read fluorescence emission values ​​at two wavelengths: a 410 nm filter for detecting Rluc8 and a 515 nm filter for detecting GFP2. The BRET ratio was calculated by dividing the fluorescence value at 515 nm by the fluorescence value at 410 nm. The net BRET value was obtained by subtracting the background BRET ratio of the vehicle group. The net BRET value was used to fit the dose-response curve. The experimental results are shown below. Figure 1 As shown in the figure, [Thr4, Gly7]OT, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 can all induce Gαq protein dissociation in OXTR in a dose-dependent manner, with EC50 values ​​of 445.8 nM, 75.10 nM, 100.6 nM, 288.7 nM, 108.8 nM, and 13.87 nM, respectively (as shown in Table 2). Furthermore, the dose-response curves of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 are significantly shifted to the left, indicating that SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 have enhanced activity compared to [Thr4, Gly7]OT, with SEQ ID NO: 5 showing the greatest increase in activity, being 32 times that of [Thr4, Gly7]OT.

[0026] Table 2: Oxytocin receptor selective cyclic peptide molecules in OXTR, V2R, and V1 A R and V1 B Functional activity on R

[0027] Example 7: Detection of SEQ ID NO: 5 against OXTR and V1 using a BRET-based method A R, V1 B Selective activation of R and V2R The operation steps are the same as in Example 6, and the experimental results are shown in Table 1. Figures 2 to 5 As shown, by Figures 2 to 5 As shown in Table 1, SEQ ID NO: 5, which selectively activates OXTR, can induce dose-dependent dissociation of the OXTR Gαq protein. SEQ ID NO: 5 can only induce a weak Gαs protein dissociation effect in V2R at high doses, and SEQ ID NO: 5 cannot induce V1. A R induces dose-dependent dissociation of the Gαq protein, and SEQ ID NO: 5 can only induce V1 at high doses. B R produces a weak dissociation effect on the Gαq protein.

[0028] Example 8: Detection of mechanical withdrawal reflex in mice using the Von Frey method (SEQ ID NO: 5) The Von Frey method was used to detect the mechanical withdrawal reflex threshold in mice after intervention with SEQ ID NO: 5. The experimental results are as follows: Figure 6 As shown in the figure, SEQ ID NO: 5 dose-dependently improved the paclitaxel-induced decrease in the mechanical withdrawal reflex threshold in mice, and 0.4 mg / kg SEQ ID NO: 5 restored the mechanical withdrawal reflex threshold in mice to normal levels. Following intravenous injection of SEQ ID NO: 5, the analgesic effect lasted up to 40 minutes, after which the effect significantly decreased.

[0029] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An oxytocin receptor-selective cyclic peptide molecule, characterized in that, The oxytocin receptor-selective cyclic peptide molecule comprises an amino acid sequence as shown in Formula I and its salts: Formula I: [CYIX1NC]X2X3G-NH2, i.e. SEQ ID NO: 6; X1 is (2S,3R)-2-amino-3-methoxybutyric acid, serine, (2S)-2-amino-3-methoxypropionic acid, threonine or (2S,3R)-2-amino-3-methoxybutyric acid. X2 is glycine or 2-(methylamino)acetic acid; X3 is leucine or (2S)-2-amino-5-methylhexanoic acid; The first cysteine ​​and the sixth cysteine ​​form an intramolecular disulfide bond.

2. The oxytocin receptor-selective cyclic peptide molecule as described in claim 1, characterized in that, The oxytocin receptor-selective cyclic peptide molecule is selected from the group consisting of the following amino acid sequences: SEQ ID NO: 1: [CYI-((2S,3R)-2-amino-3-methoxybutyric acid)NC]GLG-NH2; SEQ ID NO: 2: [CYISNC]GLG-NH2; SEQ ID NO: 3: [CYI-((2S)-2-amino-3-methoxypropionic acid)NC]GLG-NH2; SEQ ID NO: 4: [CYITNC]G-((2S)-2-amino-5-methylhexanoic acid)G-NH2; and SEQ ID NO: 5: [CYI-((2S,3R)-2-amino-3-methoxybutyric acid)NC]-(2-(methylamino)acetic acid)LG-NH2.

3. A selective cyclic peptide agonist of the oxytocin receptor, characterized in that, The selective cyclic peptide agonist of the oxytocin receptor comprises the selective cyclic peptide molecule of the oxytocin receptor as described in claim 1 or 2, or a salt thereof, or a mixture thereof.

4. The use of a selective cyclic peptide agonist of the oxytocin receptor in the preparation of a pharmaceutical composition for the prevention or treatment of neuropathic pain, characterized in that, The selective cyclic peptide agonist of the oxytocin receptor is the selective cyclic peptide agonist of the oxytocin receptor as described in claim 3.

5. The use as described in claim 4, characterized in that, The neuropathic pain mentioned is anxiety disorder or autism spectrum disorder.

6. A pharmaceutical composition for the prevention or treatment of neuropathic pain, characterized in that, The pharmaceutical composition comprises a selective cyclic peptide agonist of the oxytocin receptor as described in claim 3, and a pharmaceutically acceptable carrier.

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