Use of setd2 inhibitor ezm0414 in the preparation of a medicament for treating hsv-1 induced postherpetic neuralgia

Abstract

The application belongs to the technical field of biological medicine, and provides application of a SETD2 inhibitor EZM0414 in preparation of a medicine for treating HSV-1 induced postherpetic neuralgia, and the application is proved through a mouse model experiment of HSV-1 induced postherpetic neuralgia that the SETD2 inhibitor EZM0414 can significantly relieve mechanical hyperalgesia of the model mouse, and can significantly inhibit release of proinflammatory factors TNF-alpha, IL-1beta and CCL2 of spinal cord microglia cells, and can up-regulate expression of anti-inflammatory factors TGF-beta, IL-10 and IL-4, and can be used for preparing a medicine for treating HSV-1 induced postherpetic neuralgia.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the SETD2 inhibitor EZM0414 in the preparation of a drug for treating HSV-1-induced postherpetic neuralgia. Background Technology

[0002] Herpes simplex virus type 1 (HSV-1) infection is a common viral infectious disease in clinical practice. After infection, it often induces persistent and intractable postherpetic neuralgia. The core clinical manifestations are mechanical hyperalgesia, spontaneous burning pain and chronic inflammation of the dorsal horn of the spinal cord, which seriously reduces the patient's quality of life and may even lead to mental disorders such as anxiety and depression.

[0003] Currently, the drugs used in clinical practice to treat postherpetic neuralgia are mainly symptomatic relief, including antiepileptic drugs (such as pregabalin and gabapentin), antidepressants, and opioid analgesics. These drugs generally have core limitations such as limited efficacy, large individual differences, significant side effects, inability to block disease progression, and inability to target the cause. Approximately 40% of patients cannot achieve effective pain relief, indicating a huge unmet clinical need for treatment.

[0004] SETD2 (SET domain-containing 2) is a methyltransferase that specifically catalyzes the trimethylation of histone H3K36 in vivo. Existing studies have only reported its role in tumorigenesis and immune cell activation. As a specific small molecule inhibitor of SETD2, EZM0414 has only been disclosed in existing technologies for its potential anti-tumor effects in hematologic malignancies and solid tumors. No studies have ever revealed the correlation between EZM0414 and HSV-1-induced postherpetic neuralgia, nor has it been disclosed that EZM0414 can be used to prepare drugs for the treatment of this disease.

[0005] The existing technology, "Intravenous lidocaine alleviates postherpetic neuralgia inrats via regulation of neuroinflammation of microglia and astrocytes," published in February 2021, discloses that intravenous lidocaine can alleviate mechanical hyperalgesia and thermal hypoalgesia in a rat PHN model. The mechanism is related to the inhibition of microglia and astrocyte activation in the dorsal horn of the spinal cord and the downregulation of TNF-α and IL-1β expression. This literature also discloses methods for constructing the PHN model (resin toxin RTX induction), methods for detecting the mechanical pain threshold (von Frey cilia), and methods for detecting inflammatory factors in the dorsal horn of the spinal cord. Although lidocaine in this literature can inhibit microglia activation and the release of pro-inflammatory factors, it is a symptomatic treatment using sodium channel blockers, and pain is prone to recurrence after drug withdrawal.

[0006] The existing technology, "GCN5L1 Aggravates Postherpetic Neuralgia Through Regulating Microglial Mitochondrial Fission–Fusion Homeostasis," published in September 2025, discloses that GCN5L1 is significantly upregulated in spinal cord dorsal horn microglia in an HSV-1-induced PHN mouse model. Knockdown of GCN5L1 via intrathecal injection of AAV-shGCN5L1 inhibits microglial activation, reduces neuroinflammatory responses, and alleviates mechanical hyperalgesia. Mechanistically, GCN5L1 regulates mitochondrial fission-fusion homeostasis by promoting DRP1 acetylation. However, this study uses AAV-mediated GCN5L1 gene knockdown, which requires intrathecal injection of a viral vector, posing risks of immunogenicity, insertional mutations, and inconvenient administration.

[0007] Therefore, there is an urgent need to explore new therapeutic drugs that can target and regulate the inflammatory homeostasis of spinal cord nerves and address the core pathological mechanisms of postherpetic neuralgia. Summary of the Invention

[0008] The first aspect of the present invention provides the use of the SETD2 inhibitor EZM0414 in the preparation of a medicament for treating HSV-1-induced postherpetic neuralgia.

[0009] Specifically, the concentration of EZM0414 is 10-100 μM.

[0010] More specifically, the concentration of EZM0414 is 50-100 μM.

[0011] Optionally, the injection volume of the drug is 5-10 μL.

[0012] Specifically, the SETD2 inhibitor treats postherpetic neuralgia by reducing the expression and secretion levels of pro-inflammatory factors and increasing the expression and secretion levels of anti-inflammatory factors.

[0013] More specifically, the pro-inflammatory factors include, but are not limited to, any one or more of TNF-α, IL-1β, and CCL2.

[0014] More specifically, the anti-inflammatory factors include, but are not limited to, any one or more of TGF-β, IL-10, and IL-4.

[0015] Specifically, the dosage form of the drug includes liquid dosage form, gas dosage form, solid dosage form, or semi-solid dosage form.

[0016] More specifically, the liquid dosage form includes: solvent-based, aromatic aqueous solutions, tinctures, elixirs, colloidal solutions, pastes, suspensions, or emulsifiers.

[0017] More specifically, the gaseous dosage form includes: aerosol or spray.

[0018] More specifically, the solid dosage form includes: powder, pill, tablet or film.

[0019] More specifically, the semi-solid dosage form includes: ointment, suppository or paste.

[0020] Specifically, the routes of administration of the drug include oral, injection, implantation, external application, spraying, or inhalation.

[0021] More specifically, the routes of administration of the drug include intrathecal injection, intravenous injection, subcutaneous injection, oral administration, or local administration.

[0022] Specifically, the treatment targets a mouse model of postherpetic neuralgia induced by HSV-1.

[0023] More specifically, the HSV-1 is HSV-1 7401H strain.

[0024] Optionally, the mouse is a male C57BL / 6J mouse.

[0025] In certain specific embodiments of the present invention, the method for constructing the mouse postherpetic neuralgia model is as follows: S1 and C57BL / 6J mice, weighing 20-25g, were housed in an animal room with constant temperature and humidity and a 12h / 12h light-dark cycle, with free access to food and water. S2. A mouse model was established using the HSV-1 7401H strain. Hair was removed from the hind limbs of mice, and superficial scratches were made on the hind limb skin. The mice were then inoculated with 10 μL of a solution containing 1×10⁻⁶ HSV-17401H. 6 A postherpetic neuralgia model was constructed using HSV-1 suspension of PFU. S3. On day 14 after inoculation, von Frey cilia were used to detect mechanical hyperalgesia in mice. Mice exhibiting persistent mechanical hyperalgesia were considered to have successfully established a mouse model of postherpetic neuralgia.

[0026] In certain specific embodiments of the present invention, the drug is 10 μM, 50 μM or 100 μM EZM0414, the volume of the drug is 5 μL, the administration method is intrathecal injection, and the subject of administration is the above-mentioned mouse postherpetic neuralgia model.

[0027] A second aspect of the present invention provides a pharmaceutical composition for treating postherpetic neuralgia, the pharmaceutical composition comprising a therapeutically effective amount of a SETD2 inhibitor and pharmaceutically acceptable excipients.

[0028] Specifically, the SETD2 inhibitors include, but are not limited to, EZM0414.

[0029] Specifically, the pharmaceutically acceptable excipients include, but are not limited to, any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

[0030] A third aspect of the invention provides a method for treating postherpetic neuralgia, the method comprising administering a therapeutically effective amount of a SETD2 inhibitor to a subject.

[0031] The term "subject" includes living organisms (e.g., mammals) that can elicit an immune response. Examples of subjects include humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0032] The term "treatment" refers to the medical management of a subject's disease, condition, or undesirable illness. Benefits of treatment or prevention include improved clinical outcomes; reduction or alleviation of symptoms associated with the disease, condition, or undesirable illness; reduced symptom occurrence; improved quality of life; longer disease-free status; reduction in the severity of the disease, condition, or undesirable illness; stabilization of the disease state; delay in disease progression; remission; survival; prolonged survival; or any combination thereof.

[0033] The term "therapeutic effective dose" refers to a pharmaceutically considered effective dosage, that is, an amount of active drug sufficient to significantly improve the condition without causing serious side effects. Dosage depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality, and individual response of the patient or animal, the route of administration, frequency of administration, and therapeutic purpose; therefore, the dosage of this invention can vary widely.

[0034] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first to discover the new drug use of EZM0414 in treating HSV-1-induced postherpetic neuralgia, filling the technical gap in targeted therapy for this disease and possessing outstanding novelty; (2) This invention has demonstrated through in vivo experiments that EZM0414 can simultaneously achieve the dual therapeutic effects of dose-dependent pain relief and restoration of neuroinflammatory homeostasis, breaking through the limitation of existing clinical drugs that only provide symptomatic relief. Its efficacy is clear and its mechanism of action is well understood. (3) This invention provides core candidate compounds for the development of targeted therapies for HSV-1-induced postherpetic neuralgia, which have important clinical translational value and application prospects. Attached Figure Description

[0035] Figure 1 The curves showing the change in mechanical pain threshold over time in HSV-1 infected mice after administration of different doses of EZM0414, as measured by 0.6g von Frey. * indicates p<0.05. Figure 2 The curves showing the change of mechanical pain threshold over time in HSV-1 infected mice after administration of different doses of EZM0414, as measured by 2g von Frey assay. * indicates p<0.05. Figure 3 To detect the effects of different doses of EZM0414 on the expression of pro-inflammatory factors Tnf-α, Il-1β, and Ccl2 mRNA in the dorsal horn of the spinal cord of model mice by qPCR, * indicates p<0.05; Figure 4 To detect the effects of different doses of EZM0414 on the protein levels of pro-inflammatory factors TNF-α, IL-1β, and CCL2 in the dorsal horn of the spinal cord of model mice using ELISA, * indicates p<0.05; Figure 5 To detect the effect of different doses of EZM0414 on the expression of anti-inflammatory factors Tgf-β, Il-10, and Il-4 in the dorsal horn of the spinal cord of model mice by qPCR, * indicates p<0.05; Figure 6The effect of different doses of EZM0414 on the protein levels of anti-inflammatory factors TGF-β, IL-10, and IL-4 in the dorsal horn of the spinal cord of model mice was detected by ELISA. * indicates p<0.05. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1: Establishment of an HSV-1-induced mouse model of postherpetic neuralgia 1. Experimental animals: SPF grade C57BL / 6J male mice, weighing 20-25g, were housed in an SPF grade animal room with constant temperature and humidity and a 12h / 12h light-dark cycle, with free access to food and water.

[0038] 2. Model Construction: The HSV-1 7401H strain was used to construct the model. Hair was removed from the hind limbs of mice. Three days later, superficial scratches were made on the skin of the right hind limb, and 10 μL of a solution containing 1×10⁻⁶ mg / L of HSV-1 7401H was injected. 6 A postherpetic neuralgia model was constructed using a suspension of HSV-1 from PFU.

[0039] 3. Model validation: On day 14 after inoculation, 0.6g and 2g von Frey cilia were used to detect mechanical hyperalgesia in mice. Mice exhibiting persistent mechanical hyperalgesia were considered to have successfully established the model and were used for subsequent drug administration experiments.

[0040] The method for detecting mechanical hyperalgesia was as follows: Before the test, mice were placed in a transparent acrylic cage (10cm×10cm×15cm) with a metal mesh bottom for 2 hours to acclimatize. The skin on the soles of the feet, lesions, and abdomen was vertically stimulated with 0.6g and 2g of von Frey fibers, respectively. Each stimulation lasted 2-3 seconds, and each site was repeated 6 times with a 60-second interval between stimulations. Contact with hair and scar tissue was avoided during stimulation. Scoring criteria: 0 points = no response; 1 point = rapid lifting of the stimulated area; 2 points = shaking or licking of the stimulated area. Pain-related score (%) = (total score / 12) × 100%. The experiment was conducted in a double-blind manner, and the experimenters were unaware of the group assignments.

[0041] Example 2: Validation of the analgesic efficacy of EZM0414 in HSV-1-induced postherpetic neuralgia mice 1. Experimental grouping: Mice with successfully constructed models were randomly divided into 4 groups, with 6 mice in each group: Solvent control group (Vehicle): Intrathecal injection of an equal volume of physiological saline; EZM0414 low-dose group: 10μM / 5μL, intrathecal injection; EZM0414 medium-dose group: 50μM / 5μL, intrathecal injection; EZM0414 high-dose group: 100μM / 5μL, intrathecal injection.

[0042] 2. Administration method: Start administration on day 14 after HSV-1 inoculation, once daily, for 3 consecutive days via intrathecal injection.

[0043] 3. Behavioral testing: On days 14, 21, 24, 28, and 35 post-vaccination, 0.6g and 2g von Frey cilia were used to test the mechanical hyperalgesia of mice. The testing method was the same as in Example 1. Each mouse was tested 6 times at each site, and the pain-related score (%of max score) was calculated.

[0044] 4. Experimental Results: Figure 1 The results of the 0.6g von Frey assay showed that, compared with the solvent control group, the mechanical hyperalgesia of mice in the EZM0414 administration group was significantly relieved in a dose-dependent manner, and the high-dose group had the most significant analgesic effect. Figure 2 The results of the 2g von Frey assay were consistent with those of the 0.6g assay. EZM0414 significantly reduced pain-related scores and increased mechanical pain threshold in model mice in a dose-dependent manner.

[0045] This embodiment confirms that EZM0414 can significantly relieve HSV-1-induced postherpetic neuralgia and has a clear analgesic effect.

[0046] Example 3: Regulation of pro-inflammatory factors in the spinal cord of model mice by EZM0414 1. Sample collection: On day 24 after HSV-1 inoculation, mice were anesthetized and sacrificed, and L4-L5 spinal cord dorsal horn tissue was collected to extract total RNA and total protein.

[0047] Total RNA extraction method: Take approximately 20 mg of spinal cord dorsal horn tissue, add 1 mL of TRIzol reagent, and homogenize thoroughly on ice using a homogenizer. Incubate at room temperature for 5 minutes, add 200 μL of chloroform, shake vigorously for 15 seconds, and incubate at room temperature for 3 minutes. Centrifuge at 12000g for 15 minutes at 4℃, transfer the upper aqueous phase to a new EP tube, add an equal volume of isopropanol, mix well, and incubate at room temperature for 10 minutes. Centrifuge at 12000g for 10 minutes at 4℃, discard the supernatant, wash the precipitate twice with 75% ethanol, dry at room temperature, and dissolve in 20 μL of DEPC water. Determine RNA concentration and purity (A260 / A280 between 1.9 and 2.1) using Nanodrop 2000.

[0048] Total protein extraction method: Add RIPA lysis buffer containing protease inhibitor cocktail (1:100 ratio) to the dorsal horn tissue of the spinal cord, homogenize thoroughly on ice, centrifuge at 12000g for 15 minutes at 4℃, and collect the supernatant.

[0049] 2. Detection method: The mRNA expression levels of pro-inflammatory factors TNF-α, Il-1β, and Ccl2 were detected by qPCR, with β-actin as an internal reference, and the relative expression levels were calculated using the 2-ΔΔCt method.

[0050] Take 1 μg of total RNA and reverse transcribe it into cDNA using the PrimeScript RT kit. Reaction system: 4 μL 5×PrimeScript Buffer, 1 μL PrimeScript RT Enzyme Mix I, 1 μL Oligo dT Primer (50 μM), 1 μL Random 6 mers (100 μM), 1 μg total RNA, and RNase-free dH2O to a final volume of 20 μL. Reaction conditions: 37℃ for 15 minutes, 85℃ for 5 seconds, and store at 4℃. Q-PCR was used to detect HSV-1 TK gene expression. Reaction system: 10 μL SYBR Premix Ex Taq II, 0.8 μL each of forward and reverse primers (10 μM), 0.4 μL ROX Reference Dye II, 2 μL cDNA template, 6 μL dH2O, total volume 20 μL. Reaction conditions: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds, 60℃ annealing and extension for 34 seconds, 40 cycles. Detection was performed on an ABI QuantStudio 5 real-time quantitative PCR instrument. β-actin was used as an internal control, and 2... -ΔΔCT The relative expression level is calculated using this method.

[0051] TNF-α primer sequence: Positive 5'-GTAGCCCACGTCGTAGCAAA-3' (SEQ ID NO.1); Reverse 5'-ACAAGGTACAACCCATCGGC-3' (SEQ ID NO.2); IL-1β primer sequence: Positive 5'-AGAGCCCATCCTCTGTGACT-3' (SEQ ID NO.3); Reverse 5'-GCTCATATGGGTCCGACAGC-3' (SEQ ID NO.4); CCL2 primer sequence: Positive 5'-GCTACAAGAGGATCACCAGCAG-3' (SEQ ID NO.5); Reverse 5'-GTCTGGACCCATTCCTTCTTGG-3' (SEQ ID NO.6); β-actin primer sequence: Positive 5'-AGAGGGAAATCGTGCGTGAC-3' (SEQ ID NO.7); Reverse 5'-CAATAGTGATGACCTGGCCGT-3' (SEQ ID NO.8).

[0052] The protein release levels of pro-inflammatory factors TNF-α, IL-1β, and CCL2 were detected by ELISA, normalized to the total protein concentration, and the protein concentration was determined by BCA method. The protein concentration of each sample was adjusted to 1 mg / mL.

[0053] Follow the instructions for the Mouse TNF-α ELISA Kit (Invitrogen, BMS607-3), Mouse IL-1β ELISA Kit (Invitrogen, BMS6002), and Mouse CCL2 ELISA Kit (Sigma-Aldrich, RAB0055): Add 100 μL of standard or sample to each well and incubate at 37°C for 90 minutes; wash the plate 4 times, add 100 μL of biotinylated antibody working solution, and incubate at 37°C for 60 minutes; wash the plate 4 times, add 100 μL of enzyme conjugate working solution, and incubate at 37°C in the dark for 30 minutes; wash the plate 4 times, add 100 μL of chromogenic substrate, and incubate at 37°C in the dark for 15-20 minutes; add 100 μL of stop solution, and immediately measure the OD value at 450 nm using a microplate reader. Calculate the concentrations of TNF-α, IL-1β, and CCL2 in each sample based on the standard curve.

[0054] 3. Experimental Results: Figure 3 Compared with the solvent control group, the mRNA expression levels of TNF-α, IL-1β, and Ccl2 in the dorsal horn of the spinal cord of mice in the EZM0414-treated group were significantly reduced in a dose-dependent manner. Figure 4 Compared with the solvent control group, the protein levels of TNF-α, IL-1β, and CCL2 in the dorsal horn of the spinal cord of mice in the EZM0414-treated group decreased significantly in a dose-dependent manner.

[0055] This embodiment demonstrates that EZM0414 can significantly inhibit the abnormal increase of HSV-1 infection-induced pro-inflammatory factors in the spinal cord and improve the neuroinflammatory microenvironment.

[0056] Example 4: Regulatory effect of EZM0414 on anti-inflammatory factors in the spinal cord of model mice 1. Sample collection: Same as in Example 3, take the dorsal horn tissue of mouse L4-L5 spinal cord and extract total RNA and total protein.

[0057] 2. Detection method: The mRNA expression levels of anti-inflammatory factors Tgf-β, Il-10, and Il-4 were detected by qPCR, following the same procedure as in Example 3. The primer sequences are as follows: Tgf-β primer sequence: Positive 5'-CACTGATACGCCTGAGTG-3' (SEQ ID NO.9); Reverse 5'-GTGAGCGCTGAATCGAAA-3' (SEQ ID NO.10); Il-10 primer sequence: Forward 5'-GCTCTTACTGACTGGCATGAG-3' (SEQ ID NO.11); Reverse 5'-CGCAGCTCTAGGAGCATGTG-3' (SEQ ID NO.12); Il-4 primer sequence: Forward 5'-GGTCTCAACCCCCAGCTAGT-3' (SEQ ID NO.13); Reverse 5'-GCCGATGATCTCTCTCAAGTGAT-3' (SEQ ID NO.14); The protein levels of anti-inflammatory factors TGF-β, IL-10, and IL-4 were detected by ELISA, following the same procedure as in Example 3. The detection kits for TGF-β, IL-10, and IL-4 were TGF-β1 Mouse ELISA Kit (Beyotime, PT878), IL-10 Mouse ELISA Kit (Sigma-Aldrich, RAB0245), and IL-4 Mouse ELISA Kit (Sigma-Aldrich, RAB0299), respectively.

[0058] 3. Experimental Results: Figure 5 Compared with the solvent control group, the mRNA expression levels of Tgf-β, Il-10, and Il-4 in the dorsal horn of the spinal cord of mice in the EZM0414-treated group were significantly upregulated in a dose-dependent manner. Figure 6 Compared with the solvent control group, the protein levels of TGF-β, IL-10, and IL-4 in the dorsal horn of the spinal cord of mice in the EZM0414-treated group were significantly increased in a dose-dependent manner.

[0059] This embodiment demonstrates that EZM0414 can significantly restore the expression of anti-inflammatory factors in the spinal cord after HSV-1 infection and rebuild neuroinflammatory homeostasis.

[0060] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of SETD2 inhibitor EZM0414 in the preparation of drugs for treating HSV-1-induced postherpetic neuralgia.

2. The application according to claim 1, characterized in that, The concentration of EZM0414 is 10-100 μM.

3. The application according to claim 1, characterized in that, The SETD2 inhibitor treats postherpetic neuralgia by reducing the expression and secretion levels of pro-inflammatory factors and increasing the expression and secretion levels of anti-inflammatory factors.

4. The application according to claim 3, characterized in that, The pro-inflammatory factors include TNF-α, IL-1β, and CCL2.

5. The application according to claim 3, characterized in that, The anti-inflammatory factors include TGF-β, IL-10, and IL-4.

6. The application according to any one of claims 1-5, characterized in that, The dosage form of the drug includes liquid dosage form, gas dosage form, solid dosage form or semi-solid dosage form.

7. The application according to any one of claims 1-5, characterized in that, The routes of administration of the drug include oral, injection, implantation, external application, spraying, or inhalation.

8. The application according to claim 7, characterized in that, The routes of administration for the drug include intrathecal injection, intravenous injection, subcutaneous injection, oral administration, or local administration.

9. A pharmaceutical composition for treating postherpetic neuralgia, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of a SETD2 inhibitor and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that, Pharmaceutically acceptable excipients include binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

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