Application of orphan receptor GPR88 as target spot in preparation of medicine for treating neuropathic pain

Abstract

The invention discloses application of an orphan receptor GPR88 as a target spot in developing or preparing a medicine for treating neuropathic pain. The invention proposes that the orphan receptor GPR88 is a target of a medicine for treating neuropathic pain for the first time, the expression characteristic of the orphan receptor GPR88 at the spinal cord level is verified for the first time, the pain behavior of a mouse is observed through intrathecal administration of a GPR88 receptor agonist, the definite analgesic effect after the GPR88 receptor is activated is verified, and the effect is possibly generated by inhibiting a cAMP / PKA / CREB signal channel, so that the effect is obvious. And thinking and theoretical basis are provided for research and development of analgesic drugs for targeting the spinal cord GPR88 receptor.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of an orphan receptor GPR88 as a target in the preparation of a drug for treating neuropathic pain. Background Art

[0002] The International Association for the Study of Pain (IASP) defines chronic pain as pain that persists or recurs for more than three months. Chronic pain has no clear physiological significance and often persists after the disease is cured. It is the third largest health problem in the world after cardiovascular and cerebrovascular diseases and tumors, affecting nearly one-third of the global population. Neuropathic pain (NP) is the most common chronic pain, affecting approximately 500 million to 700 million people worldwide, of which my country currently has 90 million NP patients. Currently, conventional treatments for NP, such as surgery, nerve blocks, and opioid analgesics, are less than ideal and are accompanied by varying degrees of adverse reactions. Therefore, promoting the research of new treatment methods for NP and exploring new drug targets have important clinical significance and social value.

[0003] At the spinal cord level, targeting GPCRs has great potential in finding new targets for pain treatment.

[0004] G protein-coupled receptors (GPCRs) are the largest family of cell membrane receptors in mammals, with over 800 different types of GPCRs discovered in humans. Their widespread distribution throughout the peripheral and central nervous systems makes them an important new target for pain treatment and a key area of drug development. As of 2023, there will be approximately 500 GPCR-targeted drugs in the global pipeline, accounting for 34% of FDA-approved drugs.

[0005] Orphan G protein-coupled receptors (oGPCRs), also known as orphan receptors, are GPCRs whose endogenous ligands are unknown and whose functions remain incompletely elucidated. In recent years, a growing number of studies have demonstrated the significant research value and potential of oGPCRs in pain research. GPR88, a class A GPCR, is known to be specifically expressed in the brain, particularly the striatum. Studies have shown that the GPR88 receptor can modulate GABA and glutamate signaling pathways and regulate the activity of other GPCRs, such as dopamine and opioid receptors. Transcriptional analysis and mouse knockout studies have demonstrated that GPR88 plays an important role in regulating brain function and behavior, such as cognition, emotion, reward learning, and motor control. However, whether GPR88 is involved in the regulation of neuropathic pain remains unclear, and no reports on this topic have been reported. Summary of the Invention

[0006] The purpose of the present invention is to provide an orphan receptor GPR88 as a target for the development or preparation of drugs for treating neuropathic pain, so as to overcome the shortcomings of the existing technology.

[0007] Another object of the present invention is to provide a pharmaceutical composition for treating neuropathic pain, which comprises an agonist of the orphan receptor GPR88 or its encoding gene, and a pharmaceutically acceptable carrier.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The use of orphan receptor GPR88 as a target in the development or preparation of drugs for the treatment of neuropathic pain.

[0010] The drug for treating neuropathic pain can at least increase the mechanical pain threshold of normal mice.

[0011] The drug for treating neuropathic pain can at least increase the thermal pain threshold of normal mice.

[0012] The drug for treating neuropathic pain can alleviate the neuropathic pain in mice induced by SNI.

[0013] The neuropathic pain drug relieves neuropathic pain by inhibiting the cAMP / PKA / CREB signaling pathway.

[0014] The drug for treating neuropathic pain is a drug that activates GPR88 receptors at the spinal cord level.

[0015] A pharmaceutical composition for treating neuropathic pain, comprising an agonist of the orphan receptor GPR88 or its encoding gene, and a pharmaceutically acceptable carrier.

[0016] The agonist is selected from one of 2-PCCA, RTI-13951-33 or BI-9805 or a mixture of two or more thereof.

[0017] The dose of the agonist is 10-100 nmol.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. This invention proposes for the first time that the orphan receptor GPR88 is a target for drugs treating neuropathic pain. It also demonstrates the expression of the orphan receptor GPR88 at the spinal cord level. By intrathecally administering a GPR88 receptor agonist and observing pain behavior in mice, it confirms a clear analgesic effect after activating the GPR88 receptor, likely through inhibition of the cAMP / PKA / CREB signaling pathway.

[0020] 2. Experiments have shown that activation of the GPR88 receptor significantly inhibits the cAMP / PKA / CREB signaling pathway, increasing mechanical and thermal allodynia in normal mice. Knocking down GPR88 gene expression at the spinal cord level significantly lowers the basal pain threshold in normal mice. These results suggest that activation of the GPR88 receptor has a significant alleviating effect on neuropathic pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the distribution of GPR88 receptors in the spinal cord;

[0022] Figure 2 Schematic diagram of the analgesic effect of GPR88 receptor agonist 2-PCCA on normal mice;

[0023] Figure 3 Schematic diagram of the analgesic effect of GPR88 receptor agonist 2-PCCA on SNI neuropathic pain mice;

[0024] Figure 4 Schematic diagram of the expression changes of gpr88 mRNA in the SNI model;

[0025] Figure 5 Schematic diagram of behavioral changes in mice after intrathecal injection of siRNA to knock down the gpr88 gene;

[0026] Figure 6 Schematic diagram of the effect of activating the spinal cord GPR88 receptor on the cAMP / PKA / CREB signaling pathway. DETAILED DESCRIPTION

[0027] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0028] The term "comprising" and other equivalent descriptions in the description and claims of this application are intended to cover non-exclusive inclusions, including both the contents clearly described in the description and claims and the steps or units that are not described in the description and claims but are inherent in the product, method or structure.

[0029] Specifically, this embodiment provides the use of the orphan receptor GPR88 as a target in the development or preparation of drugs for treating neuropathic pain.

[0030] As an optional embodiment, the drug for treating neuropathic pain can at least increase the mechanical pain threshold of normal mice.

[0031] As an optional embodiment, the drug for treating neuropathic pain can at least increase the thermal pain threshold of normal mice.

[0032] As an optional embodiment, the drug for treating neuropathic pain can alleviate neuropathic pain in mice induced by SNI.

[0033] As an optional embodiment, the neuropathic pain drug relieves neuropathic pain by inhibiting the cAMP / PKA / CREB signaling pathway.

[0034] As an optional embodiment, the drug for treating neuropathic pain is a drug that activates the orphan receptor GPR88 at the spinal cord level.

[0035] This embodiment also provides a pharmaceutical composition for treating neuropathic pain, which includes an agonist of the orphan receptor GPR88 or its encoding gene, and a pharmaceutically acceptable carrier.

[0036] As an optional embodiment, the agonist is selected from one of 2-PCCA, RTI-13951-33 or BI-9805, or a mixture of two or more thereof.

[0037] As an optional embodiment, the dosage of the agonist is 10-100 nmol.

[0038] As a preferred embodiment, the agonist is 2-PCCA, and the dosage is 10-100 nmol.

[0039] This example first investigated the distribution of the GPR88 receptor in the spinal cord. By analyzing human spatial transcriptome data, we observed that the GPR88 receptor is concentrated in the dorsal horn of the human spinal cord. Furthermore, we used RNAscope technology to further verify the distribution of the GPR88 receptor in the mouse spinal cord. This also confirmed that the receptor is specifically highly expressed in the dorsal horn of the mouse spinal cord.

[0040] To further investigate the function of GPR88 receptors at the spinal cord level, the GPR88 receptor-specific agonist 2-PCCA was injected intrathecally. The pain behavior of normal mice and mice with neuropathic pain (SNI model) was examined after activation of the GPR88 receptor at the spinal cord level. Mechanical allodynia in mice with neuropathic pain was examined using the Von-Frey test, while thermal allodynia was examined using the hot plate method. The results showed that intrathecal injection of 2-PCCA (10 nmol and 0.1 μmol it) alleviated SNI-induced mechanical and thermal allodynia. Furthermore, intrathecal injection of transfected RNA (siRNA) knocked down the expression of the gpr88 gene at the spinal cord level. The results suggested that knocking down the expression of the spinal GPR88 receptor significantly decreased the basal pain threshold of normal mice.

[0041] This example further validated the effect of spinal GPR88 receptor activation on the cAMP / PKA / CREB signaling pathway. Western blot and qPCR experiments were performed to investigate changes in the cAMP / PKA / CREB pathway following 2-PCCA activation of the spinal cord GPR88 receptor. The results showed that intrathecal 2-PCCA injection significantly inhibited the cAMP / PKA / CREB signaling pathway. These results further reveal the regulatory mechanism of the GPR88 receptor in neuropathic pain.

[0042] The above results show that activating the GPR88 receptor at the spinal cord level can clearly alleviate SNI-induced pain behavior in mice and inhibit the cAMP / PKA / CREB signaling pathway at the spinal cord level.

[0043] The relevant experimental results and analysis of this embodiment are as follows:

[0044] Example 1: Distribution characteristics of orphan receptor GPR88 in the spinal cord

[0045] 1. RNAscope (fluorescence in situ hybridization) experiment

[0046] Mice were anesthetized by intraperitoneal injection of 0.4% sodium pentobarbital (50 mg / kg) and fixed by transcardial perfusion with 4% paraformaldehyde. Thoracic opening was performed and PBS was perfused through the left ventricle to flush out blood. Following tissue pre-fixation with 4% paraformaldehyde (PFA), spinal cord tissue from lumbar vertebrae L4-L6 was removed. Tissues were post-fixed with 4% paraformaldehyde at 4°C for 24 hours and then dehydrated with 20% and 30% sucrose for 24 hours, respectively, until sedimented. After dehydration, tissues were embedded in OCT and sectioned at 20 μm using a cryostat. After elution of OCT, tissues were dehydrated with gradient ethanol and treated with hydrogen peroxide and protease III. Gene probes were labeled according to the Advanced Cell Diagnostics protocol. Finally, fluorescence development was performed using a PerkinElmer fluorescence development kit (NEL760001KT), and images were obtained using a laser confocal microscope.

[0047] 2. Experimental Results

[0048] Human spatial transcriptome data were analyzed and the distribution characteristics of GPR88 in the mouse spinal cord were observed. Figure 1 As shown in A, GPR88 is concentrated in the dorsal horn of the spinal cord. In addition, the distribution characteristics of GPR88 receptors were further verified at the spinal cord level using RNAscope technology. Figure 1 As shown in Figures B and C, gpr88 mRNA (red fluorescence) is specifically and highly expressed in the dorsal horn of the spinal cord of mice. Given the important role of the dorsal horn in the ascending pain pathway and the distribution characteristics of the GPR88 receptor, these results provide important evidence for further research into the function of the GPR88 receptor.

[0049] Example 2: Effects of Activating Spinal GPR88 Receptors on Behavior of Normal Mice

[0050] 1. Animal Grouping and Dosing

[0051] Male C57 mice were randomly divided into three groups (n=8): a control group (saline), a low-dose 2-PCCA group (10 nmol), and a high-dose 2-PCCA group (0.1 μmol). After intrathecal injection, the pain behavior of the mice was observed using the Von Frey test at 10, 30, 60, and 120 minutes, respectively.

[0052] Male C57 mice were randomly divided into four groups (n=8): a control group (saline), a naloxone group (Nalo), a 2-PCCA group, and a naloxone + 2-PCCA group (Nalo + 2-PCCA). Thirty minutes after administration, the pain behavior of the mice was observed using the Von Frey test.

[0053] 2. VonFrey Experiment

[0054] The animals were first placed in a transparent box with a wire mesh bottom. Mice were acclimated to the test environment for 30 minutes before the test began. Mice withdrew their feet in response to mechanical stimulation from a needle prick, and those that were overly sensitive or insensitive to mechanical stimulation were removed. Mice were placed on a wide-gauge wire mesh surface, and Von Frey filaments were applied from the underside of the mesh to the plantar surface of the mouse's hind paw. The mechanical pain threshold of the mice was measured using the up-and-down method.

[0055] 3. Experimental Results

[0056] like Figure 2 As shown in A, 30 minutes after 2-PCCA administration, the mechanical pain threshold of mice in the 2-PCCA (10 nmol and 0.1 μmol) groups was significantly increased compared to the saline group (P < 0.01), and there was a certain dose-dependency. This analgesic effect can last up to 60 minutes after administration. It has been reported that GPR88 receptors have a certain regulatory effect on opioid receptors. In order to verify whether the analgesic effect of 2-PCCA is mediated by opioid receptors at the spinal cord level, the opioid receptor agonist naloxone was administered intrathecally. Figure 2 As shown in Figure B, the analgesic effect of 2-PCCA was not reversed. The above research results suggest that using 2-PCCA to activate spinal GPR88 receptors has a clear analgesic effect, and this effect is independent of opioid receptors.

[0057] Example 3: Effects of Activating Spinal GPR88 Receptors on the Behavior of Mice with SNI Neuropathic Pain I. Animal Grouping and Modeling

[0058] Male C57 mice were randomly divided into two groups (n=8): a control group (saline) and a 2-PCCA administration group (0.1 μmol). After intrathecal injection, the pain behavior of the mice was observed using the Von Frey test before modeling, after modeling, and at 0.5, 1, and 2 hours after administration.

[0059] Female C57 mice were randomly divided into two groups (n=8): a control group (saline) and a 2-PCCA administration group (0.1 μmol). After intrathecal injection, the pain behavior of the mice was observed using the Von Frey test before modeling, after modeling, and 0.5, 1, and 2 hours after administration.

[0060] SNI model construction: Adult C57BL / 6J mice (20-30 g, 8 weeks) were anesthetized and fixed in a prone position. After disinfection, the skin was incised at the upper edge of the hind limb. The biceps femoris was separated, and the sciatic nerve trunk and tibial, common peroneal, and sural nerves were exposed. The tibial and common peroneal nerves were ligated with 5-0 silk sutures and then transected distally, preserving and avoiding injury to the sural nerve. The muscles and skin were then sutured layer by layer.

[0061] 2. Experimental Methods

[0062] The Von Frey test method was the same as in Example 2. Hot plate test: The hot plate temperature was set at 52 ± 0.5°C, and the cold plate temperature was set at 4 ± 0.5°C. The mouse was placed on the hot / cold plate. Timing was started from the moment the mouse was placed on the hot plate until it licked its hind paw or jumped. The pain threshold (s) of the mouse was recorded.

[0063] 3. Experimental Results

[0064] The effects of intrathecal injection of GPR88-specific agonist 2-PCCA on pain behavior in mice with sciatic nerve injury (SNI) model were observed. Figure 3 As shown in Figure 3, intrathecal administration of 2-PCCA significantly alleviated SNI-induced mechanical and thermal hyperalgesia in mice. These results indicate that activation of GPR88 receptors at the spinal cord level can effectively alleviate SNI-induced neuropathic pain.

[0065] Example 4: Changes in the expression of spinal GPR88 receptors in SNI neuropathic pain mice

[0066] 1. Animal grouping and modeling are the same as in Example 3

[0067] 2. Experimental Methods

[0068] The RNAscope (fluorescence in situ hybridization technique) experiment was the same as in Example 1.

[0069] 3. Experimental Results

[0070] Given the analgesic effect of activating the spinal GPR88 receptor on SNI model mice, and the significant downregulation of gpr88 mRNA in SNI model mice as seen in qPCR results, RNAscope technology was further used to verify the expression changes of gpr88 mRNA in spinal Tac1-positive neurons under the SNI model. Figure 4 As shown in the results, compared with the sham group, the expression of gpr88 mRNA in Tac1-positive neurons in the dorsal horn of the spinal cord of SNI mice was significantly downregulated (P < 0.05). These results further clarified the expression of GPR88 receptors in the SNI model.

[0071] Example 5: Effects of knocking down the gpr88 gene on mouse behavior

[0072] 1. Animal Grouping and Dosing

[0073] Male C57 mice were randomly divided into two groups (n=6): a control group (siRNA-sham) and a gpr88 knockdown group (siRNA-gpr88). Following intrathecal injection of RNA, pain behavior was assessed using the Von Frey test on days 5, 5, and 7. Following the behavioral experiment, spinal cord tissue was obtained and Western blot analysis was performed to examine GPR88 protein expression in the spinal cord.

[0074] 2. Experimental Methods

[0075] 1. Spinal / Intrathecal Viral Injection

[0076] After rapid anesthesia with isoflurane, the mouse is placed on the operating table and its mouth and nose are placed in the hose of the anesthesia system to maintain anesthesia. Chlortetracycline ointment is applied to the mouse's eyes to protect them. Firmly grasp the rodent's iliac crest (hip bone) with your thumb and index finger. Trace the spine from the lumbar vertebrae to the sacral vertebrae. Gently press down on the spine from the lumbar to the sacrum while very slightly pulling on the spine. This opens the space between the vertebrae to facilitate needle insertion. Insert the needle of the microsyringe at a 70-degree angle into the lumbar vertebrae at the midline of the spine. When you feel the needle hit the bone, lower the angle of the needle to approximately 30 degrees and then insert the needle into the rodent's lumbar vertebrae. Observe the mouse's reflexive tail flick. Depress the plunger of the syringe and inject 10 μl of injection solution into the subarachnoid space. Wait a moment after the virus injection, then remove the syringe from the vertebral body and wipe the skin with an alcohol cotton ball.

[0077] 2. Western blot experiment

[0078] After anesthetizing mice, fresh spinal cord tissue was collected and placed in a 1.5 mL centrifuge tube with 150 μL of strong RIPA. The tissue was disrupted by sonication and allowed to rest on ice for 30 minutes. The tissue was then centrifuged (13,000 rpm) at 4°C for 15 minutes. The supernatant was transferred to a fresh, labeled 1.5 mL EP tube. Tissue protein was quantified using BCA assay, with 10 μL added to each well. Electrophoresis was performed at a constant voltage of 90 V for 120 minutes. A PVDF membrane was cut to the same size as the separating gel and soaked in methanol for 30 seconds to fully activate it. Transfer was performed at a constant current of 350 mA for 120 minutes. The membrane was blocked with 5% skim milk for 1 hour at room temperature or overnight at 4°C, followed by three 5-minute rinses in TBST. Incubate with the primary antibody GPR88 (1:200) at room temperature for 1 hour, or overnight at 4°C, followed by three 5-minute rinses in TBST. Incubate with the secondary antibody (1:2000) at room temperature for 1 hour, followed by three 5-minute rinses in TBST. Add an appropriate amount of developer solution based on the membrane size, allow to react for approximately 30 seconds, and then allow the membrane to stand for exposure. Grayscale analysis of the exposure results was performed using Image J software.

[0079] 3. Experimental Results

[0080] like Figure 5 As shown in A, 7 days after intrathecal siRNA injection, the expression of GPR88 receptor protein at the spinal cord level of mice was significantly downregulated (P<0.05). Before siRNA injection and on days 3, 5, and 7 after injection, the mechanical and thermal allodynia of mice were tested, as shown in Figure 5 As shown in Figures C and D, after intrathecal siRNA injection to knock down GPR88 receptor expression, the mechanical and thermal pain thresholds of mice decreased significantly compared to the blank control group, particularly on day 7 (P < 0.001). These results suggest that the GPR88 receptor in the spinal cord of normal mice has a pain-regulating function, and knocking down its expression significantly reduces the mice's basal pain threshold.

[0081] Example 6: Effect of activating the spinal cord GPR88 receptor on the cAMP / PKA / CREB signaling pathway.

[0082] 1. Animal Grouping and Dosing

[0083] Male C57 mice were randomly divided into three groups (n=6): a saline control group, a SNI model group, and an SNI model + 2-PCCA (0.1 μmol) group. 2-PCCA was administered intrathecally for four consecutive days starting on day 3 of SNI model induction. On day 7 after SNI induction (experimental day 7), spinal cord tissue was obtained from each group and cAMP / PKA / CREB protein and gene expression was measured.

[0084] Experimental methods

[0085] 1. Western blot experimental method is the same as in Example 5

[0086] 3. Experimental Results

[0087] like Figure 6 As shown in A, compared with the SNI model group, after intrathecal injection of 2-PCCA to activate GPR88 receptor, the phosphorylated PKA and CREB proteins at the spinal cord level were significantly downregulated. Figure 6 As shown in Figures DF, qPCR results show that activation of the GPR88 receptor significantly downregulated the expression of cAMP, p-PKA, and p-CREB mRNA in the spinal cord. This suggests that activation of the GPR88 receptor has a certain inhibitory effect on the cAMP / PKA / CREB signaling pathway. These experimental results further reveal the regulatory mechanism of the GPR88 receptor in neuropathic pain.

[0088] This study clarified the distribution of GPR88 receptors in the spinal cord and their expression changes in a pain model, examining the analgesic effects and functions of GPR88 receptor activation and the effects of spinal GPR88 receptor activation on the cAMP / PKA / CREB signaling pathway. The results of this study provide insights and theoretical basis for the development of analgesic drugs targeting spinal GPR88 receptors.

[0089] Based on the disclosure and teachings of the above description, those skilled in the art may also make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and modifications and variations of the present invention should also fall within the scope of protection of the claims of the present invention. In addition, although certain specific terms are used in this description, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims

1. Application of the orphan receptor GPR88 as a target in the development or preparation of drugs for the treatment of neuropathic pain.

2. The use according to claim 1, characterized in that: The drug for treating neuropathic pain can at least increase the mechanical pain threshold of normal mice.

3. The use according to claim 1, characterized in that: The drug for treating neuropathic pain can at least increase the thermal pain threshold of normal mice.

4. The use according to claim 1, characterized in that: The drug for treating neuropathic pain can alleviate the neuropathic pain in mice induced by SNI.

5. The use according to claim 4, characterized in that: The neuropathic pain drug relieves neuropathic pain by inhibiting the cAMP / PKA / CREB signaling pathway.

6. The use according to claim 1, characterized in that: The drug for treating neuropathic pain is a drug that activates GPR88 receptors at the spinal cord level.

7. A pharmaceutical composition for treating neuropathic pain, characterized in that: The invention comprises an agonist of the orphan receptor GPR88 or its encoding gene, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition for treating neuropathic pain according to claim 7, characterized in that: The agonist is selected from one of 2-PCCA, RTI-13951-33 or BI-9805 or a mixture of two or more thereof.

9. The pharmaceutical composition for treating neuropathic pain according to claim 8, characterized in that: The dose of the agonist is 10-100 nmol.

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