Preparation of hepcidin-targeting biological agent and application of hepcidin-targeting biological agent in relieving neuropathic pain

Abstract

The invention belongs to the field of biological medicine, and provides preparation of a targeting hepcidin biological agent and application of the targeting hepcidin biological agent in relieving neuropathic pain. According to the invention, a new analgesic target, namely, Hamp, in the spinal cord is found for the first time, and the Hamp is abnormally highly expressed in the spinal cord of a pain model rat. A small interfering RNA (siRNA) targeting Hamp is designed, when the substance is applied to the spinal cord, cell ferroptosis and neuroinflammation in the spinal cord of a rat can be remarkably inhibited, and the substance has a remarkable analgesic effect on mechanical pain sensitivity and crymodynia sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, relates to the discovery of a new analgesic target in the spinal cord and its functional verification, and prepares a biological preparation based on the target for the treatment of neuropathic pain. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] NP (Neuropathic pain) is pain caused or resulted from primary damage or dysfunction of the nervous system, manifested as persistent spontaneous pain, ectopic pain or hyperalgesia. It is one of the most common clinical diseases, lasts for a long time and is difficult to cure, seriously affecting the quality of life of patients. The pathogenesis of NP is complex, involving multiple aspects of the peripheral and central nervous systems, and has not yet been fully elucidated. In recent years, a large number of studies have found that spinal cord cell dysfunction is the main cause of the occurrence and persistence of NP. Currently, additional pain therapies are needed worldwide, and neuropathic pain has developed into a major health problem for the general population.

[0004] Small interfering RNA has strict sequence specificity, high efficiency and biological inheritance. It can specifically and efficiently block the expression of homologous genes in vivo, promote the degradation of homologous mRNA, and induce cells to show a phenotype of specific gene deletion. It is simple to operate and has high specificity. At present, biological agents with analgesic effects have been reported, for example: previously (The transcription factor C / EBPβin the dorsal root ganglion contributes to peripheral nerve trauma-induced nociceptive hypersensitivity, DOI:10.1126 / scisignal.aam5345, published on July 11, 2017) disclosed that the transcription factor C / EBPβ contributes to the nociceptive hypersensitivity induced by peripheral nerve trauma, and si-Cebpb (catalog number SC-29862) solution can block the increase of C / EBPβ in the ipsilateral DRG induced by CCI in mice and reduce CCI-induced mechanical allodynia, thermal hyperalgesia and cold allodynia, but does not affect the basal response to acute pain and motor activity, thereby treating neuropathic pain. However, the pathogenesis of neuropathic pain is still unclear and the clinical application of analgesics is still difficult. Summary of the invention

[0005] In view of the current unclear pathogenesis of neuropathic pain and the difficulties in the clinical application of analgesics, the present invention provides a biological preparation targeting hepcidin and its application in relieving neuropathic pain. The present invention discovered for the first time a new analgesic target in the spinal cord, Hamp, which is abnormally highly expressed in the spinal cord of pain model rats. A small interfering RNA (siRNA) targeting Hamp was designed. The spinal administration of this substance can significantly inhibit cell ferroptosis and neuroinflammation in the rat spinal cord, and has a significant analgesic effect on mechanical allodynia and cold allodynia.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a use of a reagent targeting a biomarker in the preparation of a drug for relieving / treating neuropathic pain, wherein the biomarker is Hamp.

[0008] In some embodiments, the agent targeting a biomarker comprises an inhibitor of Hamp.

[0009] In some embodiments, the Hamp inhibitor comprises siRNA of the Hamp gene.

[0010] In some embodiments, the siRNA comprises si-Hamp;

[0011] The nucleic acid sequences of the sense strand and antisense strand of the si-Hamp are shown in SEQ ID NO.1 and SEQ ID NO.2:

[0012] GCAUAACAUAGAGAGCCAATT SEQ ID NO.1,

[0013] UUGGCUCUCUAUGUUAUGCTT SEQ ID NO.2.

[0014] In some embodiments, the dosage form of the agent includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.

[0015] The second aspect of the present invention provides a siRNA for inhibiting the expression of Hamp gene, wherein the siRNA comprises si-Hamp;

[0016] The nucleic acid sequences of the sense strand and antisense strand of the si-Hamp are shown in SEQ ID NO.1 and SEQ ID NO.2:

[0017] GCAUAACAUAGAGAGCCAATT SEQ ID NO.1,

[0018] UUGGCUCUCUAUGUUAUGCTT SEQ ID NO.2.

[0019] The third aspect of the present invention provides a pharmaceutical composition for relieving / treating neuropathic pain, comprising: the above-mentioned siRNA solution and a pharmaceutically acceptable carrier.

[0020] In some embodiments, the concentration of the siRNA solution is 20-80 μM.

[0021] In some embodiments, the volume ratio of the siRNA solution to the pharmaceutically acceptable carrier is 1:0.5-1.5, or 1:1.

[0022] More specifically, the ratio of the siRNA to the pharmaceutically acceptable carrier is: 20 μM siRNA solution (μl) to carrier solution (μl), the volume ratio is 1: 1. If the concentration of the siRNA is 40 μM, the volume of the siRNA should be halved; if the concentration of the siRNA is 80 μM, the volume of the siRNA should be divided by 4, and so on.

[0023] In some embodiments, the pharmaceutically acceptable carrier comprises at least one of a liposome, a cationic polymer, and a nanomaterial.

[0024] Beneficial effects of the present invention

[0025] (1) The present invention discovered for the first time a new analgesic target in the spinal cord, Hamp, which is abnormally highly expressed in the spinal cord of pain model rats. A small interfering RNA (siRNA) targeting Hamp was designed. Spinal administration of this substance can significantly inhibit cell ferroptosis and neuroinflammation in the rat spinal cord, and has a significant analgesic effect on mechanical allodynia and cold allodynia.

[0026] (2) Since there are no reports on Hamp and pain, this phenomenon / biological pathway is discovered for the first time by the present invention, and therefore, no hepcidin inhibitor has been used to relieve pain. Therefore, the present invention provides a biomarker-targeted reagent for use in the preparation of a drug for relieving / treating neuropathic pain. The biomarker-targeted reagent (si-Hamp) knocks down the expression of the target gene Hamp, significantly inhibits spinal cord cell ferroptosis and neuroinflammation, thereby exerting its efficacy in relieving / treating neuropathic pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] Figure 1 Experimental analysis of intrathecal injection of si-Hamp significantly improved the hyperalgesia of CCI model rats to mechanical and cold stimulation. (A) Experimental flow chart; (B) Intrathecal injection of si-Hamp significantly knocked down the expression level of Hamp; Mechanical pain threshold (C) and the number of responses to cold stimulation (D) of rats in each group at different time points after modeling and intrathecal injection. (Data are expressed as mean ± standard error, ** , *** Compared with the Sham group, P < 0.01 and P < 0.001, respectively; # , ## , ### Compared with the CCI+si-NC group, P<0.05, P<0.01, P<0.001).

[0029] Figure 2 The abnormally high expression of Hamp gene in the spinal cord of pain model animals, and the correlation analysis between its expression level and pain intensity. (A) Mechanical paw withdrawal threshold of rats in Sham and CCI groups at different time points after surgery. (B, C) Hamp mRNA levels in the modeling side and contralateral spinal cord of rats in Sham and CCI groups at different time points after surgery. (D) Correlation analysis results of Hamp-mRNA levels in the spinal cord of the modeling side of CCI rats at various time points and mechanical paw withdrawal threshold. (The data shown are mean ± standard error, **, *** indicate P<0.01 and P<0.001 compared with the Sham group; ### indicates P<0.001 compared with CCI-1days; ΔΔΔ indicates P<0.001 compared with CCI-3days). DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0031] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0032] Example 1

[0033] (1) synthesizing si-Hamp according to the sequence list;

[0034] (2) Mix 10 μl of si-Hamp (20 μM) with 10 μl of in vivo transfection reagent and let stand at room temperature for 15 min to form a stable complex. Then, inject 20 μl of the mixture intrathecally into the rat for 3 consecutive days ( Figure 1 A), the experimental animals were divided into 5 groups (Sham, CCI, CCI+si-NC, Sham+si-Hamp, CCI+si-Hamp). Behavioral test results showed that si-Hamp effectively alleviated the mechanical and cold allodynia of the model rats ( Figure 1 In C and D), the response to cold stimulation, in particular, returned to the same level as the sham-operated group.

[0035] The present invention first discovered that Hamp was significantly overexpressed in the spinal cord of CCI model rats ( Figure 2 B), while the expression of Hamp in the control spinal cord did not change significantly at any time point before and after modeling ( Figure 2 C). Pearson correlation analysis showed that the expression level of Hamp in the spinal cord of the modeling side was significantly negatively correlated with the mechanical pain threshold ( Figure 2 Middle D) suggests that there is a correlation between Hamp and neuropathic pain.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a reagent targeting a biomarker in the preparation of a drug for relieving / treating neuropathic pain, characterized in that: The biomarker is Hamp.

2. The use according to claim 1, characterized in that The biomarker-targeting agent includes an inhibitor of Hamp.

3. The use according to claim 1, characterized in that The Hamp inhibitor includes siRNA of Hamp gene.

4. The use according to claim 1, characterized in that The siRNA includes si-Hamp; The nucleic acid sequences of the sense strand and antisense strand of the si-Hamp are shown in SEQ ID NO.1 and SEQ ID NO.2: GCAUAACAUAGAGAGCCAATT SEQ ID NO.1, UUGGCUCUCUAUGUUAUGCTT SEQ ID NO.

2.

5. The use according to claim 1, characterized in that The dosage form of the reagent includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.

6. A siRNA for inhibiting Hamp gene expression, characterized in that: The siRNA includes si-Hamp; The nucleic acid sequences of the sense strand and antisense strand of the si-Hamp are shown in SEQ ID NO.1 and SEQ ID NO.2: GCAUAACAUAGAGAGCCAATT SEQ ID NO.1, UUGGCUCUCUAUGUUAUGCTT SEQ ID NO.

2.

7. A pharmaceutical composition for relieving / treating neuropathic pain, characterized in that: include: The siRNA solution according to claim 6 and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition for relieving / treating neuropathic pain according to claim 7, characterized in that: The concentration of the siRNA solution is 20-80 μM.

9. The pharmaceutical composition for relieving / treating neuropathic pain according to claim 7, characterized in that: The volume ratio of the siRNA solution to the pharmaceutically acceptable carrier is 1:0.5-1.5, or 1:

1.

10. The pharmaceutical composition for relieving / treating neuropathic pain according to claim 7, characterized in that: The pharmaceutically acceptable carrier includes at least one of liposomes, cationic polymers and nanomaterials.

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