Application of Ryk-Fc fusion protein in preparation of anti-cancer pain drugs

Abstract

The invention discloses application of Ryk-Fc fusion protein in preparation of anti-cancer pain drugs, and belongs to the technical field of biological medicines. The invention provides a new application of the Ryk-Fc fusion protein, the new application is an application of the Ryk-Fc fusion protein in preparation of drugs for treating cancer pain, the Ryk-Fc fusion protein realizes a function of treating cancer pain by inhibiting expression of TRPV1 protein in dorsal root node neurons, and the Ryk-Fc fusion protein has strong pertinence in a treatment process, has a remarkable treatment effect, and can be used for preparing drugs for treating cancer pain. Side effects are low. The invention provides a theoretical basis for research and development of drugs for treating cancer pain, and provides a feasible scheme for preparation of novel drugs for treating cancer pain.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of Ryk-Fc fusion protein in the preparation of anti-cancer pain medicine. Background Art

[0002] Cancer pain is a chronic pain that is difficult to cure. The pain symptoms are severe and accompany the patient throughout life. It is one of the serious diseases that long-term threatens human health. With the increase in the incidence of cancer worldwide and the worsening trend of cancer chronicity, the medical demand for the treatment of cancer pain and the related financial burden are also increasing. It is reported that about 60% to 90% of patients with advanced cancer suffer from varying degrees of pain, of which about 30% suffer from persistent, severe and unrelieved pain. The prevention and treatment of cancer pain is a thorny issue in clinical pain management and a medical need that needs to be urgently met.

[0003] There is no reliable specific drug in clinical practice. At present, the drugs used to treat cancer pain mainly include non-steroidal anti-inflammatory drugs, opioids and bisphosphonates. These drugs are not effective in treating moderate and severe cancer pain, cannot fully relieve pain, and have adverse reactions and side effects such as safety, addiction and drug tolerance. The "three-step therapy" recommended by the World Health Organization is the most influential treatment guideline for cancer pain, but there are still 10% to 20% of cancer pain patients who cannot effectively control pain through this therapy. Therefore, it is crucial to provide new drugs for the treatment of cancer pain. Summary of the invention

[0004] The purpose of the present invention is to provide the use of Ryk-Fc fusion protein in the preparation of anti-cancer pain drugs to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is the use of Ryk-Fc fusion protein in the preparation of anti-cancer pain drugs.

[0007] The second technical solution of the present invention is an anti-cancer pain drug, including Ryk-Fc fusion protein.

[0008] The third technical solution of the present invention is a pharmaceutical composition, comprising Ryk-Fc fusion protein.

[0009] Based on the above technical solution, the present invention has the following technical effects:

[0010] The present invention provides a new application of Ryk-Fc fusion protein, which is the application of Ryk-Fc fusion protein in the preparation of a drug for treating cancer pain. The Ryk-Fc fusion protein achieves the function of treating cancer pain by inhibiting the expression of TRPV1 protein in dorsal root ganglion neurons, and has strong pertinence in the treatment process, significant treatment effect, and low side effects. The present invention provides a theoretical basis for the research and development of therapeutic drugs for cancer pain and provides a feasible solution for the preparation of a new drug for treating cancer pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0012] Figure 1 A single administration of Ryk-Fc can inhibit the mechanical pain (A) and thermal pain behaviors (B) induced by TCI model.

[0013] Figure 2 A single administration of Ryk-Fc can inhibit the spontaneous pain behavior caused by TCI modeling. Where A is the number of spontaneous paw withdrawals and B is the lameness score.

[0014] Figure 3 Multiple administrations of Ryk-Fc can inhibit pain behaviors caused by TCI modeling. Where A is the mechanical withdrawal threshold and B is the thermal withdrawal threshold.

[0015] Figure 4 The expression level of Ryk is significantly increased in the dorsal root ganglion of rats with bone cancer pain. A is an example of Ryk protein expression in the dorsal root ganglion, B is the expression statistics of Ryk protein in the FL segment, C is the expression statistics of Ryk protein in the CTF segment, and D is the expression statistics of Ryk protein in the ICD segment.

[0016] Figure 5 Intrathecal injection of Ryk-Fc fusion protein can regulate the protein expression level of TRPV1 in dorsal root ganglion neurons of rats with bone cancer pain. A is an example of TRPV1 protein expression in dorsal root ganglia, and B is the statistical expression of TRPV1 protein. DETAILED DESCRIPTION

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0020] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0021] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0022] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0023] The embodiment of the present invention provides the use of Ryk-Fc fusion protein in the preparation of anti-cancer pain drugs.

[0024] In some specific embodiments, the cancer pain includes moderate to severe pain caused by tumor compression or tumor infiltration.

[0025] In some specific embodiments, the moderate to severe pain manifests as allodynia, hyperalgesia or spontaneous pain.

[0026] The embodiment of the present invention also provides an anti-cancer pain drug, including Ryk-Fc fusion protein.

[0027] In some specific embodiments, a pharmaceutically acceptable excipient is also included.

[0028] In some specific embodiments, the drug is in the form of tablets, powders, granules, capsules, injections, sprays, films, suppositories, nasal drops or pills.

[0029] In some specific embodiments, the drug is administered by intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

[0030] An embodiment of the present invention further provides a pharmaceutical composition, comprising a Ryk-Fc fusion protein.

[0031] The present invention discovers for the first time that Ryk-Fc fusion protein has the effect of relieving cancer pain and can be used to prepare drugs for treating cancer pain. Ryk-Fc fusion protein achieves the above function by inhibiting the expression of TRPV1 in dorsal root ganglion neurons, wherein TRPV1 is a non-selective cation channel, which is abundantly expressed in peripheral nociceptors and plays an important role in the peripheral sensitization of bone cancer pain. Nerve damage caused by tumor compression or infiltration promotes the expression of TRPV1 in dorsal root ganglion neurons, causing peripheral sensitization and pain. Therefore, Ryk-Fc fusion protein has the potential to prepare drugs for treating cancer pain, and has targeted treatment of pain with significant efficacy.

[0032] Example 1

[0033] 1TCI Modeling

[0034] TCI modeling, tumor cells were transplanted into the tibia to induce bone cancer pain, producing cancer-related thermal hyperalgesia, mechanical allodynia, and spontaneous and movement-induced pain behaviors. Tumor cells were extracted from the ascites of rats that had previously received Walker256 breast cancer cells. Tumor cells (1×10 5 Bone cancer pain (BCP) was induced in rats by injecting 5 mL of 5 mL of 1% tibial medullary fluid (50 mL / mL, 5 mL) into the left tibial medullary cavity. Briefly, rats were anesthetized with isoflurane (1.2%), and a superficial incision of approximately 1 cm long was made near the knee to expose the patellar ligament. A 23-gauge needle was inserted into the tibial cavity, and the needle was connected to a 25 mL microsyringe containing 5 mL of air, 5 mL of tumor cell suspension (5 × 10 5 ) and 5 ml of air. The contents of the syringe were slowly injected into the tibial cavity and the needle was kept for 2 minutes to allow the cells to spread. The needle hole was then sealed with bone wax when the needle was removed. The bone surface of the injection site was sealed with dental cement to prevent tumor cell leakage. The injection of boiled tumor cells served as a sham control.

[0035] Pain-like behaviors, including spontaneous pain, pain induced by mechanical and thermal stimuli, were assessed in TCI- and sham-operated control-treated rats. Mechanical allodynia was defined as a significant decrease in the paw withdrawal threshold to mechanical indentation on the plantar surface of each hindpaw. Withdrawal was measured using von Frey filaments (Anesthesia, Ugo, Italy) in a similar manner as described previously. Briefly, the animals were individually placed in an inverted ventilated cage equipped with a metal mesh floor. The von Frey filament was applied vertically from below to the mid-plantar surface of each hindpaw until the paw was withdrawn. Each stimulus lasted approximately 1–2 s, with an interstimulus interval of approximately 10–15 s. Thermal hyperalgesia was determined by a significant decrease in the paw withdrawal latency in response to thermal stimulation. An analgesia machine (model 390G; IITC Life Sciences, Woodland Hills, CA) was used to provide the heat source. The protocol was similar to that described previously. In brief, each rat was individually placed in an inverted cage (22–15 cm) with a smooth, temperature-controlled glass floor. The heat source was focused on a portion of the hind paw and delivered radiant heat stimulation to that site. When the hind paw was moved, the stimulation was automatically turned off (or after 25 s to prevent tissue damage). The intensity of the heat stimulation was kept constant in all experiments. Heat stimulation was applied four times per hind paw with an interval of 5–8 min. Spontaneous and motor-induced pain-like behaviors were also analyzed. Spontaneous aggressive behavior was assessed by measuring the number of hind paw withdrawals or licking within 2 min. Motor-induced pain was assessed by measuring limb use during autonomous walking, with a score ranging from 0 to 4: 0–5, normal use; 15, mild lameness; 25, marked lameness; 3, limb immobility (partial); 45, limb immobility.

[0036] Example 2

[0037] To investigate the therapeutic effect of a single intrathecal administration of Ryk-Fc fusion protein on pain caused by TCI modeling:

[0038] Twelve female rats were randomly divided into Ryk-Fc group and Vehcle group. On the 7th day after TCI modeling, Ryk-Fc fusion protein (2 μg / rat, Cat#: 4649-RK, ​​R&D, Minneapolis, MN) and solvent control were injected intrathecally according to the grouping, and the mechanical allodynia, heat / cold allodynia and spontaneous pain behavior changes within 24 hours and 21 days before modeling and on the seventh day after modeling were detected to clarify the short-term therapeutic effect of Ryk-Fc fusion protein on bone cancer pain. The TCI modeling and pain behavior detection methods are shown in Example 1.

[0039] The experimental results are as follows Figure 1 and 2As shown, after TCI modeling, compared with the solvent group, a single intrathecal administration of Ryk-Fc in the early stage of modeling can significantly alleviate the mechanical allodynia, thermal allodynia and spontaneous pain behavior of rats, indicating that Ryk-Fc fusion protein significantly inhibits the occurrence and development of cancer pain.

[0040] Example 3

[0041] To investigate the therapeutic effect of multiple intrathecal administration of Ryk-Fc fusion protein on pain caused by TCI modeling:

[0042] Twelve female rats were randomly divided into Ryk-Fc group and Vehicle group. Ryk-Fc fusion protein (2 μg / rat, Cat#: 4649-RK, ​​R&D, Minneapolis, MN) and solvent control were intrathecally injected on days 7, 8, and 9 after TCI modeling. The changes in mechanical allodynia and heat / cold allodynia behavior before and within 21 days after modeling were detected to clarify the long-term therapeutic effect of Ryk-Fc fusion protein on bone cancer pain. The TCI modeling and pain behavior detection methods are shown in Example 1.

[0043] The experimental results are as follows Figure 3 As shown, after TCI modeling, three consecutive intrathecal administrations of Ryk-Fc can long-term inhibit the mechanical allodynia, thermal allodynia and spontaneous pain behavior of rats compared with the solvent group, indicating that continuous administration of Ryk-Fc fusion protein can long-term inhibit the development of cancer pain.

[0044] Example 4

[0045] Detection of Ryk protein expression in rat dorsal root ganglia by immunoblotting

[0046] Twenty female rats were randomly divided into a sham operation group and a TCI model group. The rats were killed by cervical dislocation 1, 5, 7 and 14 days after modeling. The dorsal root ganglion tissues corresponding to the L3-L5 spinal cord segments of the rats were removed, and the expression of Ryk protein in the dorsal root ganglion was detected by immunoblotting.

[0047] Experimental Results Figure 4 As shown in the figure, compared with the Sham group, the expression level of Ryk protein in the dorsal root ganglia of rats in the TCI group was significantly increased after TCI modeling, indicating that after the rats were inoculated with tumor cells, the expression level of Ryk protein in their dorsal root ganglia would be significantly increased, while the inoculation of inactivated tumor cells would not lead to an increase in its content.

[0048] Example 5

[0049] Detection of TRPV1 expression in rat dorsal root ganglia by immunoblotting

[0050] Twelve female rats were randomly divided into Sham+IgG group, TCI+IgG group and TCI+Ryk-Fc group. 7-9 days after TCI modeling (the steps are as mentioned above), the TCI+Ryk-Fc group was intrathecally injected with Ryk-Fc fusion protein (2 μg / rat, as above), and the other two groups were injected with the same amount of IgG. Then the rats were killed by cervical dislocation, and the dorsal root ganglion tissues corresponding to the L3-L5 spinal cord segments of the rats were taken out, and the expression of TRPV1 protein in the dorsal root ganglion was detected by immunoblotting.

[0051] Experimental Results Figure 5 As shown, compared with the TCI+IgG group, the expression of TRPV1 protein in DRG of rats in the TCI+Ryk-Fc group was significantly decreased after medication, indicating that intrathecal injection of Ryk-Fc fusion protein into rats can regulate the expression of TRPV1 protein in dorsal root ganglia.

[0052] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of Ryk-Fc fusion protein in the preparation of anti-cancer pain drugs.

2. The use according to claim 1, characterized in that: The cancer pain includes moderate to severe pain caused by tumor compression or tumor infiltration.

3. The use according to claim 2, characterized in that: The moderate to severe pain may manifest as allodynia, hyperalgesia or spontaneous pain.

4. An anticancer pain drug, characterized in that: Including Ryk-Fc fusion protein.

5. The anticancer pain drug according to claim 4, characterized in that: Pharmaceutically acceptable excipients are also included.

6. The anticancer pain drug according to claim 4, characterized in that: The dosage form of the medicine is tablet, powder, granule, capsule, injection, spray, film, suppository, nasal drops or pills.

7. The anticancer pain drug according to claim 4, characterized in that: The administration route of the drug is intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

8. A pharmaceutical composition, characterized in that Including Ryk-Fc fusion protein.

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