Long-acting analgesic fusion protein and application thereof

Abstract

The invention discloses a long-acting analgesic fusion protein and application thereof.The analgesic peptide and the C terminal of human serum albumin (HSA) are fused through a flexible linker (GGGGS) n to form the fusion protein, n is 1, 2 or 3, meanwhile, an isolation sequence Pro or Gly-Pro is used for preventing Cys wrong disulfide bond formation so as to effectively mask exogenous epitopes, and therefore the long-acting analgesic fusion protein is obtained. The fusion protein retains the active conformation of the Cys1-Cys10 disulfide bond in the analgesic peptide fragment, and meanwhile, the half-life period is prolonged and the immunogenicity is reduced by using human serum albumin (HSA). According to the fusion protein of the analgesic peptide and the human serum albumin, the plasma half-life period of the human analgesic peptide can be prolonged, and the fusion protein can be used for preparing human analgesic peptide drugs or drugs for treating pains.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a long-acting analgesic fusion protein and applications thereof. Background Art

[0002] Currently commonly used analgesics, such as opioid peptides, are easily degraded by proteases, have short half-lives, and are prone to tolerance and dependence. The immunogenicity of exogenous peptides can lead to antibody neutralization, resulting in insufficient efficacy and unpredictable immunotoxic side effects. Therefore, the search for analgesics that extend the half-life of analgesic peptides while eliminating their immunogenicity is of great medical significance and clinical need.

[0003] Patent document CN202410583285.X discloses a GABA with analgesic activity A Receptor-targeting peptide DZ2, its synthesis method, and applications. The amino acid sequence of DZ2, a cyclic peptide analgesic targeting the GABAA receptor, is: Cys-Asn-Thr-Glu-Arg-Pro-Gly-Met-Leu-Asp-Phe-Lys-Gly-Lys-Ala-Lys-Trp-Asp-Ala-Trp-Cys, wherein the N-terminal Cys and the C-terminal Cys form a ring via a disulfide bond, with a molecular weight of 2454.81 g / mol. This cyclic peptide can be obtained via classic Fmoc-solid-phase synthesis and plays an important role in the development of drugs targeting the GABAA receptor. The compounds provided by the present invention can activate the GABAA receptor to produce a GABA-like inward current. DZ2 exhibits a strong analgesic effect in a mouse pain model. However, there is no record of this targeting peptide DZ2 achieving an analgesic effect by synergistically activating the μ opioid receptor and inhibiting voltage-gated sodium channel 1.8.

[0004] Patent document CN200510127627.4 discloses a transduction peptide-human brain-derived neurotrophic factor fusion protein and its applications. It relates to the transduction peptide-human brain-derived neurotrophic factor fusion protein and a nucleic acid encoding the fusion protein, as well as a pharmaceutical composition containing the transduction peptide-human brain-derived neurotrophic factor fusion protein. The pharmaceutical composition can be used to treat neurodegenerative diseases, diabetes, brain damage, auditory and optic nerve damage, and analgesia. However, the patent document does not describe the analgesic effect achieved by synergistically activating μ-opioid receptors and inhibiting voltage-gated sodium channel 1.8, nor does it describe the fusion of a synergistic analgesic peptide with human albumin. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a long-acting analgesic fusion protein with low immunogenicity and its application. The core innovation is firstly the HSAC end fusion design, that is, the C-terminus of human serum albumin HSA (away from the FcRn binding domain) is connected to the analgesic peptide through a flexible linker (GGGGS)_n; secondly, the flexible linker (GGGGS)_n provides spatial freedom; thirdly, the isolation sequence Pro or Gly-Pro is used to prevent the formation of erroneous disulfide bonds of Cys; fourthly, the natural conformation of the Cys1-Cys10 disulfide bond in the analgesic peptide is retained. The analgesic peptide contains an opioid receptor ligand and a NaV1.8 binding region. The synergistic effect of multiple targets can achieve a high-efficiency, long-term, low-toxic analgesic effect and reduce the risk of medication. Therefore, the fusion protein can be used to prepare drugs for treating or relieving pain.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions: a long-acting analgesic fusion protein, characterized in that: an analgesic peptide is fused to the C-terminus of human serum albumin HSA via a flexible linker (GGGGS)_n to form a fusion protein, wherein n is 1, 2 or 3, and an isolation sequence Pro or Gly-Pro is used to prevent the formation of an erroneous disulfide bond of Cys to effectively mask the exogenous epitope. The fusion protein retains the active conformation of the Cys1-Cys10 disulfide bond in the analgesic peptide segment, and uses human serum albumin HSA to extend the half-life and reduce immunogenicity; the amino acid sequence of the analgesic peptide is the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence with equivalent function by substitution, deletion and / or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; the Cys is cyclized in the amino acid sequence shown in SEQ ID NO: 2 using a disulfide bond. 1 With Cys 10 A cyclic peptide structure is formed to stabilize the relative positions of the N-terminus and the C-terminus, which is used to improve enzyme resistance and structural stability, thereby resisting protease degradation and prolonging the half-life, achieving efficient analgesia by synergistically activating the μ opioid receptor and inhibiting the voltage-gated sodium ion channel 1.8.

[0007] Preferably, the structure of the analgesic peptide is:

[0008]

[0009] A long-acting analgesic fusion protein, characterized by comprising:

[0010] The mature sequence of human serum albumin HSA, the amino acid sequence of which is shown in SEQ ID NO: 1;

[0011] Flexible linker: (GGGGS)_n, n=1, 2 or 3;

[0012] Isolating sequence: Pro or Gly-Pro;

[0013] Analgesic peptide sequence: the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having equivalent function by substitution, deletion and / or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2.

[0014] Preferably, the amino acid sequence of the flexible linker (GGGGS)_2 is as shown in SEQ ID NO:3.

[0015] A long-acting analgesic fusion protein, characterized in that: the amino acid sequence of the fusion protein is the amino acid sequence shown in SEQ ID NO: 4 (HSA-(GGGGS)_2-PC YGGF LR RAC KPSW RAD), or an amino acid sequence shown in SEQ ID NO: 4 with substitution, deletion and / or addition of one or more amino acids and having equivalent function, or an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4.

[0016] The long-acting analgesic fusion protein of the present invention is used in the preparation of analgesics, wherein the pain includes but is not limited to postoperative pain, neuropathic pain (peripheral neuropathy (diabetes, HIV, chemotherapy, radiotherapy), somatic pain (burns, fractures, incisions, wounds, cellulitis, herpes zoster, arthritis, gout, musculoskeletal pain), visceral pain (tumor infiltration, obstruction (intestines, ureters, bile ducts), colic, angina pectoris, pancreatitis), nociceptive pain (fibromyalgia, complex regional pain) type 1 pain syndrome, irritable bowel syndrome, bladder pain syndrome) and mixed pain (cancer pain, lower back pain, osteoarthritis pain, persistent postoperative pain).

[0017] A pharmaceutical composition for treating or alleviating pain, comprising the aforementioned long-acting analgesic fusion protein and a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition can be formulated using pharmaceutically acceptable carriers and / or excipients according to methods readily accomplished by one of ordinary skill in the art, and provided as a unit dosage form or in multi-dose vials. The formulation can be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, granules, gel, suppository, powder, granule, tablet, or capsule. The formulation may additionally contain a dispersant or stabilizer.

[0018] The pharmaceutical composition for treating or relieving pain of the present invention is prepared into an injectable preparation, which is a reconstituted lyophilized formulation or a liquid preparation provided in a ready-to-inject (RTI) form.

[0019] The pharmaceutical composition for treating or alleviating pain of the present invention is administered as a single therapeutic agent or in combination with other therapeutic agents. The pharmaceutical composition can be administered sequentially or simultaneously with conventional therapeutic agents.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: the present invention discloses an expression system for expressing a fusion protein of an analgesic peptide and human serum albumin, and obtains a CHO monoclonal cell line that stably and efficiently expresses a human recombinant protein by electroporating a plasmid carrying a fusion gene of an analgesic peptide and human serum albumin into CHO S cells. The monoclonal cell line of the present invention can secrete a fusion protein expressing an analgesic peptide and human serum albumin, and the fusion protein can prolong the plasma half-life of the human analgesic peptide, and can be used to prepare human analgesic peptide drugs or drugs for treating pain. The dual-functional domain design of the analgesic peptide can act on the opioid domain and the sodium channel domain simultaneously, and can inhibit the peripheral NaV1.8 channel to block pain signal transmission, and can also activate the μ opioid receptor to downregulate central pain sensitization, that is, achieve efficient analgesia by synergistically activating the μ opioid receptor and inhibiting the NaV1.8 channel. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Predicting Cys for fusion protein structures 1 -Cys 10 Diagram of the disulfide bond-formed cyclic peptide structure, where the fusion protein structure simulation range is GGGGSGGGGSP-C1 YG GF LR RAC10 KP SW RAD. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. The specific embodiments should be understood as merely illustrative and not restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0023] Example 1

[0024] Fusion protein preparation:

[0025] The fusion protein (amino acid sequence shown in SEQ ID NO: 4) is human serum albumin HSA mature protein-(GGGGS)2-Pro-CYGGFLRRACKPSWRAD.

[0026] According to conventional methods, the gene sequence corresponding to the amino acid sequence of the above fusion protein was cloned into the pcDNA3.1(+) vector, and the codons were optimized using CHO-S cell preferred codons (e.g., TGT instead of TGC was used for Cys codon), and the GC content was adjusted to 60%-65% (to avoid mRNA secondary structure).

[0027] Functional elements can be added to the above vectors, such as a Kozak sequence (GCCACC) at the 5' end, a 6×His tag (for purification) and a termination codon (TAA) at the 3' end, and a CMV-enhanced promoter.

[0028] CHO S cells can be transfected by electroporation, and after 14 days of methotrexate (MTX) pressure selection, the fusion protein can be captured by Ni-NTA column and His tag removed, and then purified by molecular sieve.

[0029] Example 2

[0030] Fusion protein immunogenicity testing:

[0031] A multifactorial assay was used to assess the risk of the fusion protein inducing a cytokine storm in vivo. The assay will examine changes in the levels of key inflammatory factors following administration.

[0032] The experimental animals were SPF grade C57BL / 6 mice, 10 per group, and the mice were randomly divided into the following experimental groups: A. fusion protein group (0.5 mg / kg, intravenous injection twice a week); B. free peptide C 1 YGGFLRRAC 10 KPSWRAD group (0.5 mg / kg, intravenous injection twice a week); C. HAS fusion protein control group (0.5 mg / kg, intravenous injection twice a week); D. PBS negative control group (equal volume, twice a week); E. positive control group (LPS 1 mg / kg, intraperitoneal injection only once, as a positive cytokine storm induction). Serum was collected 6 hours after the first dose (acute phase of the cytokine peak window) and 48 hours after the second dose at the end of the fourth week (memory response assessment) to measure the levels of the above cytokines.

[0033] Table 1 Serum cytokine concentrations 6 hours after the first dose (pg / mL, Mean ± SD)

[0034]

[0035]

[0036] Table 2 Serum cytokine concentrations during the 48-hour memory response period after administration at week 4 (pg / mL, Mean ± SD)

[0037]

[0038] The fusion protein did not induce significant cytokine release at any time point, and its safety profile met the FDAICHS6(R1) immunotoxicity requirements for therapeutic proteins.

[0039] Example 3

[0040] Analgesic effect of the fusion protein on the acetic acid writhing pain model in mice:

[0041] To gain a deeper understanding of the analgesic activity of the fusion protein, the animal experiments in this example were conducted in accordance with animal ethics requirements. The classic acetic acid writhing animal pain model was used to study its analgesic effect. The results showed that the fusion protein exhibited good analgesic activity in both animal pain models, as reported below:

[0042] The experimental animals were SPF grade C57BL / 6 mice, 10 mice per group, and the mice were randomly divided into the following experimental groups: A. fusion protein group (0.5 mg / kg, IP); B. free peptide C 1 YGGFLRRAC 10 KPSWRAD group (0.5 mg / kg, IP); C. HAS fusion protein control group (0.5 mg / kg, IP); D. PBS negative control group (equal volume, IP); E. positive drug group (aspirin 50 mg / kg IP).

[0043] According to the above grouping, the drug was slowly and uniformly injected into the left abdominal cavity of mice. 30 minutes later, 250 μL of 0.6 wt% acetic acid solution was injected into the right abdominal cavity of each group of mice. The number of writhings in the mice was recorded within 30 minutes after the acetic acid injection. The writhing reduction rate was calculated as [1 - (number of writhings in the treatment group / number of writhings in the PBS group)] × 100%. The half-life was calculated using the drug efficacy decay curve.

[0044] Table 3 Comparison of analgesic effects of fusion proteins on acetic acid writhing pain model in mice

[0045]

[0046]

[0047] The fusion protein took effect quickly within 30 minutes after a single intraperitoneal injection of 0.5 mg / kg, indicating that the fusion protein also had a good analgesic effect in a short period of time.

[0048] Example 4

[0049] Analgesic effect of the fusion protein on the carrageenan paw swelling model in mice:

[0050] To further investigate the long-lasting analgesic activity of the fusion protein, a carrageenan paw swelling model was used, and the analgesic effect was evaluated using the Hargreaves method to measure the thermal pain threshold. Mice were divided into groups according to the above protocol, and the drug was slowly and uniformly injected into the left abdominal cavity. Half an hour after administration, 30 μL of 1 wt% carrageenan was injected into the right hind paw of each group. The paw was then irradiated with an infrared heat source (50°C), and the paw withdrawal latency (in seconds) was recorded at 0, 6, 24, 48, and 72 hours.

[0051] Table 4 Analgesic effect of fusion protein on carrageenan paw swelling model in mice Thermal allodynia latency

[0052]

[0053] The fusion protein has an analgesic effect of up to 72 hours after a single intraperitoneal injection of 0.5 mg / kg, demonstrating an ultra-long-lasting and effective analgesic effect.

[0054] The basic principles, main features and advantages of the present invention are shown and described above. Without departing from the spirit and scope of the present invention, the present invention may also be subject to various changes and improvements, which fall within the scope of the present invention to be protected.

[0055] SEQ ID NO: 1:

[0056] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLY EIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPCLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEK PLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0057] SEQ ID NO:2:

[0058] CYGGFLRRAKPSWRAD

[0059] SEQ ID NO:3:

[0060] GGGGSGGGGSP

[0061] SEQ ID NO:4:

[0062] -GGGGSGGGGSP-CYGGFLRRAKPSWRAD

Claims

1. A long-acting analgesic fusion protein, characterized in that: A fusion protein is formed by fusing an analgesic peptide to the C-terminus of human serum albumin (HSA) via a flexible linker (GGGGS)_n, wherein n is 1, 2, or 3, and utilizing an isolation sequence Pro or Gly-Pro to prevent erroneous disulfide bond formation of Cys to effectively mask exogenous epitopes. The fusion protein retains the active conformation of the Cys1-Cys10 disulfide bond within the analgesic peptide segment, while utilizing human serum albumin (HSA) to extend the half-life and reduce immunogenicity. The amino acid sequence of the analgesic peptide is the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence with equivalent function by substitution, deletion, and / or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 2; Cys is cyclized in the amino acid sequence shown in SEQ ID NO: 2 using a disulfide bond. 1 With Cys 10 A cyclic peptide structure is formed to stabilize the relative positions of the N-terminus and the C-terminus, which is used to improve enzyme resistance and structural stability, thereby resisting protease degradation and prolonging the half-life, achieving efficient analgesia by synergistically activating the μ opioid receptor and inhibiting the voltage-gated sodium ion channel 1.

8.

2. The long-acting analgesic fusion protein according to claim 1, characterized in that The structure of the analgesic peptide is:

3. A long-acting analgesic fusion protein, characterized in that Include: The mature sequence of human serum albumin HSA, the amino acid sequence of which is shown in SEQ ID NO: 1; Flexible linker: (GGGGS)_n, n=1, 2 or 3; Isolating sequence: Pro or Gly-Pro; Analgesic peptide sequence: the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having equivalent function by substitution, deletion and / or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

4. The long-acting analgesic fusion protein according to claim 3, characterized in that: The amino acid sequence of the flexible linker (GGGGS)_2 is shown in SEQ ID NO:

3.

5. A long-acting analgesic fusion protein, characterized in that: The amino acid sequence of the fusion protein is the amino acid sequence shown in SEQ ID NO:4 (HSA-(GGGGS)_2-PC YGGF LR RAC KPSW RAD), or an amino acid sequence in which one or more amino acids are substituted, deleted and / or added to the amino acid sequence shown in SEQ ID NO:4 and has equivalent function, or an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

4.

6. Use of the long-acting analgesic fusion protein according to any one of claims 1 to 5 in the preparation of analgesic drugs.

7. A pharmaceutical composition for treating or relieving pain, characterized in that: The pharmaceutical composition comprises the long-acting analgesic fusion protein according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier and / or excipient.

8. The pharmaceutical composition according to claim 7, characterized in that: The dosage form of the pharmaceutical composition is a lyophilized preparation, an injection, a granule preparation, a gel preparation, a suppository, a powder, a granule, a tablet, a capsule, a solution preparation, a suspension preparation or an emulsion preparation.

9. The pharmaceutical composition according to claim 7, characterized in that: The pharmaceutical composition is administered as a single therapeutic agent or in combination with other therapeutic agents. The pharmaceutical composition is administered sequentially or simultaneously with conventional therapeutic agents.

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