Double-target cyclic peptide as well as synthesis method and medical application thereof

Abstract

The invention discloses a double-target-point cyclic peptide and a synthesis method and medical application thereof.The amino acid sequence of the double-target-point cyclic peptide is Cys-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Alaa-Cys-Lys-Pro-Ser-Trp-Arg-Alaa-Asp, Cys1 and Cys10 form a cyclic peptide structure through disulfide bond cyclization, the relative positions of the N terminal and the C terminal are stabilized, the enzyme resistance and the structural stability are improved, and the double-target-point cyclic peptide has the advantages that the double-target-point cyclic peptide is obtained; further, protease degradation is resisted, the half-life period is prolonged, and efficient analgesia is realized by synergistically activating a mu opioid receptor and inhibiting a voltage-gated sodium ion channel 1.8. The double-target cyclopeptide designed by the invention has the beneficial characteristics of simple structure, convenience in artificial synthesis, low production cost and the like, and has huge medical industrial applicability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a dual-target cyclic peptide, a synthesis method thereof, and medical uses thereof. The dual-target cyclic peptide is a polypeptide molecule that achieves high-efficiency analgesia by synergistically activating μ opioid receptors and inhibiting voltage-gated sodium ion channels 1.8 (NaV1.8). Background Art

[0002] Pain, as a form of actual or potential tissue damage, severely impacts people's quality of life. Currently, commonly used analgesics, such as nonsteroidal anti-inflammatory drugs (NSAIDs), can cause complications such as bleeding, immunosuppression, and gastric irritation. Opioids have central nervous system side effects (such as addiction) and visceral side effects (such as intestinal obstruction), and are prone to developing tolerance and dependence. Single-target sodium channel inhibitors (such as lidocaine) have a short duration of action and insufficient efficacy. Therefore, the search for new, highly effective, and less toxic analgesics is of great medical significance and clinical need.

[0003] The patent document CN202110272260.4 discloses a class of opioid / neuropeptide FF receptor multi-target cyclic peptide molecules and their preparation and application, and designs a new class of peripherally restricted multi-target cyclic peptide molecules or pharmaceutically acceptable salts thereof for opioid receptors and neuropeptide FF receptors. That is, using DN-9 as a chemical template, the opioid peptide and neuropeptide FF pharmacophores are structurally optimized using polypeptide chemical strategies such as amino acid replacement and cyclization modification to obtain a series of cyclic peptide molecules. This cyclic peptide molecule can simultaneously activate opioid receptors and NPFF receptors. Its analgesic activity and analgesic duration are greatly improved compared with the parent DN-9 molecule, and opioid side effects such as analgesic tolerance, constipation and addiction are lower. However, this multi-target cyclic peptide molecule does not have any relevant records about achieving analgesic effects by synergistically activating μ opioid receptors and inhibiting voltage-gated sodium ion channels 1.8.

[0004] Patent document CN202410583285.X discloses a GABA with analgesic activity AReceptor-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. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a dual-target cyclic peptide and a synthesis method thereof. The structural innovation of the dual-target cyclic peptide lies in the dual-functional domain design, namely, it contains opioid receptor ligands and NaV1.8 binding regions. The multi-target synergy is aimed at achieving a high-efficiency and low-toxic analgesic effect and reducing the risk of medication. Therefore, it can be used to prepare drugs for treating or relieving pain.

[0006] The present invention adopts the following technical solution to solve the above technical problems. The dual-target cyclic peptide has an amino acid sequence as shown in SEQ ID NO: 1. The specific amino acid sequence is:

[0007] Cys-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ala-Cys-Lys-Pro-Ser-Trp-Arg-Ala-Asp

[0008] Among them, disulfide bond cyclization is used to cyclize Cys 1 With Cys 10 The formation of a cyclic peptide structure stabilizes the relative position of the N-terminus and the C-terminus, which is used to enhance enzyme resistance and improve structural stability, thereby resisting protease degradation and prolonging half-life. It achieves efficient analgesia by synergistically activating the μ opioid receptor and inhibiting the voltage-gated sodium ion channel 1.8. The structure of this dual-target cyclic peptide is:

[0009]

[0010] The full-sequence amino acid structure of the dual-target cyclic peptide designed by the present invention was searched and compared with databases such as NCBI, and no identical polypeptide sequences were found. The structural innovation of the dual-target cyclic peptide lies in the dual-functional domain design that can act on the opioid domain and the sodium channel domain simultaneously. Specifically, the dual-target cyclic peptide designed by the present invention with analgesic function 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, by synergistically activating the μ opioid receptor and inhibiting the NaV1.8 channel to achieve efficient analgesia. Therefore, even patients receiving opioid antagonists can use the dual-target cyclic peptide with analgesic function designed by the present invention.

[0011] One aspect of the applicability of the present invention is that the dual-target cyclic peptide designed by the present invention can be used to prepare a drug for treating or alleviating pain, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the cyclic peptide and a pharmaceutically acceptable carrier to a patient in need.

[0012] The scalability of the present invention lies in: the design uses a dual-target cyclic peptide as an innovative core structure, and based on the special amino acid epitopes of this structure, more technical application scenarios can be developed. For example, the dynamic visualization of pain foci can be achieved through near-infrared second region (NIR-II) labeling; or combined with 99 mTc / 18 F radionuclide imaging allows for precise anatomical positioning, in order to achieve an integrated diagnosis and treatment strategy (such as the treatment of bone metastasis pain).

[0013] The industrial applicability of the present invention lies in that the dual-target cyclic peptide designed by the present invention has the advantageous characteristics of simple structure, convenient artificial synthesis, low production cost, etc., and has great applicability in the pharmaceutical industry.

[0014] The synthesis method of the dual-target cyclic peptide designed in the present invention is as follows: the full sequence is synthesized in the C→N direction using an automatic peptide synthesizer, the peptide chain is cut, cold ether precipitation is performed, and disulfide bonds are formed by oxidation using NH4HCO3 (0.1M, pH 8.0) solution. 1 -Cys 10 Selective pairing formed a cyclic peptide structure, and the dual-target cyclic peptide was obtained by desalting and purification via HPLC reverse phase column chromatography.

[0015] In certain embodiments, the cyclic peptide is packaged for delivery in a titratable dosage form. In certain embodiments, the cyclic peptide is packaged so that delivery targets an area selected from the group consisting of: sublingual; buccal; parenteral; oral; rectal, nasal, skin; and pulmonary system. In certain embodiments, the cyclic peptide is in a form selected from the group consisting of: gel; gel spray; tablet; liquid; capsule and vaporization.

[0016] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all pharmaceutically compatible solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffers, lubricants, sustained-release agents, sustained-release agents, diluents, emulsifiers, humectants, lubricants, dispersion media, coatings, antibacterial or antifungal peptides, isotonic and absorption-delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. In certain embodiments, pharmaceutically acceptable carriers include serum albumin and nanoscale drug carriers.

[0017] The formulation of the pharmaceutical composition of the present application is compatible with its intended route of administration. The example of route of administration includes parenteral administration, such as intra-arterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical) and transmucosal administration. In certain embodiments, the pharmaceutical composition is directly applied to tumor tissue.

[0018] Solutions or suspensions for parenteral, intradermal or subcutaneous application may include the following components: a sterile diluent, such as water for injection, physiological saline, fixed oils, polyethylene glycols, glycerol; propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfate; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose.

[0019] Injectable preparations can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).

[0020] Oral compositions typically include an inert diluent or edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, wherein the compound in the liquid carrier is taken orally and swished into the mouth and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvants can be part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate; slip agents such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as mint, methyl salicylate, or orange flavor.

[0021] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0022] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are well known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished by the use of nasal sprays or suppositories. For transdermal administration, the pharmaceutical composition is formulated into an ointment, salves, gel, or cream as is generally known in the art.

[0023] In certain embodiments, the pharmaceutical compositions are formulated for sustained or controlled release of the active ingredient. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, liposomal suspensions, and nanoparticles can be used. Methods for preparing such formulations will be readily apparent to those skilled in the art.

[0024] The International Association for the Study of Pain defines pain as "an unpleasant sensory and emotional experience associated with or resembling actual or potential tissue damage." The pain described in this case 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 (intestinal, ureter, bile duct), colic, angina, pancreatitis), nociceptive pain (fibromyalgia, complex regional pain syndrome type 1, irritable bowel syndrome, painful bladder syndrome), and mixed pain (cancer pain, low back pain, osteoarthritis pain, and persistent postoperative pain). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Predicting Cys for dual-target cyclic peptide protein structures 1 -Cys 10 Diagram of the structure of a cyclic peptide formed by disulfide bonds. DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1

[0028] The dual-target cyclic peptide provided by the present invention has an amino acid sequence as follows:

[0029] Cys-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ala-Cys-Lys-Pro-Ser-Trp-Arg-Ala-Asp

[0030] Among them, disulfide bond cyclization is used to cyclize Cys 1 With Cys 10 Forming a cyclic peptide structure.

[0031] The synthesis method is as follows:

[0032] The full sequence was synthesized in the C→N direction using an automatic peptide synthesizer. The peptide chain was cut, precipitated with cold ether, and oxidized with NH4HCO3 (0.1 M, pH 8.0) solution to form a disulfide bond. Cys 1 -Cys 10 The selective pairing formed a cyclic peptide structure, and the dual-target cyclic peptide was obtained by desalting and purification via HPLC reverse phase column chromatography.

[0033] Determination of analgesic activity of dual-target cyclic peptides:

[0034] To gain a deeper understanding of the analgesic activity of analgesic peptides, the present invention conducted animal experiments in accordance with ethical requirements (approval number: XYIL-20250124) using the classic acetic acid writhing pain model, the hot plate method animal pain model, and the formalin animal pain model. The results showed that the analgesic peptides exhibited good analgesic activity in all of the above animal pain models, as described below:

[0035] 1. Analgesic effect of analgesic peptides on the acetic acid writhing pain model in mice

[0036] Four-week-old SPF Kunming mice (weighing 18-22 g) were used as experimental animals. The mice were randomly divided into the following experimental groups: A-control group (normal saline), B-positive drug group (aspirin 50 mg / kg IP), C-high-dose analgesic peptide group (0.5 mg / kg IP), D-naloxone blocking group (naloxone 2 mg / kg IP + analgesic peptide 0.5 mg / kg IP), E-protoxin-I blocking group (protoxin-I 0.1 mg / kg IP + analgesic peptide 0.5 mg / kg IP), F-double blocking group (naloxone 2 mg / kg IP + protoxin-I 0.1 mg / kg IP + analgesic peptide 0.5 mg / kg IP), G-instrumental drug control group (protoxin-I 0.1 mg / kg IP alone, verified to have no analgesic effect by itself), H-naloxone alone group (2 mg / kg IP, verified to have no analgesic effect by itself), with 10 mice in each group.

[0037] The drug was slowly and evenly injected into the left abdominal cavity of mice according to the above grouping. 30 minutes later, 250 μL of 0.6 wt% acetic acid solution was injected into the right abdominal cavity of each group. The number of writhing events in the mice within 20 minutes after acetic acid injection was recorded, and the inhibition rate was calculated. The inhibition rate was calculated as follows: (average number of writhing events in the control group - average number of writhing events in the drug group) / average number of writhing events in the control group × 100%.

[0038] Table 1 Analgesic effect of analgesic peptides on the acetic acid writhing pain model in mice

[0039]

[0040] As shown in Table 1, the analgesic peptide can reduce the number of acetic acid-induced writhings in mice, indicating that the analgesic peptide has a significant analgesic effect on the pain caused by acetic acid in animals.

[0041] Quantitative analysis of target contribution:

[0042] μ receptor contribution rate = (Protoxin-I blocking group inhibition rate - dual blocking group inhibition rate) / analgesic peptide group inhibition rate = (55.1% - 8.5%) / 75.2% = 61.97%.

[0043] NaV1.8 contribution rate = (naloxone blocking group inhibition rate - double blocking group inhibition rate) / analgesic peptide group inhibition rate = (22.8% - 8.5%) / 75.2% = 19.01%.

[0044] Synergistic enhancement efficiency = 100% - μ receptor contribution rate - NaV1.8 contribution rate = 100% - 61.97% - 19.01% = 19.02%. That is, the dual-target synergistic analgesic enhancement efficiency of the present invention targeting μ opioid receptors and voltage-gated sodium ion channel 1.8 (NaV1.8) is about 19.02%.

[0045] Calculation of synergy effect quantitative index:

[0046] Dual-target analgesic synergy index = measured effect / (μ receptor effect×NaV1.8 effect) = 75.2% / (22.8%×55.1%) = 5.99.

[0047] 2. Analgesic effect of analgesic peptides on the hot plate pain model in mice

[0048] 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, mice in each group were placed on a hot plate apparatus at 55 ± 0.5°C. The latency (in seconds) for the mice to lick their hind paws or jump was recorded, with a cutoff time of 30 seconds. The results are shown in the following table (mean ± SD, in seconds).

[0049] Table 2 Analgesic effect of analgesic peptides on the hot plate pain model in mice

[0050]

[0051] As shown in Table 2, analgesic peptides can increase and prolong the time that mice lick their hind paws, indicating that analgesic peptides have analgesic effects on pain caused by high temperature or thermal stimulation.

[0052] Calculation of synergy effect quantitative index:

[0053] Dual-target analgesia synergy index = improvement rate of analgesic peptide group / (improvement rate of naloxone blocking group×improvement rate of Protoxin-I blocking group-) = 198.8% / (47.5%×109.8%) = 3.81.

[0054] 3. Experiment on the effect of analgesic peptides on formalin-induced pain

[0055] 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, 20 μL of a 5 wt% formalin solution was injected into the right hind paw of each group of mice. The duration of paw licking during phase 1 (0-10 minutes) and phase 2 (15-60 minutes) was recorded. The results are shown in the following table (mean ± SD, unit: seconds).

[0056] Table 3-1 Analgesic effect of analgesic peptides on formalin-induced pain Phase I in mice

[0057]

[0058] The synergy effect quantitative index is calculated as shown in Table 3-1:

[0059] Phase I analgesic synergy index = analgesic peptide group inhibition rate / (naloxone blocking group inhibition rate × protoxin-I blocking group inhibition rate) = 66.1% / (22.9% × 52.9%) = 5.46

[0060] Table 3-2 Analgesic effect of analgesic peptides on formalin-induced pain Phase II in mice

[0061]

[0062] The synergy effect quantitative index is calculated as shown in Table 3-2:

[0063] The analgesic synergy index of Phase II = 71.5% / (24.0%×42.1%) = 7.07.

[0064] The experimental results showed that the paw licking time of mice in phase I and phase II can be significantly reduced, indicating that the analgesic peptide has a good analgesic effect.

[0065] In summary, the present invention has obtained a novel dual-target cyclic peptide, and through various mouse pain experimental animal model experiments, it is proved that the dual-target cyclic peptide has a significant analgesic effect and can be used in the preparation of drugs for treating or relieving pain.

[0066] 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 as claimed.

[0067]

Claims

1. Dual-target cyclic peptide, characterized by: The amino acid sequence of the dual-target cyclic peptide is shown in SEQ ID NO:

1. The specific amino acid sequence is: Cys-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ala-Cys-Lys-Pro-Ser-Trp-Arg-Ala-Asp Among them, disulfide bond cyclization is used to cyclize Cys 1 With Cys 10 The formation of a cyclic peptide structure stabilizes the relative position of the N-terminus and the C-terminus, which is used to enhance enzyme resistance and improve structural stability, thereby resisting protease degradation and prolonging half-life. It achieves efficient analgesia by synergistically activating the μ opioid receptor and inhibiting the voltage-gated sodium ion channel 1.

8. The structure of this dual-target cyclic peptide is:

2. A method for synthesizing the dual-target cyclic peptide according to claim 1, characterized in that The specific synthesis process is: using an automatic peptide synthesizer to synthesize the full sequence in the C→N direction, cutting the peptide chain, cold ether precipitation, using NH4HCO3 solution to oxidize to form a disulfide bond, and then converting Cys 1 -Cys 10 Selective pairing formed a cyclic peptide structure, and the dual-target cyclic peptide was obtained by desalting and purification via HPLC reverse phase column chromatography.

3. Use of the dual-target cyclic peptide according to claim 1 in the preparation of a drug for treating or alleviating pain.

4. The use according to claim 3, characterized in that: The medicine comprises a pharmaceutical composition containing a therapeutically effective amount of a dual-target cyclic peptide and a pharmaceutically acceptable carrier.

5. The use according to claim 3, characterized in that: The dosage form of the drug is oral preparation, injection, transdermal patch, aerosol or nanoparticle preparation.

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