Polypeptide and use thereof

Abstract

Provided in the present application are a polypeptide and the use thereof. The polypeptide has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, wherein the amino acid at position 12 in the amino acid sequence of the polypeptide is alanine or glutamic acid; the amino acid at position 19 in the amino acid sequence of the polypeptide is methionine or leucine; the amino acid at position 28 in the amino acid sequence of the polypeptide is lysine or isoleucine; and the amino acid at position 29 in the amino acid sequence of the polypeptide is selected from one of isoleucine, leucine and tryptophan.

Description

A polypeptide and its application Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a polypeptide and its application. Background Art

[0002] Voltage-gated sodium channels (VGSCs) are multi-subunit transmembrane glycoproteins expressed on the cell membrane. They are composed of α subunits (functional units) and β subunits. The α subunit is composed of four homologous transmembrane domains (I to IV), each of which contains six transmembrane hydrophobic α helices (S1 to S6). The positively charged S4 segment has the function of a voltage sensor, which can regulate the hydrophilic channel between S5 and S6 that allows sodium ions to pass through, causing cell depolarization or hyperpolarization, and completing transmembrane signal transmission.

[0003] Compared to other sodium channel subtypes, Nav1.7 channels have rapid activation and inactivation, but slow inactivation kinetics. They can be activated by small stimuli, promoting further depolarization of the cell membrane. Nav1.7 is the predominant sodium channel expressed in peripheral neurons, and its expression is more pronounced in nociceptive sensory neurons than in non-nociceptive ones.

[0004] Nav1.7 is a sodium ion channel that was previously found to be involved in the sensation of pain (i.e., nociception) through genetic studies of patients with rare pain disorders. Neurons use electrical currents to send signals to the brain and throughout the body, and sodium ion channels are critical to the cells' ability to generate these currents. When a neuron is stimulated, the Nav1.7 channel opens, allowing positively charged sodium ions to cross the cell membrane and enter the previously negatively charged cell. The change in charge on the cell membrane generates an electric current, increases the excitability of the neuron, and initiates a chain of events that leads to pain. Therefore, the development of a peptide that can selectively inhibit the activation of the Nav1.7 channel is of extremely important clinical significance for pain disorders.

[0005] Application Contents

[0006] The present application provides a polypeptide and its application, which has good analgesic function and can selectively inhibit the activation of Nav1.7 channels, thereby inhibiting pain, and has extremely important clinical significance for pain diseases.

[0007] According to the first aspect of the present application, a polypeptide is provided, the amino acid sequence of the polypeptide being as shown in SEQ ID NO: 1 or SEQ ID NO: 2; the 12th amino acid in the amino acid sequence of the polypeptide is alanine or glutamic acid; the 19th amino acid in the amino acid sequence of the polypeptide is methionine or leucine; the 28th amino acid in the amino acid sequence of the polypeptide is lysine or isoleucine; and the 29th amino acid in the amino acid sequence of the polypeptide is selected from one of isoleucine, leucine, and tryptophan.

[0008] The polypeptide provided in this application has good analgesic function and can selectively inhibit the activation of Nav1.7 channels, thereby inhibiting pain, and has extremely important clinical significance for pain diseases.

[0009] Preferably, the amino acid sequence of the polypeptide is selected from one of SEQ ID NOs: 3 to 18.

[0010] According to the second aspect of the present application, a nucleic acid molecule is provided, which comprises a nucleotide sequence encoding the above-mentioned polypeptide.

[0011] According to the third aspect of the present application, a recombinant expression vector is provided, which comprises the above-mentioned nucleic acid molecule.

[0012] According to the fourth aspect of the present application, a recombinant host cell is provided, which contains the above-mentioned recombinant expression vector.

[0013] According to the fifth aspect of the present application, there is provided the use of the above-mentioned polypeptide in the preparation of a drug having analgesic function.

[0014] Preferably, the above-mentioned drug can inhibit the activation of voltage-gated sodium ion channels.

[0015] Preferably, the voltage-gated sodium ion channel includes a Nav1.7 channel.

[0016] Preferably, the preparation type of the above-mentioned drug with analgesic function includes at least one of a colloidal solution type, an emulsion type or a suspension type, an oral fast-dissolving film, an oral solution, a capsule, an injection or a transdermal absorption preparation.

[0017] According to the sixth aspect of the present application, a pharmaceutical composition is provided, wherein the active ingredient in the pharmaceutical composition contains the above-mentioned polypeptide.

[0018] According to the seventh aspect of the present application, the use of the above-mentioned polypeptide in analgesia is provided.

[0019] The polypeptide provided in this application can selectively inhibit the activation of the voltage-gated Nav1.7 sodium ion channel, thereby inhibiting pain. The polypeptide provided in this application is used in the preparation of analgesic drugs. The prepared drugs have good analgesic effects and are of extremely important clinical significance for pain diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 is a graph showing the effect of the polypeptide provided in this application on the current trajectory of HEK293 cells overexpressing Nav1.7.

[0021] Figure 2 shows the effect of polypeptide PPN-01 on the current trajectory of HEK293 cells overexpressing Nav1.4, Nav1.5, Nav1.6, and Nav1.7.

[0022] Figure 3 shows the current trajectory of Nav1.7 under the addition of different concentrations of peptide PPN-01.

[0023] FIG4 is a graph showing the inhibition results of Nav1.7 peak current under the addition of different concentrations of peptide PPN-01.

[0024] FIG5 is a graph showing the results of voltage-dependent inhibition of Nav1.7 channel current by polypeptide PPN-01.

[0025] FIG6 is a graph showing the effects of the polypeptides provided in this application on the time of first writhing and the number of writhing times in mice.

[0026] FIG7 is a graph showing the effects of different doses of the polypeptide PPN-01 provided in this application on the number of writhing times in mice.

[0027] FIG8 is a graph showing the effects of different concentrations of the polypeptide PPN-01 on the electrical signals of the sciatic nerve of rats provided by the present application. DETAILED DESCRIPTION

[0028] The following is a further clear and complete description of the technical features of the technical solution provided by this application in conjunction with specific implementation methods. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making any creative efforts are within the scope of protection of this application.

[0029] Example 1

[0030] This embodiment provides a polypeptide, whose amino acid sequence is shown in SEQ ID NO: 1, wherein the 12th amino acid in the amino acid sequence of the polypeptide is alanine or glutamic acid; the 19th amino acid in the amino acid sequence of the polypeptide is methionine or leucine; the 28th amino acid in the amino acid sequence of the polypeptide is lysine or isoleucine; and the 29th amino acid in the amino acid sequence of the polypeptide is selected from one of isoleucine, leucine, and tryptophan.

[0031] The amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 represent the general formula of the polypeptide provided in this application. In SEQ ID NO: 1 and SEQ ID NO: 2, amino acids at positions 12, 19, 28, and 29 are all represented by X.

[0032] The difference between SEQ ID NO: 1 and SEQ ID NO: 2 is that SEQ ID NO: 1 contains a total of 29 amino acids, while SEQ ID NO: 2 has an additional amino acid (W) at the N-terminus of the amino acid sequence of SEQ ID NO: 1.

[0033] Table 1 Amino acid sequence of polypeptide

[0034] Table 1 of this example lists several polypeptides that meet the above requirements and their corresponding amino acid sequences. The polypeptides involved in this application are all prepared using the following steps:

[0035] 1. Using 4-toluenehydroamine resin (MBHA Resin, 1.0 mmol / g) at S = 0.3 mmol / g, the Fmoc synthesis process was used to sequentially condense amino acid links from the C-terminus to the N-terminus of the peptide until a linear peptide was condensed to obtain a resin peptide. Acetic anhydride and pyridine were then added to acetylate the N-terminus of the peptide. The peptide was then cleaved from the resin using a cleavage solution (calculated by volume: trifluoroacetic acid (TFA) : thioanisole : phenol : EDT : water = 87.5:5:2.5:2.5:2.5) to obtain a linear peptide.

[0036] 2. Add the linear peptide to the acetonitrile solution (acetonitrile: water = 1:1 by volume) and mix well to obtain a peptide solution with a concentration of 5 mg / mL; mix 60 mg of oxidized glutathione, 30 mg of reduced glutathione, and 2.58 g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and adjust the pH of the mixture to 7.5 with 5 M NaOH to obtain a folding solution;

[0037] 3. Take 30 mL of the above-mentioned polypeptide solution, add 90 mL of folding solution, mix well, and place in a 4°C refrigerator to react for 48 hours. Sampling and detection of the sample by high performance liquid chromatography. After the reaction is basically completed, purification is carried out by preparative liquid chromatography according to the conditions shown in Table 2.

[0038] Table 2 Preparative liquid phase purification conditions

[0039] Example 2 Inhibitory effects of different peptides on Nav1.7 channels

[0040] The purpose of this example is to use patch clamp technology to study the inhibitory effects of different polypeptides on Nav1.7 channels.

[0041] The recording electrode used in this embodiment is a glass microelectrode, which is prepared according to the following method: a glass electrode blank (outer diameter 1.50 mm, inner diameter 0.84 mm) is drawn into a single-tube glass microelectrode with a tip diameter of 1 to 2 μm in four steps using a P-1000 microelectrode horizontal drawing instrument for subsequent experiments. When drawing the electrode, be careful not to touch the heated platinum sheet in the middle, and be careful not to touch the drawn electrode tip to prevent the tip from breaking. In addition, the tip of the glass microelectrode is very easy to absorb dust, so it is generally used immediately after being drawn and should not be left overnight.

[0042] The method of using the glass microelectrode is as follows: the glass microelectrode must be cleaned before use, and then all the glass microelectrodes must be placed in a large beaker, and anhydrous ethanol must be added to completely immerse the glass microelectrode in the anhydrous ethanol, and the beaker must be placed on an ultrasonic cleaner for ultrasonic vibration for 30 minutes; then the anhydrous ethanol must be poured out, and the glass microelectrode must be cleaned 3 to 4 times with ultrapure water, and then covered with another small beaker to prevent dust from contaminating the electrode, and then placed in an oven to dry the glass microelectrode. After drying, the glass microelectrode must be placed in a clean box for later use.

[0043] In patch-clamp experiments, measured sodium currents were corrected for leakage current. Raw data were recorded using Clampfit 10.6, and data acquisition was performed using pCLAMP software. Three to five sweeps (a term used in patch-clamp software and consisting of a series of sampling points) during which the current was stable before peptide addition were selected for analysis. The average peak current was calculated from this value and used as the control current amplitude. Three to five sweeps during which the current was stable after peptide addition were then selected for analysis, and the average residual peak current was calculated as the residual current amplitude.

[0044] The glass microelectrodes prepared as described above were used to study the electrical stimulatory effects of polypeptides PPN-01 to PPN-16 on Nav1.7-overexpressing HEK293 cells (n=1). In the voltage clamp mode, the clamping voltage was maintained at -90 mV, and a square wave stimulation was applied with a depolarization from -90 mV to +40 mV and a duration of 30 ms. The concentration of each drug administration (peptide) was 300 nM, and 6 polypeptides with significant effects were repeatedly verified (n=2).

[0045] In the voltage clamp mode, the clamping voltage was maintained at -90 mV, and a square wave stimulus of depolarization from -90 mV to +40 mV with a duration of 30 ms was given. The experimental results are shown in Figure 1. Compared with the negative control (no polypeptide was added), the 16 polypeptides provided in Example 1 (PPN-01, PPN-02, PPN-03, PPN-04, PPN-05, PPN-06, PPN-07, PPN-08, PPN-09, PPN-10, PPN-11, PPN-12, PPN-13, PPN-14, PPN-15, PPN-16) all had an inhibitory effect on Nav1.7-overexpressing HEK293 cells, among which PPN-01, PPN-04, PPN-06, PPN-11, PPN-12, and PPN-16 all had a significant inhibitory effect on Nav1.7-overexpressing HEK293 cells.

[0046] Example 3 Selectivity of Peptide PPN-01

[0047] The purpose of this example is to study the selectivity of the polypeptide PPN-01 provided in Example 1 for voltage-gated sodium ion channels Nav1.4, Nav1.5, Nav1.6, and Nav1.7.

[0048] The glass microelectrode prepared in Example 2 was used to study the effect of the polypeptide PPN-01 provided in Example 1 on voltage-gated sodium ion channels using the whole-cell patch clamp technique. The specific operation was as follows:

[0049] 1. First, place the electrode liquid on ice to thaw. After thawing, use a 1mL syringe to draw an appropriate amount of electrode liquid. After putting a homemade dispenser on the head of the syringe, empty the air in the pipe so that the entire dispenser is filled with electrode liquid. Then use the tail perfusion method to pour the electrode liquid in the dispenser into the tail of the glass microelectrode. Slowly inject the electrode liquid into the glass microelectrode. The perfusion volume is generally 1 / 3 to 1 / 2 of the length of the glass microelectrode (to ensure that the electrode silver wire can fully contact the electrode liquid and to avoid electrode contamination caused by excessive electrode liquid). After the glass microelectrode is perfused with electrode liquid, gently tap the wall of the glass microelectrode tube to expel bubbles and fill the tip of the glass microelectrode with electrode liquid. The resistance of the glass microelectrode after filling is 4.0 to 6.0 MΩ. Note that the force when popping bubbles should not be too large, and the tube wall should be supported with the middle finger of the left hand, otherwise the glass microelectrode will easily break.

[0050] 2. The glass microelectrode prepared as described above was used and the whole-cell patch clamp technique was used to study the electrical stimulatory effect of the polypeptide PPN-01 provided in Example 1 on HEK293 cells overexpressing Nav1.4, Nav1.5, Nav1.6, and Nav1.7 (n=5). In the voltage clamp mode, the clamping voltage was maintained at -90 mV, and a square wave stimulation was given from -90 mV to +40 mV with a duration of 30 ms. The concentration of each drug administration (polypeptide) was 300 nM.

[0051] 3. HEK293 cells overexpressing Nav1.4, Nav1.5, Nav1.6, and Nav1.7 were added to the glass microelectrode perfused with the electrode internal solution, and the polypeptides provided in Example 1 were added thereto. In the voltage clamp mode, the clamping voltage was maintained at -90 mV, and a square wave stimulation was given from -90 mV to +40 mV with a duration of 30 ms. The experimental results showed that the polypeptide PPN-01 provided in Example 1 had a significant inhibitory effect on HEK293 cells overexpressing Nav1.7, but had no significant inhibitory effect on HEK293 cells overexpressing Nav1.4, Nav1.5, and Nav1.6. Figure 2 shows the current traces after the polypeptide PPN-01 shown in NO:3 was applied and the glass microelectrode containing HEK293 cells overexpressing Nav1.4, Nav1.5, Nav1.6, and Nav1.7 was stimulated by depolarization from -90 mV to +40 mV with a duration of 30 ms in voltage clamp mode.

[0052] As can be seen from Figure 2, HEK293 cells overexpressing Nav1.4, Nav1.5, Nav1.6, and Nav1.7 were added to the glass microelectrodes perfused with electrode internal solution (respectively recorded as Nav1.4 group, Nav1.5 group, Nav1.6 group, and Nav1.7 group), and the polypeptide PPN-01 provided in Example 1 with an amino acid sequence as shown in SEQ ID NO: 3 was added thereto. In the voltage clamp mode, the clamping voltage was maintained at -90 mV, and a square wave stimulus of depolarization from -90 mV to +40 mV with a duration of 30 ms was given. The current trajectory of the Nav1.7 group showed obvious fluctuations, while the current trajectory fluctuations of the Nav1.4 group, Nav1.5 group, and Nav1.6 group were not obvious.

[0053] The above results indicate that the polypeptide PPN-01 provided in Example 1 can selectively inhibit the activation of the voltage-gated sodium ion channel Nav1.7.

[0054] Example 4 Inhibitory effect of different concentrations of polypeptide PPN-01 on Nav1.7 channels

[0055] The purpose of this example is to use patch clamp technology to study the inhibitory effect of different concentrations of a polypeptide (whose amino acid sequence is shown in SEQ ID NO: 3, hereinafter referred to as "polypeptide PPN-01" for convenience) on Nav1.7 channels.

[0056] In the patch clamp experiment (see Example 2), the measured sodium currents were corrected for leakage current. The raw experimental data were recorded using Clampfit 10.6, and data acquisition was completed using pCLAMP software. Three to five sweeps (a sweep is a term used in patch clamp software and consists of a series of sampling points) in which the current was in a stable state before the addition of the polypeptide PPN-01 were selected for analysis. The average value of the peak current was calculated from this and used as the control current amplitude. Three to five sweeps in which the current was in a stable state after the addition of the polypeptide PPN-01 were also selected for analysis, and the average value of the residual peak current was calculated as the residual current amplitude. The inhibition rate of the Nav1.7 current by the polypeptide PPN-01 was calculated according to the following equation: Inhibition rate (%) = [1-(residual current amplitude) / (control current amplitude)] × 100%.

[0057] The dose-effect curve was fitted by the Hill equation: I drug / I control =1 / [1+(C / IC 50 ) H ];

[0058] Among them, I control is the average current amplitude of the control group, I drugis the average current amplitude at different drug concentrations C (i.e. different concentrations of peptide-17), IC 50 is the drug concentration (ie, polypeptide concentration) required to block 50% of Nav1.7 channels, and H is the Hill coefficient.

[0059] The inward sodium current curve is obtained by fitting the 90% to 10% data between the peak and baseline using a single exponential equation or a double exponential equation for the rise and decay phases:

[0060] Among them, A i and τ i are the initial current amplitude and activation / inactivation time constant, respectively, and C is the time-independent component.

[0061] The activation and inactivation curves of Nav1.7 channels were fitted with the Boltzmann equation: G = G max / [1+exp((V 1 / 2 –V m ) / k)];

[0062] Where G is the sodium conductivity, G max is the maximum value of sodium conductance, V 1 / 2 is the voltage at which half of the channel is activated, V m is the clamping voltage and k is the slope factor.

[0063] Sodium conductivity G is calculated according to the following formula: G = I / (V m -Vrev);

[0064] Where G is the sodium conductance, I is the current, and V m is the clamping voltage, V rev is the reversal potential of the sodium current. The reversal potential V of the sodium current is calculated according to the Nernst equation. rev is +66mV.

[0065] All statistics in this application were analyzed using one-way ANOVA between two groups, with P < 0.05 considered statistically significant (* indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001). All statistical data were plotted using Graphpad Prism 9.0 software.

[0066] Representative Nav1.7 current traces (n=7) recorded before and after administration of different concentrations of the peptide PPN-01 (low concentration: 30 nM; medium concentration: 100 nM; high concentration: 300 nM) induced currents from a 30 ms square wave stimulus depolarizing from a holding voltage of -90 mV to +40 mV are shown in FIG3 , where the control group indicates no drug administration.

[0067] The corresponding dose-effect curve was drawn according to the inhibition percentage of Nav1.7 peak current at each concentration of the peptide, and the data was fitted according to the above steps. The results are shown in Figure 4.

[0068] As shown in Figures 3 and 4, the IC of peptide PPN-01 inhibiting Nav1.7 peak current is 50 The value was 52.7±7.8nM, and the Hill coefficient was 1.18. The above results showed that there was no intermolecular cooperativity when the polypeptide PPN-01 interacted with the Nav1.7 channel.

[0069] Example 5 Voltage dependence of Nav1.7 channel blockade by polypeptide PPN-01

[0070] The purpose of this example is to use the patch clamp technique to study the voltage dependence of the Nav1.7 channel blockade by the polypeptide PPN-01 (whose amino acid sequence is shown in SEQ ID NO: 3).

[0071] In voltage clamp mode, the clamping voltage was -90mV, and HEK293 cells overexpressing Nav1.7 were stimulated with a square wave train from -90mV to +80mV with a step voltage of 10mV and a duration of 30ms to elicit current traces at different voltages. Among them, the experimental group was treated with 300nM peptide PPN-01, and the control group was treated without 300nM peptide PPN-01. Then, the peak amplitude of Nav1.7 current was plotted against the test potential (membrane The IV relationship curve related to the Nav1.7 current was constructed (n=6, P<0.05), and the relationship between the inhibition percentage and the test potential was plotted. The results are shown in Figure 5, wherein Figure 5 (inset A) shows the Nav1.7 current trajectory induced by a square wave train stimulation with a duration of 30 ms from the clamping voltage of -90 mV to +80 mV (step voltage of 10 mV), Figure 5 (inset B) shows the IV (current-voltage) relationship curve of the Nav1.7 channel before and after administration of the polypeptide PPN-01, and Figure 5 (inset C) shows the relationship between the current inhibition percentage caused by 300 nM polypeptide PPN-01 and the membrane potential.

[0072] As shown in Figure 5 (inset A), current inhibition was observed at all potentials tested that activated Nav1.7 channels, indicating that current blockade induced by the peptide PPN-01 occurred across the entire potential activation range. As shown in Figure 5 (inset B and C), the peptide PPN-01 produced voltage-dependent inhibition of current between -40 mV and +40 mV, with the current inhibition rate increasing from 43.57% ± 6.95% at -40 mV to 73.57% ± 6.26% at -30 mV (n = 6, P < 0.01), and then from 73.57% ± 6.26% at -30 mV to 89.59% ± 2.46% at +40 mV (n = 6, P < 0.05). These results indicate that the peptide PPN-01 has a stronger inhibitory effect on Nav1.7 channels in the open state.

[0073] Example 6 Effects of different polypeptides on the acetic acid writhing model in mice

[0074] The purpose of this example is to use patch clamp technology to study the effects of different peptides on the mouse acetic acid writhing model.

[0075] Seventy SPF-grade healthy C57BL / 6 mice (half male and half female, weighing 22-25 g and aged 8-10 weeks) were randomly selected. The 70 C57BL / 6 mice were weighed and numbered from low to high according to weight (S1 to S70). The mice were randomly divided into seven groups using a random number table: a saline group and an experimental group. The experimental groups included the PPN-01 group, the PPN-04 group, the PPN-06 group, the PPN-11 group, the PPN-12 group, and the PPN-16 group. The drug concentration in each group (i.e., the concentration of the added peptides PPN-01, PPN-04, PPN-06, PPN-11, PPN-12, and PPN-16) was 10 mg / kg, and there were 10 mice in each group.

[0076] Normal saline group: 10 healthy mice were routinely raised for one week and intraperitoneally injected with normal saline at a dose of 0.1 mL / 10 g once for 7 consecutive days. 15 minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was intraperitoneally injected and the writhing behavior of each mouse was observed.

[0077] Experimental group: 10 healthy mice were routinely housed for one week and intraperitoneally injected with 10 mg / kg of different polypeptides at a dose of 0.1 mL / 10 g for 7 consecutive days. 15 minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was intraperitoneally injected and the writhing behavior of each mouse was observed.

[0078] During the above experiment, the time when each group of mice began to writhe (writhing latency) and the number of writhing times within 15 minutes were recorded immediately after the injection of glacial acetic acid solution. A complete writhing reaction was defined as the occurrence of behavioral responses such as abdominal concavity, trunk and hind limb extension, and buttocks elevation. The results are shown in Table 3 and Figure 6.

[0079] Table 3 Effects of different peptides on the acetic acid writhing model in mice

[0080] As shown in Table 3 and Figure 6, the number of writhing times in the experimental group was less than that in the saline group. This indicates that the peptides PPN-01, PPN-04, PPN-06, PPN-11, PPN-12, and PPN-16 all have a certain analgesic effect on the acetic acid writhing model in mice, thereby significantly reducing the number of writhing times in mice.

[0081] Example 7 Effects of different concentrations of polypeptide PPN-01 on the acetic acid writhing model in mice

[0082] The purpose of this example is to use patch clamp technology to study the effects of different concentrations of polypeptide PPN-01 (its amino acid sequence is shown in SEQ ID NO: 3) on the acetic acid writhing model in mice.

[0083] Forty SPF healthy C57BL / 6 mice (half male and half female, weighing 22-25 g and aged 8-10 weeks) were randomly selected. The 40 C57BL / 6 mice were weighed and numbered from low to high weight (1 to 40). The mice were randomly divided into five groups using a random number table: saline group, low-dose group (2.5 mg / kg), medium-dose group (10 mg / kg), high-dose group (40 mg / kg), and diclofenac sodium group (30 mg / kg), with 8 mice in each group.

[0084] Normal saline group: 8 healthy mice were routinely raised for one week and intraperitoneally injected with normal saline at a dose of 0.1 ml / 10 g once for 7 consecutive days. 15 minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was intraperitoneally injected and the writhing behavior of each mouse was observed.

[0085] Low-dose group (2.5 mg / kg): 8 healthy mice were routinely housed for one week and intraperitoneally injected once daily with a 0.5 μM solution of the peptide PPN-01 at a dose of 10 mL / kg for 7 consecutive days. The mice were given a dose of 2.5 mg / kg. 15 minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was intraperitoneally injected and the writhing behavior of each mouse was observed.

[0086] Medium-dose group (10 mg / kg): 8 healthy mice were housed conventionally for one week and intraperitoneally injected once daily with a 2 μM solution of the peptide PPN-01 at a dose of 10 mL / kg for 7 consecutive days. 15 minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was intraperitoneally injected, and the writhing behavior of each mouse was observed.

[0087] High-dose group (40 mg / kg): 8 healthy mice were housed conventionally for one week and intraperitoneally injected once daily with 8 μM PPN-01 solution at a dose of 10 mL / kg. The mice were given a dose of 40 mg / kg for 7 consecutive days. 15 minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was intraperitoneally injected, and the writhing behavior of each mouse was observed.

[0088] Diclofenac sodium group (10 mL / kg): 8 healthy mice were housed conventionally for one week and intraperitoneally injected once daily with 3 mg / mL diclofenac sodium solution at a dose of 10 mL / kg for 7 consecutive days. The mice were given a dose of 30 mg / kg. 15 minutes after the last injection, 0.7% glacial acetic acid (10 mL / kg) was intraperitoneally injected, and the writhing behavior of each mouse was observed.

[0089] During the above experiment, the time to onset of writhing (writhing latency) and the number of writhings per mouse within 15 minutes were recorded immediately after injection of glacial acetic acid solution. A complete writhing reaction was defined as the occurrence of behavioral responses such as abdominal indentation, trunk and hind limb extension, and buttocks elevation. The analgesic effect of the peptide PPN-01 was evaluated using the inhibition rate (%) and analgesia rate (%).

[0090] Inhibition rate (%) = [(mean latency of drug group - mean latency of saline group) / mean latency of saline group] × 100%

[0091] Analgesia rate (%) = [(mean number of writhing times in the saline group - mean number of writhing times in the drug group) / mean number of writhing times in the saline group] × 100%

[0092] All statistical data were analyzed using one-way ANOVA between the two groups, with P < 0.05 considered statistically significant (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001). All statistical data were plotted using Graphpad Prism 9.0 software.

[0093] The writhing inhibition rates of different concentrations of polypeptide PPN-01 on mice in each group are shown in Table 4 , and the writhing times of mice in each group after injection of different concentrations of polypeptide PPN-01 are shown in FIG7 .

[0094] Table 4 Results of writhing inhibition rate of mice in each group

[0095] As shown in Table 4 and Figure 7, after intervention with diclofenac sodium or different concentrations of the peptide PPN-01, the writhing reactions caused by inflammatory pain in mice decreased. As the dosage of the peptide PPN-01 increased, the inhibition rate of mouse writhing increased, reaching a maximum of 75.7%, which was slightly lower than that in the diclofenac sodium group.

[0096] Example 8 Effects of PPN-01 on the Electrical Signals of Rat Sciatic Nerve

[0097] The purpose of this example is to study the effect of the polypeptide PPN-01 (its amino acid sequence is shown in SEQ ID NO: 3) on the electrical signals of the sciatic nerve of rats.

[0098] Four SPF-grade healthy Sprague-Dawley (SD) rats (half male and half female, weighing 180-200 g, aged 6-8 weeks) were randomly selected. The rats were first acclimated for 1 week and fasted for 12 hours before the experiment, with free access to water. Rats weighing between 180 and 200 g were anesthetized with 7% chloral hydrate solution at a dose of 0.4 mL / 100 g. Complete anesthesia was indicated by the absence of a toss-over reflex and a lack of response to pinching the tail. The anesthetized rats were then placed on an operating table with their backs facing upward and their paws secured with a rope. The skin and connective tissue of the legs were cut open in sequence, and the muscles between the biceps femoris and gluteus superficialis were bluntly dissected. The sciatic nerve was then delineated between the two muscles using a glass needle. Two sutures were passed through the two ends of the sciatic nerve to facilitate subsequent experiments.

[0099] Electrodes specifically designed to record nerve impulses were placed on the sciatic nerve. The electrical signals generated by these nerve impulses were converted into digital signals by a multi-channel physiological signal acquisition and processing system. These signals were displayed on a computer as compound action potentials (CAPs), which were then stored and analyzed later. Each experiment was performed alternately between the left and right legs. The sciatic nerve was periodically moistened with saline solution to prevent prolonged exposure to air.

[0100] In the PPN-01 group, the sciatic nerve of one rat was isolated and an electrode specifically designed to record nerve impulses was placed on it. CAP was recorded. The nerve was allowed to stabilize for 5 minutes, during which time the nerve was moistened with 0.9% saline. After 5 minutes, recording began. 20 μL of 0.9% saline was dripped onto the sciatic nerve and recorded for 5 minutes. The sole of the rat's foot was then stimulated with a syringe needle and recorded for another 5 minutes. Subsequently, 20 μL of low (30 nM), medium (100 nM), and high (300 nM) concentrations of PPN-01 were dripped onto the sciatic nerve. Each reagent was removed with a tissue before the next one was added. Recording was performed in the same manner as with saline. The other sciatic nerve was first dripped with PPN-01, rinsed, and then dripped with saline as a control. The order of drug administration and saline application was altered between different rats.

[0101] Negative control drug lidocaine group: isolate the sciatic nerve on one side of the rat and place an electrode specifically for recording nerve impulses on the sciatic nerve to record CAP. First, stabilize it for 5 minutes. During this period, the sciatic nerve needs to be moistened with 0.9% saline. After 5 minutes, start recording. Draw 20 μL of 0.9% saline and drip it onto the sciatic nerve. Record for 5 minutes. Then, stimulate the sole of the rat with a syringe needle and record for another 5 minutes. Then, drip 20 μL of lidocaine onto the sciatic nerve. Record for 5 minutes. Then, stimulate the sole of the rat with a syringe needle and record for 5 minutes. The bilateral control method for different rats is the same as above.

[0102] The rats in the polypeptide PPN-01 group and the negative control drug lidocaine group were observed, and the inhibition rate was used to evaluate the effect of each concentration of polypeptide PPN-01 on the peak amplitude of the positive and negative phases of the sciatic nerve CAP of the rats. The inhibition rate was calculated according to the following formula: inhibition rate (%) = [(peak amplitude of the drug group - peak amplitude of the negative control group) / peak amplitude of the negative control group] × 100%.

[0103] Four to six CAPs within 2 minutes after each recording period were selected for analysis, and the average peak amplitude was calculated as the representative CAP peak amplitude and duration of each group. The peak amplitude of CAP was the difference between the baseline and peak CAP levels.

[0104] The results of the effect of polypeptide PPN-01 on the electrical signals of the sciatic nerve of rats are shown in Figure 8, wherein the inner panel A of Figure 8 shows the representative trajectories of the compound action potential of the sciatic nerve of rats before and after acupuncture before and after lidocaine administration, the inner panel B of Figure 8 shows the representative trajectories of the compound action potential of the sciatic nerve of rats in each group with different concentrations of polypeptide PPN-01 before and after acupuncture, the inner panel C of Figure 8 shows the statistical analysis results of the comparison of the positive phase amplitude of CAP of rats in the different concentrations of polypeptide PPN-01 groups and lidocaine groups before and after acupuncture, and the inner panel D of Figure 8 shows the statistical analysis results of the comparison of the negative phase amplitude of CAP of rats in the different concentrations of polypeptide PPN-01 groups and lidocaine groups before and after acupuncture. The above data are expressed as mean ± standard deviation (x ± s); “*”, “**” and “***” indicate P < 0.05, P < 0.01 and P < 0.001, respectively, compared with the normal saline group before acupuncture; “###” indicates P < 0.001 compared with the normal saline group after acupuncture; “$” indicates P < 0.05 compared with the lidocaine group after acupuncture.

[0105] As shown in Figure 8, the peak amplitude of the normal saline group before and after acupuncture was the largest. After drug intervention, the peak amplitude of each group was reduced to varying degrees. Compared with the negative control group, all concentrations of PPN-01 groups before and after acupuncture could significantly reduce the peak amplitude of CAP (P<0.05). Among them, the high-dose PPN-01 group had the largest inhibition rate on the positive and negative phase peak amplitudes, and the low-dose PPN-01 group had the smallest inhibition rate. Compared with the positive control drug lidocaine, the high-dose PPN-01 group before acupuncture had the largest inhibition rate, and the lidocaine group after acupuncture had the largest inhibition rate. Compared with the high-dose PPN-01 group, the low-dose PPN-01 group and the medium-dose PPN-01 group had smaller inhibition rates before and after acupuncture, but the difference between the two and the high-dose PPN-01 group was not statistically significant (P>0.05). The above results show that the polypeptide PPN-01 can effectively reduce the peak amplitude of the positive and negative phases of the CAP in rat sciatic nerve. This effect is similar to that of lidocaine (P>0.05), and the higher the drug dose, the more obvious its effect.

[0106] In summary, the polypeptide provided in this application can selectively inhibit the activation of the voltage-gated Nav1.7 sodium ion channel, thereby inhibiting pain. The polypeptide provided in this application is used in the preparation of analgesic drugs. The prepared drugs have good analgesic effects and are of extremely important clinical significance for pain diseases.

[0107] The above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present application can be modified or replaced by equivalents, but these modifications or replacements are all within the scope of protection of the present application.

Claims

1. A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; The 12th amino acid in the amino acid sequence of the polypeptide is alanine or glutamic acid; The 19th amino acid in the amino acid sequence of the polypeptide is methionine or leucine; The 28th amino acid in the amino acid sequence of the polypeptide is lysine or isoleucine; The 29th amino acid in the amino acid sequence of the polypeptide is selected from one of isoleucine, leucine and tryptophan.

2. The polypeptide according to claim 1, wherein: The amino acid sequence of the polypeptide is selected from one of SEQ ID NOs: 3 to 18.

3. A nucleic acid molecule, wherein: The nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide according to claim 1 or 2.

4. A recombinant expression vector, wherein: The recombinant expression vector comprises the nucleic acid molecule according to claim 3.

5. A recombinant host cell, wherein: The recombinant host cell contains the recombinant expression vector according to claim 4.

6. Use of the polypeptide according to claim 1 or 2 in the preparation of a drug having analgesic function.

7. Use of the polypeptide according to claim 6 in the preparation of a drug with analgesic function, wherein the drug can inhibit the activation of voltage-gated sodium ion channels.

8. Use of the polypeptide according to claim 7 in the preparation of a drug having analgesic function, wherein: The voltage-gated sodium ion channel includes a Nav1.7 channel.

9. Use of the polypeptide according to claim 6 in the preparation of a drug with analgesic function, wherein the drug with analgesic function is in the form of a colloidal solution, an emulsion, a suspension, an oral fast-dissolving film, an oral solution, a capsule, an injection, or a transdermal absorption preparation.

10. A pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the polypeptide according to claim 1 or 2.

11. Use of the polypeptide according to claim 1 or 2 in analgesia.

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