Polypeptide capable of passing through blood-brain barrier

Abstract

The present invention provides a polypeptide capable of crossing the blood-brain barrier. In the present invention, C-terminal of the ziconotide is linked to N-terminal of a cell membrane penetrating peptide via one glycine to obtain a polypeptide capable of crossing the blood-brain barrier. The polypeptide of the present invention is suitable for intravenous, intraperitoneal or nasal administration with convenient operation and low clinical risk. It has a long pharmacological effect in vivo, excellent analgesic effect, and slight peptide side effect after intravenous, intraperitoneal or nasal administration, and is suitable for large-scale clinical applications. The polypeptide of the invention has the advantages of simple preparation, controllable preparation process and quality during the preparation, and is suitable for large-scale industrial production.

Claims

1. A polypeptide comprising ziconotide, wherein the polypeptide is able to cross blood-brain barrier, wherein the polypeptide consists of ziconotide and a cell membrane penetrating peptide, or the polypeptide consists of ziconotide, a cell membrane penetrating peptide and a linker, wherein C-terminal of the ziconotide is linked to N-terminal of a cell membrane penetrating peptide adjacently or via a linker, and wherein the ziconotide has amino acids shown in SEQ ID NO.1, or the ziconotide is a variant of the amino acids shown in SEQ ID NO.1 with less than 4 amino acid deletions, mutations or insertions.

2. The polypeptide according to claim 1, wherein the linker is one, two or three glycines.

3. The polypeptide according to claim 1, wherein the cell membrane penetrating peptide is selected from: Penetratin, TAT peptide, Pep-1 peptide, S4.sub.13-PV, Magainin 2 or Buforin 2.

4. The polypeptide according to claim 3, wherein the TAT peptide has amino acids shown in SEQ ID NO.2, or the TAT peptide is a variant of the amino acids shown in SEQ ID NO.2 with less than 4 amino acid deletions, mutations or insertions.

5. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide is shown in anyone of SEQ ID NO.3-6.

6. A pharmaceutical composition comprising the polypeptide of claim 1 and an acceptable carrier.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is to be administered intravenously, intraperitoneally or nasally, and the pharmaceutical composition is in a dosage form for intravenous, intraperitoneal or nasal administration.

8. A method for preparing a polypeptide, the method comprises synthesizing a polypeptide comprising ziconotide, wherein the polypeptide is able to cross blood-brain barrier, wherein the polypeptide consists of ziconotide and a cell membrane penetrating peptide, or the polypeptide consists of ziconotide, a cell membrane penetrating peptide and a linker, wherein C-terminal of the ziconotide is linked to N-terminal of a cell membrane penetrating peptide adjacently or via a linker, and wherein the ziconotide has amino acids shown in SEQ ID NO.1, or the ziconotide is a variant of the amino acids shown in SEQ ID NO.1 with less than 4 amino acid deletions, mutations or insertions.

9. The pharmaceutical composition according to claim 6, wherein the amino acid sequence of the polypeptide is shown in anyone of SEQ ID NO.3-6.

10. The method according to claim 8, wherein the amino acid sequence of the polypeptide is shown in anyone of SEQ ID NO.3-6.

11. A method of treating pain or pain-related diseases, comprising: administering the pharmaceutical composition according to claim 6 to a subject in need thereof.

12. The method according to claim 11, wherein the pharmaceutical composition is administered intravenously, intraperitoneally or nasally.

13. The method according to claim 11, wherein the amino acid sequence of the polypeptide is shown in anyone of SEQ ID NO.3-6.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: HPLC analysis profiles of one-step oxidation folding of MVIIA and MVIIA-a, b, c, d;

(2) FIG. 2: circular dichroism spectra of MVIIA and MVIIA-a,b,c,d, wherein the final concentration of each peptide was 35 μmol/L dissolved in phosphate buffer (10 mM, pH=7.2) solution, respectively;

(3) FIG. 3: inhibitory effects of MVIIA and MVIIA-a, b, c, d on CaV2.2 channel currents. The dose-response curve for MVIIA was shown in FIG. 3A, and the dose-response curve for MVIIA variants were shown in FIG. 3B-3E. Data of half inhibitory concentration and the slope value were shown in the figure, and data were presented as mean±S.E.M., with 5 mice in each group. As shown in FIG. F, superimposed traces of whole-cell calcium channel currents elicited by a voltage step from −80 mV to 10 mV at 10 μM L-MVIIA (blue) and 2 μM MVIIA (red); FIG. G was a summary table on the half inhibitory concentrations of MVIIA and its variants;

(4) FIG. 4: comparison results of MVIIA and MVIIA-c on hot-plate pain. In vivo antinociceptive effects after intracerebroventricular administration of MVIIA (FIG. 4A), and tail intravenous administration of MVIIA (FIG. 4B) and MVIIA-c (FIG. 4C). The antinociceptive effect was expressed as reaction latency. Data were presented as mean±S.E.M., with 6-8 mice in each group. *p<0.05, **p<0.01 and ***p<0.001 indicated comparison with the normal saline group (data were analyzed by repeated multivariate analysis of variance and Duncan's multiple range test);

(5) FIG. 5: results of MVIIA-a, b, d in hot-plate pain test. FIG. 5A-FIG. 5C showed in vivo antinociceptive effects after tail intravenous administration of MVIIA-a, b, d polypeptides. Antinociceptive effects were expressed as a percentage of the maximum possible effect (% MPE). Data were presented as mean±S.E.M., with 8-10 mice in each group. *p<0.05, **p<0.01 and ***p<0.001 indicated comparison with the normal saline group;

(6) FIG. 6: antinociceptive effects of MVILA and MVIIA-a, b, d in acetic acid-induced writhing test. The number of writhing responses was counted from 5 minutes to 20 minutes after intraperitoneal injection of 1% acetic acid; as shown in FIG. A, comparison of effects of intraperitoneal injection of 1% acetic acid at 30 minutes after intracerebroventricular administration; as shown in FIG. B, comparison of effects of intraperitoneal injection of 1% acetic acid at 30 minutes after intravenous administration; #, compared with normal saline group; *, compared with MVIIA group; &, compared with MVIIA-C group; *, #, &, p<0.05; ***, ###, &&&, p<0.001. Data were presented as mean±S.E.M., with 9-11 mice in each group;

(7) FIG. 7: effects of MVIIA and MVIIA-a, b, c, d on tremor time in mice. The peptides (0.9 nmol/kg) and normal saline were administered intracerebroventricularly to the mice in a volume of 6 μL. After 30 and 120 min, the accumulative tremor time (s) were recorded during a period of 5 min. Data were presented as mean±S.E.M. (n=12);

(8) FIG. 8: mass spectrum for MVIIA;

(9) FIG. 9: mass spectrum for MVIIA-a;

(10) FIG. 10: mass spectrum for MVIIA-b;

(11) FIG. 11: mass spectrum for MVIIA-c;

(12) FIG. 12: mass spectrum for MVIIA-d;

(13) FIG. 13: antinociceptive abilities of MVIIA and different doses of MVIIA-c after nasal administration;

(14) FIG. 14: antinociceptive abilities of MVIIA-a, b, d during nasal administration in the hot-plate pain test.

DETAILED DESCRIPTION OF THE INVENTION

(15) In order to overcome the shortcomings of ziconotide in the prior art, the inventor have discovered through long-term research that an improved ziconotide fusion peptide obtained by linking C-terminal of ziconotide with N-terminal of a membrane penetrating peptide is suitable for intravenous or abdominal administration. In order to further investigate analgesic effects of different types of improved ziconotide, the present invention has designed and synthesized a variety of fusion polypeptides with different types and structures, including, a fusion polypeptide formed by directly linking C-terminal of ziconotide and N-terminal of a cell membrane penetrating peptide without a linker; a fusion polypeptide constructed using one or more glycines as linkers. Further, structural characterization of the above different types of fusion polypeptides, cell experiment, in vivo experiment, and side effect verification experiment are performed to illustrate effects of different types of improved ziconotide

(16) In order to better understand the technical solutions of the present invention, detailed descriptions are given below in conjunction with examples.

Example 1 Preparation of Different Types of Ziconotide Fusion Peptides

(17) Four different types of fusion peptides were prepared and named as protected polypeptides MVIIA-a, MVIIA-b, MVIIA-c, and MVIIA-d. Meanwhile, ziconotide was prepared and named MVIIA as a control. F-moc automatic solid-phase synthesis method was used in this experiment with the specific steps of:

(18) Synthesis of peptides: Protected peptides and their derivatives were assembled on the resin using model 433A automatic synthesizer (ABI, Foster City, CA). The peptide resin was incubated at room temperature in a suspension for 2.5 hours to remove protecting groups. The suspension system was composed of 10 ml TFA, 0.75 g phenol, 0.25 ml 1,2-ethanedithiol, 0.5 ml thioanisole and 0.5 ml water. (fluorenylmethoxycarbonyl (Fmoc), a common alkoxycarbonyl amino protecting group). The resin was separated from the peptide deprotection mixture by filtration. The crude polypeptide was precipitated in 150 ml of pre-cooled ether solution, and chromatographic purification was carried out on a Sephadex G-25 column with 10% glacial acetic acid as the eluent. Subsequently, the peptide-containing components were pooled and lyophilized, and the purity of the crude peptide was determined to be about 80% using high performance liquid chromatography.

(19) Peptide folding: MVIIA comprised six cysteine residues to maintain its three disulfide bond structure. Folding under oxidative conditions could produce a variety of isomers. After screening for redox system, buffer, salt, concentration and temperature, two efficient folding conditions of MVIIA were selected: (a) 0.5 M NH4Ac buffer (pH 7.9), which contained 1 mM GSH, 0.1 mM GSSG, 1 mM EDTA, and 0.2 mg/mL MVIIA; (b) 0.5 M NH4Ac buffer, which contained 1 mM cysteine, 1 mM EDTA, and 0.2 mg/mL MVIIA. At 4° C., the linear polypeptide MVIIA was folded for 48-72 hours under the a condition and for 24-48 hours under the b condition.

(20) Peptide purification and characterization: After the oxidation of MVIIA, the reaction mixture was acidified (pH <4.5) with acetic acid, and then filtered. The filtrate was directly loaded onto a Zorbax 21.2×250 mm C18 liquid chromatography column, which used a preparative high performance liquid chromatography pump (Waters 2000 series, Milford, MA). The C18 column was first pre-washed with buffer A (0.1% TFA in water), followed by linear gradient elution with 10-40% buffer B (0.1% TFA in acetonitrile) at a rate of 8 mL/min for 40 minutes. The obtained fraction was a concentrate containing 90% MVIIA, which was then further purified by semi-preparative reversed-phase high performance liquid chromatography equipped with a 9.4×250 mm Zorbax C18 liquid chromatography column. Finally, 20% acetic acid solution was used as eluent in a Sephadex G-25 chromatography column to convert the final product from the TFA salt solution to the acetate solution. The purity of the peptides was evaluated by analytical reversed-phase high performance liquid chromatography. For the evaluation, a linear gradient elution with 8-40% buffer B (0.1% TFA in acetonitrile) at a flow rate of 1 ml per minute for 25 minutes was performed by using a Zorbax C18 liquid chromatography column (4.6×250 mm). Finally, the purity of the final product (i.e., the peptide) was 98%.

Example 2: Chemical Properties and Structural Characterization of Different Types of Ziconotide Fusion Peptides

(21) 1. Chemical Properties of MVIIA and its Variants

(22) At 4° C., the linear peptide was treated with buffer for 24-48 hours, and then analyzed by high performance liquid chromatography. It was found that the folding of the linear peptide resulted in a major peak and several small peaks. The buffer system contained 1 mM glutathione, 0.1 mM oxidized glutathione, 1 mM EDTA, and 0.2 mg/mL linear peptide, at pH of 7.9. The main product was purified and evaluated by analytical reverse-phase high performance liquid chromatography, and the purity of the peptide was determined to be more than 98%. The determination was made with an Ultraflex III TOF/TOF mass spectrometer (Bruker). The prepared polypeptide sequences were shown in Table 1, and their one-step oxidation folding HPLC analysis profiles were shown in FIG. 1.

(23) TABLE-US-00001 TABLE 1 Prepared peptide sequences Name Sequence MVIIA (SEQ ID NO. 1) CKGKGAKCSRLMYDCCTGSCRSGKC MVIIA-a (SEQ ID NO. 4) CKGKGAKCSRLMYDCCTGSCRSGKCYGRKKRRQRRR MVIIA-b (SEQ ID NO. 5) CKGKGAKCSRLMYDCCTGSCRSGKCGYGRKKRRQRRR MVIIA-c (SEQ ID NO. 3) CKGKGAKCSRLMYDCCTGSCRSGKCGGYGRKKRRQRRR MVIIA-d (SEQ ID NO. 6)) CKGKGAKCSRLMYDCCTGSCRSGKCGGGYGRKKRRQRRR

(24) 2. Circular Dichroism Spectroscopy

(25) Peptides were dissolved in PBS (10 mM, pH=7.2) solution to final concentration of 35 μM. At room temperature, Chirascan Plus spectropolarimeter (Applied Photophysics Ltd., Leatherhead, Surrey, UK) instrument was used to detect the circular dichroism spectroscopy in the wavelength range of 190 nm to 260 nm. Detection parameters were set as follows: step resolution 1.0 nm; speed 20 nm/min, and cell path length of 1.0 mm.

(26) As shown in FIG. 2, MVIIA presented an obvious β-sheet structure between 195 nm-205 nm. We found that TAT variants showed a similar random coil structure with an obvious negative band at about 200 nm, suggesting the secondary structure of the peptides remained unchanged when the length of the linker between MVIIA and TAT sequence expanded. The molar ellipticity of TAT variants was deeper when linker expanded, suggesting that the expansion of the linker between MVIIA and TAT sequences helped to form a random coil. The exact molecular weights of the product peptides identified by the method of mass spectrometry (using Voyager MALDI-TOF spectrometer) was shown in Table 2, and the mass spectra of MVIIA and MVIIA-a, b, c, d were shown in FIG. 8-12. The bridging pattern of disulfide bonds was assigned based on the method that partially reduced cysteine coupling and amino acid silencing. The results of high performance liquid chromatogram and circular dichroism spectroscopy of the synthesized peptides and MVIIA standard product were consistent.

(27) TABLE-US-00002 TABLE 2 Molecular weights of MVIIA and its variants Difference between Theoretical Measured theoretical value and Sample MW m/z actual measured value MVIIA 2645.54 2639.0198 6.5202 MVIIA-a 4186.0784 4180.0108 6.0676 MVIIA-b 4243.0978 4237.0300 6.0678 MVIIA-c 4299.1353 4292.0362 7.0991 MVIIA-d 4356.1568 4351.0842 5.0726

Example 3: Electrophysiological Experiment of Different Types of Ziconotide Fusion Peptides

(28) In order to further investigate electrophysiological effects and inhibitory effect on calcium (CaV2.2) channels of different types of modified ziconotide, the following experiments were carried out:

(29) HEK293T cells (expressing SV40 large T antigen) were cultured in DMEM high glucose medium (Gibco) containing 10% fetal bovine serum, 1% penicillin and streptomycin, at the incubator environment of 37° C., 5% CO.sub.2. Rat CaV2.2 channel α.sub.1B splice variant e37a, auxiliary subunit α.sub.2δ.sub.1 and β.sub.3 plasmids (Addgene plasmid #26569, #26575, #26574) were provided by Dr. Diane Lipscombe. Three plasmids (3 μg), 0.4 μg enhanced green fluorescent protein gene and liposomes were then transiently co-transfected into HEK293T cells. 24 hours after transfection, the cells were seeded on glass slides and cultured in an incubator (37° C., 5% CO.sub.2) for at least 6 hours, followed by electrophysiological recording.

(30) This study was recorded in accordance with the method of cell voltage clamp recording in previously published research literature (F. Wang et al., 2016). Briefly, recording electrodes, with a resistance of ˜3 MW, were filled with an internal solution. The internal solution contained the following: 135 mM CsCl, 10 mM NaCl, 10 mM HEPES, and 5 mM EGTA, and was adjusted to pH 7.2 with CsOH. The extracellular recording solution contained: 135 mM N-Methyl-D-glucamine, 10 mM BaCl.sub.2.Math.2H.sub.2O, 2 mM MgCl.sub.2.Math.6H.sub.2O and 10 mM HEPES, with a final solution pH of 7.4. Acquired currents were recorded at room temperature (˜22° C.) with a MultiClamp 700B amplifier (Molecular Devices, Sunnyvale, CA) and Clampex 10.3/Digidata1440A data acquisition system and digital-to-analog converter. Membrane currents were filtered at 2 kHz and sampled at 10 kHz. All data were analyzed with the data analysis system clampfit 10.3 software (Molecular Devices), presented as mean±S.E.M. Dose-response curves of toxin blocking N-type calcium ion current were obtained using GraphPad Prism (GraphPad Software, San Diego, CA) by plotting the inhibition of current amplitude as a function of drug concentration and were fitted using a hill equation.

(31) Primary amino acid sequences and electrophysiological activities of MVIIA and its variants MVIIA-a, b, c, d were shown in Table 3.

(32) TABLE-US-00003 TABLE 3 Primary amino acid sequences and electrophysiological  activities of MVIIA and its variants Peptides Primary amino acid sequences IC.sub.50 (μM)

(33) Inhibitory Effects on Calcium (CaV2.2) Channels Induced by MVIIA and its Variants

(34) It was well known that MVIIA was a selective CaV2.2 channel blocker. The inhibitory effect of 2 μM MVIIA on CaV2.2 channel was more than 90%. (F. Wang. 2016, and other articles) In this study, we recorded the peak Ca.sup.2+ current (ICa) of CaV2.2 channels (α.sub.1B, α.sub.2δ.sub.1 and β.sub.3) in the 293T cells. All currents were evoked by a 100 ms voltage step from −80 my to 10 my. MVIIA, MVIIA-a, MVIIA-b, MVIIA-c and MVIIA-d treatment at a concentration of 1 μM could reduce the peak Ca.sup.2+ current by 98.24±0.708%, 89.45±0.752%, 91.70±1.477%, 98.81±0.427% and 84.26±3.127%, respectively. We found that MVIIA-c and MVIIA had similar ability in blocking CaV2.2 channels. L-MVIIA showed a significantly reduced ability in blocking Cav2.2, with reduced ICa amplitude of 23.28±3.347% at a concentration of 10 μM. The concentration-response relationship for MVIIA inhibition of CaV2.2 channel had a half inhibitory concentration of 0.0436 μM, which was almost 5-10 folds larger than that of TAT variants. The half inhibitory concentrations of TAT variants (MVIIA-a, MVIIA-b, MVIIA-c and MVIIA-d) were 0.413, 0.379, 0.237, and 0.345 μM, respectively, as shown in FIG. 3. These results suggested that MVIIA-a, MVIIA-b, MVIIA-c and MVIIA-d had a certain inhibitory effect on Cav2.2 channels, and the length of the linker sequence between MVIIA and TAT variants could affect the binding ability to Cav2.2 channels.

Example 4: Antinociceptive Effect In Vivo of Different Types of Ziconotide Fusion Peptides

(35) 1. Hot-Plate Pain Test

(36) 1.1 Test Method

(37) In this test, a total of nine groups of 6-8 mice were intracerebroventricularly administered MVIIA (0.11, 0.33 or 1.00 nmol/kg), or were tail intravenously administered MVIIA and MVIIA-a, MVIIA-b, MVIIA-c and MVIIA-d (0.33, 1.00 or 3.00 μmol/kg). Normal saline was administered in each routes as vehicle treated groups. The animals were placed on a hot plate with a constant temperature of 55±0.5° C. The latency time was recorded from the placement on the heated surface to the first licking of the hind paws or jumping as an index of pain threshold (Eddy and Leimbach, 1953). A cut-off time of 60 s was used: the mouse was taken out after 60s to avoid tissue damage. The latency time was measured before administration as the baseline latency; subsequently, the latency time was recorded at 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 h after administration with MVIIA, MVIIA-c or Saline (intracerebroventricular or tail intravenous administration). Mice with a latency time less than 5 s or more than 20 s compared with the latency baseline time were subsequently eliminated to exclude hyposensitive or hypersensitive mice. The antinociceptive effect was expressed by latency time.

(38) 1.2 Comparison of Antinociceptive Ability

(39) As shown in FIG. 4, MVIIA (0.11, 0.33, or 1.00 nmol/kg) exhibited a maximal effect 1 h after intracerebroventricular administration, and the effect substantially disappeared 4 h after administration (FIG. 4A). But MVIIA showed no effect when administered via tail intravenously administrations at multiple doses (FIG. 4B). MVIIA-c was the TAT variant of MVIIA that had the strongest inhibitory effect on CaV2.2 channel current. As shown in FIG. 4C, MVIIA-c exhibited a maximal effect at 3 hours after administration, with the maximal effect lasting about 4 hours and the effect disappearing within 12 hours.

(40) As shown in FIG. 5, after tail intravenous injection of different doses of MVIIA-a, b, d (0.11 μmol/kg, 0.33 μmol/kg and 1.00 μmol/kg) for 1 hour, they all showed antinociceptive effects, and the strongest effect was presented at 2-3 hours, with the effect lasting about 4 hours. The effect was gradually decreased with time, and there was still a significant difference between the drug group and the saline group at 12 hours after the administration, with the effect lasting 12 hours.

(41) 2. Acetic Acid-Induced Writhing Test (Koster et al., 1959)

(42) 2.1 Test Method

(43) Animals were treated with three dosages of MVIIA-a, b, c, d peptide group (0.6, 1.8 and 5.4 nmol/kg, low, middle and high dosages in the figure), saline control group (saline), or three dosages of positive reference drug group MVIIA (0.11, 0.33 and 1.00 nmol/kg), low, middle and high dosages in the figure). For the writhing test, mice were administered MVIIA (intracerebroventricularly) or MVIIA-a,b,c,d (intracerebroventricularly) 30 minutes before intraperitoneal injection of 1% acetic acid, followed by measuring their antinociceptive activities in vivo. To test the ability of MVIIA and MVIIA-a, b, c, d to penetrate the blood brain barrier, mice were administered MVIIA and MVIIA-a, b, c, d via tail vein 3 hours before intraperitoneal injection of 1% acetic acid. Saline group was used as a blank control group (intracerebroventricular or tail intravenous administration). The number of writhing responses was recorded from 5 minutes to 20 minutes after acetic acid injection (Galeotti et al., 2008). The recorded number of writhing movements was characterized by abdominal muscles contractions accompanied with stretching of hind limbs and elongation of the body.

(44) 2.2 Comparison of Antinociceptive Ability

(45) In the acetic acid-induced writhing test, animals were treated with three dosages of MVIIA-a, b, c, d peptide group (0.6, 1.8 and 5.4 nmol/kg, low, middle and high dosages in FIG. 6), saline control group (saline), three dosages of positive reference drug group MVIIA (0.11, 0.33 and 1.00 nmol/kg, low, middle and high dosages in FIG. 6). The numbers of writhing movements of each group at three different dosages after intravenous and intracerebroventricular administration. It was found that the MVIIA-a, b, c, d peptide group and the positive reference drug group MVIIA reduced the numbers of writhing movements induced by acetic acid in a dose-dependent manner. Under the conditions of intracerebroventricular administration, MVIIA, MVIIA-a, b, c, d reduced the number of writhing movements in mice to (relative to the saline group): MVIIA 8.97%, 53.37%, 76.88%; MVIIA-A, 2.94%, 13.36%, 48.35%; MVIIA-B, 10.82%, 42.79%, 77.60%; MVIIA-C, 14.75%, 39.53%, 81.77%; MVIIA-D, 12.08%, 23.95%, 56.54%. Under the conditions of intravenous administration, the positive reference drug MVIIA did not reduce the numbers of writhing movements in mice, and MVIIA-a, b, c, d reduced the numbers of writhing movements in mice to (relative to the saline group): MVIIA-a, 10.47%, 27.82%, 30.03%; MVIIA-b, 17.08%, 45.94%, 51.79%; MVIIA-c, 19.81%, 49.30%, 62.95%; MVIIA-d, 6.33%, 35.86%, 47.57%, as shown in FIG. 6.

(46) In conclusion, from the above test results, it could be found that MVIIA-a, b, c, d peptides could show an antinociceptive effect after intravenous injection in a dose-dependent manner compared with MVIIA. In particular, in the case of middle and high dosages, MVIIA-a, b, c, d peptides could achieve good antinociceptive effects through intravenous injection and meet the needs of clinical application. Furthermore, compared with MVIIA, MVIIA-a, b, c, d showed effects up to 12 hours after intravenous injection and had a good sustained-release effect in vivo.

(47) The above antinociceptive tests were analyzed using one-way ANOVA, two-way ANOVA with repeated measures, and, between groups, Duncan or Newman-Cole test. All data were presented as mean±S.D. or S.E.M. or 95% confidence interval. The differences with p values less than 0.05 were considered statistically significant.

Example 5: Side Effects Test of Different Types of Ziconotide Fusion Peptides

(48) In order to further investigate the side effects of different types of modified ziconotide in vivo, the following test was carried out:

(49) 1. Test Method

(50) Tremor time was regarded as a typical side-effect for ziconotide. The tremortime was the total time recorded for the rhythmic oscillatory movements of the mouse limbs, head, and trunk in a period of time. The mice were randomly divided into groups: MVIIA (0.9 nmol/kg) group, MVIIA-a,b,c,d (0.9 nmol/kg) group and a normal control group (6 μL, intracerebroventricular administration; n=12, half females and half males). 30 minutes and 120 minutes after administration, the dynamic video of the mice within 5 minutes was recorded by a digital camera, and the accumulative tremor time (s) in the period of 5 minutes was scored by a blinded observer.

(51) The toxicology tests were analyzed using one-way ANOVA and Newman-Cole test. All data were presented as mean±S.D. or S.E.M. or 95% confidence interval. The differences with p values less than 0.05 were considered statistically significant.

(52) 2.1 Comparison of Side Effects

(53) As shown in FIG. 7, MVIIA induced more obvious tremor symptoms and longer tremor time 30 minutes after administration; compared with MVIIA, there were no significant differences in the tremor symptoms and longer tremor times induced by the peptides of each group 120 minutes after administration. It could be seen from the above results that there were no significant differences in side effects between MVIIA and MVIIA-a, b, c, d polypeptides. Moreover, at the beginning of administration, the side effects of MVIIA-a, b, c, d were lower than that of MVIIA. Therefore, the MVIIA-a, b, c, d polypeptides of the present application had less toxic and side effects.

Example 6: Comparison of Antinociceptive Tests for MVII-A Intracerebroventricular Administration and MVIIA-a, b, c, d Intranasal Administration

(54) 1.1 Test Method for Hot-Plate Pain

(55) Test method for hot-plate pain was as described above. In this test, a total of nine groups of 10 mice were intracerebroventricularly administered MVIIA (1.00 nmol/kg, 5 μl/10 g) as a positive control group (in the test, it was found that MVIIA intranasal administration had no effect), and the nasal cavity was administered normal saline (saline, 2 μl/10 g), MVIIA-C(3.3, 6.6 or 9.9 nmol/kg, 2 μl/10 g), respectively. The saline group served as the blank control group. The latency time was recorded 0.5, 1, 2, 3, 4, 6, 8, 10 h after intracerebroventricular administration of MVIIA, nasal administration of MVIIA-c and Saline. Mice with a latency time less than 5 s or more than 20 s compared with the latency baseline time were subsequently eliminated to exclude hyposensitive or hypersensitive mice.

(56) The antinociceptive effect was expressed as a percentage of the maximum possible effect (% MPE), and finally calculated by the following equation: % MPE=(T.sub.1−T.sub.0)×100/(T.sub.2−T.sub.0)

(57) Wherein T.sub.0 and T.sub.1 respectively represented the latency time before and after administration, and T.sub.2 was the limit time of each test.

(58) 1.2 Test Results

(59) FIG. 13 showed the antinociceptive ability of MVIIA and different dosages of MVIIA-c after nasal administration. FIG. 13 showed antinociceptive effects of MVIIA intracerebroventricular and MVIIA-c nasal administration in the hot plate pain test. After intracerebroventricular administration of MVIIA (1.00 nmol/kg), the effect lasted 4 hours. MVIIA-C (3.3, 6.6, 9.9 nmol/kg) showed immediate effect after nasal administration. The high-dose MVIIA-C lasted a long time, and it was still significantly different from the saline group at 8 hours, with the effect disappearing after 10 hours. *p<0.05, **p<0.01 and ***p<0.001 indicated comparison with the saline group.

(60) 1.3 Antinociceptive Test for MVIIA-a,b,d Intranasal Administration

(61) FIG. 14 showed the antinociceptive effects of MVIIA-a, b, d intranasal administration in hot-plate pain test. Similar to MVIIA-C, MVIIA-a,b,d (9.9 nmol/kg) showed immediate effect after nasal administration, and MVII-b was still significantly different from the saline group at 8 hours, with the effect disappearing after 10 hours. *p<0.05, ***p<0.001 indicated comparison with the saline group.

(62) In the above, the present invention has been described in detail with general instructions and specific embodiments, but on the basis of the present invention, some modifications or improvements can be made, which is obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the present invention.