CN114805481B

CN114805481B - Short peptide and slow release preparation with long-acting analgesic or/and long-acting local anesthetic effect by taking short peptide as carrier material

Info

Publication number: CN114805481B
Application number: CN202210193248.9A
Authority: CN
Inventors: 邱峰; 张文胜; 刘静; 彭飞; 吴薇薇
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-02-28
Filing date: 2022-02-28
Publication date: 2023-06-23
Anticipated expiration: 2042-02-28
Also published as: CN114805481A

Abstract

The invention belongs to the technical field of nano material drug loading, and particularly provides a short peptide and a slow release preparation taking the short peptide as a carrier material and having long-acting analgesic or/and long-acting local anesthetic effects, wherein the amino acid sequence of the short peptide is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8. The short peptide can be used as a carrier of local anesthetic, and the local anesthetic drug slow-release preparation taking the short peptide as the carrier has the advantages of large drug loading, high encapsulation efficiency, good stability, excellent nerve blocking effect, capability of remarkably prolonging the effective acting time, excellent systemic safety and local safety, and wide application prospect in preparing drugs with long-acting analgesic or/and long-acting local anesthetic effect.

Description

Short peptide and slow release preparation with long-acting analgesic or/and long-acting local anesthetic effect by taking short peptide as carrier material

Technical Field

The invention belongs to the technical field of drug carrier materials, and mainly relates to a short peptide and a slow release preparation taking the short peptide as a carrier material and having long-acting analgesic or/and long-acting local anesthetic effects.

Background

It is counted that about 3.13 million patients undergo surgery (Bull World Health Organ,2016,94 (3): 201-9F) each year worldwide, and postoperative pain is a common adverse effect in the perioperative period. About 41% of patients develop moderate to severe pain after surgery (European journal of anaesthesiology,2008,25 (4): 267-74), while 10-50% of them change from acute pain after surgery to chronic pain (The Lancet,2006,367 (9522): 1618-25). The postoperative pain not only increases the occurrence risk of adverse events such as cardiovascular diseases, and the like, but also brings certain damage to the psychological health of patients, seriously influences the prognosis of the patients, and also increases social medical burden. Therefore, good analgesic management is important to promote rapid patient recovery. Local anesthetic drugs have been used more and more clinically in recent years as a major member of multi-modal analgesic management.

Peri-operative acute pain is mainly manifested in 3 days post-operative, especially 24 hours post-operative. In the existing local anesthetic drugs commonly used in clinic, even though the long-acting bupivacaine and ropivacaine are compatible with epinephrine, the acting time after single administration is not longer than 12 hours, and the existing analgesic requirement is difficult to meet. Although continuous administration can be achieved by intrathecal or paraspinal catheterization to extend the analgesic duration, this approach is associated with the risk of infection, hematoma, etc. With the development of drug delivery systems, more and more carrier materials are applied to the field of biological medicine, and the development of local anesthetic drug slow-release preparations by using slow-release materials also starts to show important clinical significance for prolonging the analgesic action time. At present, different materials such as liposome, microsphere, hydrogel, solid lipid nanoparticle and the like, and a mixed system of the materials are used for developing a local anesthetic drug slow-release preparation.

Although the microspheres using PLA or PLGA and other polymer materials as components can effectively encapsulate local anesthetic drugs and achieve the purpose of prolonging the analgesic effect time through slow release, the degradation time of the microspheres can reach 15-50 days (Biomaterials, 1999,20 (20): 1919-24;Verfahrenstechnik eV,2003,55 (2): 229-36), and certain safety problems can exist. The PLGA-PEG-PLGA hydrogel is used for encapsulating ropivacaine, the analgesic time in a rat plantar incision pain model is as long as 48 hours (Chem Pharm Bull (Tokyo), 2017,65 (3): 229-35), but similar to the high polymer microsphere, the degradation speed of the high polymer hydrogel is too slow, residues are still visible at the injection site 14-21 days after administration (ACS Biomater Sci Eng,2019,5 (2): 696-709;Acta biomaterialia,2018,67 (99-110), and the safety hazard is brought about.

Compared with high molecular polymer microspheres, the liposome has certain advantages in biocompatibility. Bupivacaine liposome injection "Exparel" developed by Pacifia Biosciences, america, used multivesicular liposome entrapped bupivacaine base, clinical studies showed that bupivacaine liposome injection could significantly reduce pain scores within 72 hours post-surgery (Current medical research and opinion,2012,28 (10): 1609-15), approved by the FDA in 2011. However, recent two years of clinical studies have shown that the actual analgesic effect of Exparel is very limited (Annals of surgery,2020 Dec 2.doi:10.1097/SLA.000000004424; am J ObstetGynecol,2018,219 (5): 500.e1-. E8; am J ObstetGynecol,2021,224 (1): 70.e1-. E11). In addition, the liposome has the advantages of complex preparation, poor stability and high clinical popularization and application cost.

Therefore, there is a need to develop a carrier material with good biocompatibility and simple preparation as a drug delivery system for local anesthetic drugs to achieve long-acting analgesic or long-acting local anesthetic effects.

Disclosure of Invention

The invention provides a self-assembled short peptide, a local anesthetic drug slow-release drug delivery system taking the self-assembled short peptide as a carrier material and a preparation method thereof, and aims to solve the problems that a high polymer material used in the existing local anesthetic drug slow-release preparation is slow in degradation, high in toxicity, not remarkable in analgesic effect, complex in preparation and the like.

The invention specifically provides a short peptide, the amino acid sequence of which is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.

The invention also provides a nano-structure carrier which is obtained by self-assembling one or more than two of the above short peptides in aqueous solution.

Further, the aqueous solution is water or a buffer solution, preferably a phosphate buffer solution.

Further, the aqueous solution has a pH of 7.4 to 8.0.

The invention also provides the application of the short peptide or the nanostructure carrier in preparing a drug carrier of local anesthetic drugs.

The invention also provides a local anesthetic drug sustained-release preparation, which is a preparation prepared by taking local anesthetic as an active ingredient and taking the short peptide or the nanostructure carrier as a drug carrier.

Further, the local anesthetic is an amide local anesthetic or an ester local anesthetic.

Further, the amide local anesthetic or the ester local anesthetic is one or more than two of bupivacaine alkali, ropivacaine alkali, lidocaine alkali, mepivacaine alkali, prilocaine alkali, tetracaine alkali, procaine alkali, bupivacaine salt, ropivacaine salt, lidocaine salt, mepivacaine salt, prilocaine salt, tetracaine salt and procaine salt.

Further, the salt is hydrochloride.

Further, the formulation is a solid formulation or a liquid formulation.

Further, the solid preparation is freeze-dried powder, and the liquid preparation is injection preparation.

Further, in the liquid preparation, the concentration of the local anesthetic is 1.0-200mg/mL, and the concentration of the drug carrier is 1mM-10mM.

Further, in the liquid preparation, the concentration of the local anesthetic is 7.5-120mg/mL, and the concentration of the drug carrier is 5mM.

Further, the pH of the liquid formulation is between 6.0 and 9.0.

Further, the pH of the liquid formulation is between 6.0 and 8.5.

Further, the pH of the liquid formulation is between 6.0 and 7.0.

The invention also provides a method for preparing the local anesthetic drug sustained-release preparation, which comprises the following steps:

dissolving the drug carrier in aqueous solution to obtain drug carrier solution, adding local anesthetic, mixing, and adjusting pH to target value with pH regulator.

Further, the pH of the drug carrier solution is 7.0.

In the present invention, PB buffer means phosphate buffer.

In the short peptide sequences of the present invention, "Ac" refers to acetyl.

Bupivacaine base is also known as bupivacaine, ropivacaine base is also known as ropivacaine, lidocaine base is also known as lidocaine, mepivacaine base is also known as mepivacaine, prilocaine base is also known as prilocaine, tetracaine base is also known as tetracaine, procaine base is also known as procaine. Compared with the existing local anesthetic drug carrier material, the short peptide provided by the invention has the following advantages:

materials currently used as carriers for local anesthetic drug delivery formulations mainly include liposomes, microspheres, hydrogels, solid lipids, and complex systems combining the foregoing materials. These materials degrade slowly in vivo, have certain toxicity, and have complex preparation process and high cost. The short peptide provided by the invention is a kind of short peptide molecules composed of natural amino acids, can be self-assembled into various nano structures in aqueous solution under the drive of non-covalent acting forces such as hydrogen bonds, hydrophobic action, pi-pi accumulation and the like, and has good biological safety.

The existing local anesthetic salt solution clinically used has the problems of high toxicity and short acting time. The local anesthetic drug sustained-release preparation taking the short peptide as a carrier material has the following advantages:

1. the local anesthetic drug sustained-release preparation has large drug loading capacity and high encapsulation efficiency;

2. the local anesthetic drug sustained-release preparation has excellent nerve blocking effect, and the effective action time is obviously prolonged;

3. the local anesthetic sustained-release preparation has good stability, and can obviously prolong the analgesic action time after long-time storage;

4. the local anesthetic drug sustained-release preparation has low toxicity and side effects, and has excellent systemic safety and local safety;

5. the local anesthetic drug sustained-release preparation disclosed by the invention is simple in preparation method and has a good application prospect in the field of nano biological medicines.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

Fig. 1: appearance pictures and transmission electron microscope pictures of 5mM GQY short peptide nanoparticle carrier solution.

Fig. 2: GQY carries the appearance diagram of the bupivacaine alkali system with different concentrations.

Fig. 3: appearance diagram of GQY ropivacaine-carrying alkali system.

Fig. 4: appearance diagram of GQY-carried mepivacaine alkali system.

Fig. 5: appearance diagram of 5mM GQY-Ac short peptide nanoparticle carrier solution, GQY-Ac-entrapped ropivacaine base system and GQY-Ac-entrapped bupivacaine base system.

Fig. 6: appearance of YQ5T entrapped procaine base system.

Fig. 7: appearance diagram of G2YQ4YG2 entrapped lidocaine base system.

Fig. 8: appearance of GQ5YG and the entrapped bupivacaine base system.

Fig. 9: GQY carries scanning electron microscope images of different concentrations of bupivacaine alkali systems.

Fig. 10: XRD pattern of GQY bupivacaine base system.

Fig. 11: GQY carries in vitro release profiles of different concentrations of bupivacaine base system.

Fig. 12: the GQY bupivacaine alkali system ischial nerve block model sensory blocking time period.

Fig. 13: GQY bupivacaine alkali system ischial nerve block model movement blocking time period.

Fig. 14: GQY is a curve when different concentrations of bupivacaine base system sciatic nerve resistance lags plasma drug.

Fig. 15: GQY carries general appearance and HE staining 4 days after SNB administration of different concentrations of bupivacaine base system.

Fig. 16: GQY carries general appearance and HE staining 14 days after SNB administration of different concentrations of bupivacaine base system.

Detailed Description

The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.

Example 1: synthesis of short peptides of the invention

The following short peptides were synthesized by conventional polypeptide solid-phase synthesis methods, respectively. The purity of the obtained short peptides is more than 98 percent.

GQY short peptide

The sequence is Gly-Gln-Gln-Gln-Gln-Gln-Tyr (SEQ ID NO. 1).

GQY-Ac short peptides

The sequence is Ac-Gly-Gln-Gln-Gln-Gln-Gln-Tyr (SEQ ID NO. 2).

YQ5T short peptide

The sequence is Tyr-Gln-Gln-Gln-Gln-Gln-Thr (SEQ ID NO. 3).

G2YQ4YG2 short peptide

The sequence of the catalyst is Ac-Gly-Gly-Tyr-Gln-Gln-Gln-Tyr-Gly-Gly-NH ₂ (SEQ ID NO.4)。

GQ5YG short peptide

The sequence is Gly-Gln-Gln-Gln-Gln-Gln-Tyr-Gly (SEQ ID NO. 5).

TY 4Y4 short peptide

The sequence is Ac-Thr-Tyr-Thr-Tyr-Thr-Tyr-Thr-Tyr-NH ₂ (SEQ ID NO.6)。

TYQ short peptide

The sequence is Ac-Thr-Tyr-Gln-Thr-Tyr-Gln-Thr-Tyr-Gln-NH ₂ (SEQ ID NO.7)。

yQT short peptide

The sequence is Ac-Tyr-Gln-Thr-Tyr-Gln-Thr-Tyr-Gln-Thr-NH ₂ (SEQ ID NO.8)。

Example 2: preparation of the short peptide nanoparticle Carrier of the invention

1. Preparation of GQY short peptide nanoparticle carrier

Precisely weighing a certain amount of GQY short peptide, adding a certain amount of 10mM PB buffer solution (pH 7.4-8.0), fully blowing and uniformly mixing, and performing water bath ultrasonic treatment for 15-20 minutes to obtain a GQY short peptide nanoparticle carrier solution, wherein the final concentration of the short peptide is 5mM.

The GQY short peptide nanoparticle carrier solution was observed by Transmission Electron Microscopy (TEM), and it was seen that the GQY short peptide self-assembled to form nanoparticles, and the appearance and TEM image of the GQY short peptide nanoparticle carrier solution are shown in FIG. 1.

2. Preparation of GQY-Ac short peptide nanoparticle carrier

Referring to the method of step 1, the GQY short peptide is replaced by GQY-Ac short peptide, so as to obtain GQY-Ac short peptide nanoparticle carrier solution, wherein the final concentration of the short peptide is 5mM.

3. Preparation of YQ5T short peptide nanoparticle carrier

Referring to the method of step 1, the GQY short peptide is replaced by YQ5T short peptide, and YQ5T short peptide nanoparticle carrier solution is obtained, wherein the final concentration of the short peptide is 5mM.

4. Preparation of G2YQ4YG2 short peptide nanoparticle carrier

Referring to the method of step 1, the GQY short peptide was replaced with the G2YQ4YG2 short peptide to obtain a G2YQ4YG2 short peptide nanoparticle carrier solution, wherein the final concentration of the short peptide was 5mM.

5. Preparation of GQ5YG short peptide nanoparticle Carrier

Referring to the method of step 1, the GQY short peptide was replaced with GQ5YG short peptide to obtain GQ5YG short peptide nanoparticle carrier solution, wherein the final concentration of short peptide was 5mM.

6. Preparation of T4Y4 short peptide nanoparticle carrier

Referring to the method of step 1, the GQY short peptide is replaced by a T4Y4 short peptide, so that a T4Y4 short peptide nanoparticle carrier solution is obtained, wherein the final concentration of the short peptide is 5mM.

7. Preparation of TYQ short peptide nanoparticle carrier

Referring to the method of step 1, the GQY short peptide is replaced by TYQ short peptide, so as to obtain TYQ short peptide nanoparticle carrier solution, wherein the final concentration of the short peptide is 5mM.

8. Preparation of YQT short peptide nanoparticle carrier

Referring to the method of step 1, the GQY short peptide is replaced by the YQT short peptide, so that the YQT short peptide nanoparticle carrier solution is obtained, wherein the final concentration of the short peptide is 5mM.

Example 3: preparation of sustained-release preparation of the invention

1.GQY bupivacaine base system for preparing different BUP concentrations

Precisely weighing quantitative bupivacaine alkali (Dalianmei, purity is more than or equal to 98.5%, BUP is short for short), adding the GQY short peptide nanoparticle carrier solution prepared in example 2 according to different BUP final concentrations in table 1, magnetically stirring for 20 hours, adjusting pH to 7.4 by using NaOH and HCl, and performing water bath ultrasonic treatment for 15-20 minutes to obtain BUP-GQY preparations with different BUP concentrations: BG007, BG015, BG030, BG060, BG120.

TABLE 1 BUP-GQY formulation formulations at different BUP concentrations

The appearance pictures of BUP-GQY preparations are shown in figure 2, and it can be seen that bupivacaine alkali with different concentrations can be fully dispersed in 5mM GQY short peptide nanoparticle carrier solution to form suspension with uniform milky color.

2.GQY bupivacaine-carrying alkali system for preparing different pH values

TABLE 2 BG060 formulations with different pH values

Formulation number	pH
		BG06060	6.0
BG06064	6.4
		BG06070	7.0
BG06074	7.4
		BG06084	8.4

Taking the prepared BG060 preparation, and respectively adjusting the pH of the BG060 to 6.0, 6.4, 7.0, 7.4 and 8.4 by using 1-6M HCl and NaOH to obtain BUP-GQY preparations with different pH values: BG06060, BG06064, BG060670, BG06074, BG06084.

3. Preparation of GQY ropivacaine-carrying alkali systems with different pH values

Precisely weighing a certain amount of ropivacaine alkali (purchased from microphone, purity 98%, abbreviated as ROP), adding the ropivacaine alkali into 5mM GQY short peptide nanoparticle carrier solution according to the final concentration of the ROP of 60mg/mL, adjusting the pH value to the target value in table 3 by NaOH and HCl, magnetically stirring for 20 hours, and carrying out water bath ultrasonic treatment for 15-20 minutes to obtain ROP-GQY preparations with different pH values: RG06060, RG06070, RG06085.

TABLE 3 ROP-GQY formulation formulations at different pH

As shown in fig. 3, ropivacaine base can be fully dispersed in GQY short peptide nanoparticle carrier solution to form a milky suspension with uniform properties.

4. Preparation of GQY entrapped mepivacaine base system

Precisely weighing quantitative mepivacaine (purchased from Alatine, 97% in purity and abbreviated as MEP), adding the MEP to 5mM GQY short peptide nanoparticle carrier solution according to the final concentration of 60mg/mL, adjusting pH to 8.2 with NaOH and HCl, magnetically stirring for 20 hours, and performing water bath ultrasonic treatment for 15-20 minutes to obtain MEP-GQY preparation: MG06082.

TABLE 4 MEP-GQY formulation

As shown in FIG. 4, the mepivacaine alkali can be fully dispersed in the GQY short peptide nanoparticle carrier solution to form a milky suspension with stable properties.

5. Preparation of GQY-Ac-entrapped ropivacaine base and bupivacaine base system

TABLE 5 formulation of GQY-Ac-entrapped ropivacaine base and GQY-Ac-entrapped bupivacaine base system

Precisely weighing a certain amount of ropivacaine alkali or bupivacaine alkali, adding the ropivacaine alkali or bupivacaine alkali into 5mM GQY-Ac short peptide nanoparticle carrier solution, adjusting pH to a target value by using NaOH and HCl, magnetically stirring for 20 hours, and carrying out water bath ultrasonic treatment for 15-20 minutes to respectively obtain a GQY-Ac-entrapped ropivacaine alkali system: RGAc06070 and GQY-Ac inclusion bupivacaine base system: BGAc06074.

As can be seen from FIG. 5, both ropivacaine base and bupivacaine base can be fully dispersed in GQY-Ac short peptide nanoparticle carrier solution to form a milky suspension with stable properties.

6. Preparation of YQ 5T-entrapped procaine base system

Precisely weighing a certain amount of procaine alkali (purchased from microphone, purity 98%, PRO for short), adding into 5mM YQ5T short peptide nanoparticle carrier solution, adjusting pH to a target value by using NaOH and HCl, magnetically stirring for 20 hours, and performing water bath ultrasonic treatment for 15-20 minutes to obtain a YQ5T entrapped procaine alkali system: PY06090.

TABLE 6 YQ5T procaine loaded base System formulation

As shown in fig. 6, procaine base can be sufficiently dispersed in the YQ5T short peptide nanoparticle carrier solution to form a milky suspension.

7. Preparation of G2YQ4YG 2-entrapped lidocaine base System

Precisely weighing a certain amount of lidocaine alkali (purchased from Alatidine, the purity is more than or equal to 99 percent, and is abbreviated as LIDO), adding the lidocaine alkali into a 5mM G2YQ4YG2 short peptide nanoparticle carrier, adjusting the pH value to a target value by using NaOH and HCl, magnetically stirring the mixture for 20 hours, and carrying out water bath ultrasonic treatment for 15-20 minutes to obtain a G2YQ4YG2 entrapped lidocaine alkali system: LGYG06085.

TABLE 7 G2YQ4YG2 Lidocaine base System formulation

As shown in fig. 7, lidocaine base was well dispersed in the G2YQ4YG2 short peptide nanoparticle carrier solution to form a milky suspension.

8. Preparation of GQ5YG entrapped bupivacaine base system

Precisely weighing a certain amount of bupivacaine alkali, adding the bupivacaine alkali into 5mM GQ5YG short peptide nanoparticle carrier solution, adjusting the pH value to a target value by using NaOH and HCl, magnetically stirring for 20 hours, and carrying out water bath ultrasonic treatment for 15-20 minutes to obtain a GQ5YG coated bupivacaine alkali system: GG03074.

Table 8 GQ5YG bupivacaine base formulation

As can be seen from fig. 8, bupivacaine base can be sufficiently dispersed in GQ5YG short peptide nanoparticle carrier solution to form a milky suspension.

The preparation method shows that the local anesthetic sustained-release preparation can be prepared from the short peptide and local anesthetic alkali as raw materials.

9. Preparation of T4Y 4-entrapped bupivacaine base system

Weighing quantitative T4Y4 freeze-dried powder according to the final concentration of 5mM, adding 10mM PB, and performing water bath ultrasonic treatment for 15-20 minutes; weighing quantitative bupivacaine hydrochloride (purchased from microphone, purity 99%, BUP-HCl for short) and adding into the T4Y4 short peptide nanoparticle carrier solution, regulating pH of the system to a target value, and continuing magnetic stirring for 20 hours to obtain a T4Y4 entrapped bupivacaine alkali system: BHT03074.

TABLE 9T 4Y4 coated bupivacaine base system formulation

The T4Y4 entrapped bupivacaine alkali system is a milky suspension with uniform properties.

10. Preparation of TYQ-entrapped ropivacaine base system

Weighing quantitative TYQ freeze-dried powder according to the final concentration of 5mM, adding 10mM PB, and performing water bath ultrasonic treatment for 15-20 minutes; weighing quantitative ropivacaine hydrochloride (purchased from microphone, purity 99%, abbreviated as ROP-HCl) and adding into TYQ short peptide nanoparticle carrier solution, regulating pH of the system to a target value, and continuing magnetic stirring for 20 hours to obtain TYQ-entrapped ropivacaine alkali system: RHTYQ04070.

Table 10.TYQ entrapped ropivacaine base system formulation

The TYQ entrapped ropivacaine base system presents a milky suspension with uniform appearance.

11. Preparation of YQT entrapped ropivacaine base system

Table 11.YQT entrapped ropivacaine base system formulation

Formulation number	Carrier material/vehicle	ROP-HCl(mg/mL)	pH
				RHYQT04070	5mM YQT		40	7.0

Weighing quantitative YQT freeze-dried powder according to the final concentration of 5mM, adding 10mM PB, and performing water bath ultrasonic treatment for 15-20 minutes; weighing quantitative ropivacaine hydrochloride (purchased from microphone, purity 99%, abbreviated as ROP-HCl) and adding into YQT short peptide nanoparticle carrier solution, regulating pH of the system to a target value, and continuing magnetic stirring for 20 hours to obtain a YQT ropivacaine base-carrying system: RHYQT04070.

The YQT ropivacaine-carrying alkali system has uniform appearance and milky suspension.

The preparation method shows that the local anesthetic sustained-release preparation can also be prepared from the short peptide and local anesthetic salt of the invention.

Control example: control sample for preparing BUP-GQY preparation

As controls, free bupivacaine hydrochloride (microphone, 99% purity, abbreviated as BUP-HCl), 0.9% ns (physiological saline), 5mM GQY short peptide nanoparticle carrier solution (ph 7.0), and bupivacaine base powder not encapsulated with short peptide material were used at different concentrations.

Table 12.BUP-GQY System control formula

The following experiments prove the beneficial effects of the invention.

Experimental example 1: observation of microscopic morphology of BUP-GQY formulations

In the samples prepared in example 3, 2. Mu.L of BG007, BG015, BG030, BG060 and BG120 were respectively uniformly and thinly distributed on clean glass slides with the size of 5X 5mm, and the samples were naturally dried. Microscopic morphology was observed under a scanning electron microscope (scanning electron microscope, SEM, zeiss Evo 10) after the metal spraying. The sample of comparative example 5 was used as a control.

As shown in FIG. 9, it is seen under SEM that BUP-GQY is in the size of 0.5-2.5 μm and the particle diameter tends to increase with increasing concentration of bupivacaine base in the system; the bupivacaine base which is not coated by the short peptide is agglomerated into large particles with the particle size of more than 5 mu m, which indicates that the bupivacaine base can be fully dispersed in the GQY carrier.

Experimental example 2: x-ray diffraction analysis of BUP-GQY structure

Powder X-Ray Diffraction analysis (PXRD) was performed using 2mL of lyophilized Powder of the BG060, control 2 and control 4 samples prepared in example 3, and the lyophilized Powder of the control 5 sample as a reference. Test parameters: maximum tube pressure: 60kV; maximum tube flow: 60mA,2 theta angle range: 5 ° -50 °, angular reproduction: +/-0.0001 °, step size: 0.0262606 °, detector count matrix: 256×256pixcel, pixel size: 55mm x 55mm, resolution: fwhm=0.028 °.

The results showed that BG060 was identical to the characteristic peak height of comparative example 5, indicating that in BG060, the carrier material GQY was not chemically bonded to bupivacaine base and no eutectic phenomenon occurred (fig. 10).

Experimental example 3: BUP-GQY drug-Loading (LC) and Encapsulation Efficiency (EE) determination

In the samples prepared in example 3, 1mL of each BUP-GQY preparation such as BG007, BG030, BG060 and BG120 was collected, and the supernatant was obtained by filtration through a filter having a pore size of 220. Mu.m, 3 parts of each preparation was prepared, and the concentration of bupivacaine in the supernatant was measured by a high performance liquid chromatograph (high performance liquid chromatograph, HPLC), thereby calculating the drug loading and the encapsulation efficiency of the BUP-GQY system.

Chromatographic conditions:

instrument: shimadzu LC-20AD

Chromatographic column: swell Chromplus C18 (4.6 mm. Times.150 mm,5 μm);

mobile phase: a:0.1% tfa, c: acetonitrile, c=65:35;

run time: 5min;

flow rate: 1mL/min;

sample injection amount: 5uL.

TABLE 13 drug delivery parameters of BUP-GQY formulations

System numbering	LC(％)	EE(％)
			BG007	60.26	88.84
BG015	87.09	98.84
			BG030	93.11	99.05
BG060	96.45	99.52

The results show that the GQY short peptide can effectively encapsulate bupivacaine alkali, and the BUP-GQY preparation provided by the invention has the advantages of high drug loading capacity and high encapsulation efficiency (Table 13).

Experimental example 4: in vitro Release study of BUP-GQY formulations

Test sample: BUP-GQY samples prepared in example 3 at different concentrations were compared with control example 2. 1mL of each sample is taken in a dialysis device Float-A-Lyzer G2 (Shanghai source leaf), the dialysis device is closed and then placed in a 50mL centrifuge tube, the dialysis solution is 40mL of phosphate buffer system (PBS, pH 7.4), the samples are respectively taken at 0.17h, 0.5h, 1h, 2h, 4h, 8h, 12h and 24h by shaking in a constant-temperature water bath at 37 ℃ for 70 times/min, 20mL of the dialysis solution is taken out each time every 24h after 24h, then an equal amount of fresh PBS buffer solution after rewarming is added, 1mL of the sample is reserved each time, and the concentration of bupivacaine in the reserved samples is detected by using HPLC. Chromatographic conditions were the same as in experimental example 3.

The result shows that the BUP-GQY preparation has obvious in-vitro slow release effect in an in-vitro pH7.4 phosphate buffer solution; compared with comparative example 2, no burst phenomenon occurred (see fig. 11).

Based on the experimental results, the invention further utilizes the following in vivo experiments to test the in vivo long-acting analgesic or local anesthetic effect of the pharmaceutical preparation prepared by taking the short peptide of the invention as a carrier.

Experimental example 5: long-acting local anesthesia analgesic effect test

Long-acting local anesthesia analgesic effect of BUP-GQY preparation

1. Experimental method

The local tingling and pain relieving effects of BUP-GQY were examined using a sciatic nerve block (sciatic nerve block, SNB) model. Healthy male SD rats (supplied by Chengdu laboratory animal Co., ltd.) with weights of 250-300g were selected, kept for 1-2 weeks strictly according to American laboratory animal care and use guidelines, and the rats were monitored for weights 3 days before the start of the experiment and screened according to hot plate rundown time (paw withdraw latency, PWL), eliminating rats that had rundown time of more than 4 seconds and significantly reduced weight on hot plates at 56 ℃. The qualified rats were selected from 64 rats, randomly divided into 8 groups of 8 rats each, and the groups were as follows: GB007, GB030, GB060, GB120 and comparative examples 1-4.

Placing the rat into a self-made airtight plastic box, performing anesthesia induction by using 4% isoflurane, taking out the rat after the rat is calm, placing the rat on an experiment table to enable the rat to be in a lateral position, ventilating by using a self-made mask, performing anesthesia maintenance by using 2% isoflurane, removing hair at the tail of the sacrum of the experiment side, inserting a needle at the midpoint of a connecting line between a femoral greater trochanter and a ischial tuberosity, enabling the needle tip to contact the bone, uniformly giving 0.2mL of each sample, and placing the sample into a cage after the cotton swab is pressed for hemostasis.

The sensory function of the rats was evaluated using an improved hotplate test, the hotplate (Chengdu Tech Co., ltd.) was set to 56℃at a constant temperature, the rats were lifted vertically to pedal on the hotplate after the drug administration side, the time for the feet to shrink was read with a stopwatch, the average value was taken after 3 times each time point, and the hindfoot of the rats was allowed to leave the hotplate to avoid scalding if the rats were not yet shrunk after 10 seconds each time interval, for example, after 12 seconds. Foot contraction times above 7 seconds are considered effective sensory retardation.

The hind limb pedaling test (postural extensor thrust, PET) was used to evaluate the motor function of the rats, with one hand holding the head of the rats and the other hand fixing the tail of the rats, so that the hind limb pedaling on the administration side of the rats was done on an electronic day table, the body was in a line, and the reading was recorded after the balance measurement was stable. Pedaling force less than half of baseline is considered effective motion retardation.

Rats were evaluated for sensory and motor function every 2h, and 24h, 28h, 32h, 36h, 40h, 44h, 48h, 52h, 56h, 72h, respectively, 10min, 30min, 1h,12h post-dose, and observed closely for adverse effects (systemic toxicity: dysphoria, tremors, convulsions and even death; local irritation: muscle cramps, injection site necrosis, self-disability, paw licking, and hair erection).

2. Experimental results

The results are shown in Table 14 and FIGS. 12 and 13. Neither comparative example 3 nor comparative example 4 had a nerve block phenomenon occurred, and no adverse reaction was observed. Compared with the comparative examples 1 and 2, the BUP-GQY preparation has equivalent action time, but obviously prolonged action time; and the nerve block action time is obviously increased along with the increase of the bupivacaine concentration in the BUP-GQY preparation.

The BUP-GQY rats and the rats in the control 1 group showed no significant adverse reaction, while the rats in the control 2 group showed listlessness within half an hour after administration, and the adverse reaction disappeared 2-4 hours after administration.

TABLE 14 SNB Effect of BUP-GQY System

Note that: oset represents the time of Onset; offset represents the effective blocking time.

The experimental result shows that the BUP-GQY preparation prepared by taking the GQY short peptide as a carrier has excellent nerve blocking effect, obviously prolongs the effective acting time, and can be used for preparing medicines for long-acting analgesia or long-acting local anesthesia.

Long-acting local anesthesia analgesic effect of (II) MEP-GQY preparation

The SNB model was used to evaluate the narcotic analgesic effect of MEP-GQY formulations in the same manner as part (one). The mepivacaine hydrochloride is purchased from the Alatidine, the purity is more than or equal to 98 percent, and the MEP-HCl is called for short.

TABLE 15 MEP-GQY formulation and SNB Effect

Compared with free mepivacaine hydrochloride, the sciatic nerve block analgesia time of the MEP-GQY preparation is obviously prolonged, and serious poisoning reaction is not seen (Table 15).

Long-acting local anesthesia analgesic effect of GQY-Ac-entrapped ropivacaine base and bupivacaine base system

The SNB model was used to evaluate the narcotic analgesic effect of GQY-Ac-entrapped ropivacaine base and bupivacaine base system as described in section (one).

The sciatic nerve blocking effect of the GQY-Ac ropivacaine-carrying alkali system and the GQY-Ac bupivacaine-carrying alkali system is obviously prolonged compared with that of the free ropivacaine hydrochloride and the free bupivacaine hydrochloride, and no obvious poisoning reaction is observed. And, the analgesic effect of the GQY-Ac-carried ropivacaine base was better than that of the GQY-Ac-carried bupivacaine base system (table 16).

TABLE 16 GQY-Ac-entrapped ropivacaine base and bupivacaine base System formulation and SNB Effect

(IV) Long-acting local anesthesia analgesic effect of YQ5T entrapped procaine alkali system

The SNB model was used to evaluate the narcotic analgesic effect of the YQ 5T-entrapped procaine base system in the same manner as part (one). Procaine hydrochloride is purchased from microphone with 99% purity, PRO-HCl for short.

Table 17 YQ5T-entrapped procaine base system formula and SNB effect

The sciatic nerve block analgesia time of the YQ5T entrapped procaine base system was prolonged to 8-10 hours compared to free procaine hydrochloride and no significant toxic reaction was observed (table 17).

(five) long-acting local anesthesia analgesic effect of G2YQ4YG2 entrapped lidocaine base system

The SNB model was used to evaluate the narcotic analgesic effect of the G2YQ4YG2 entrapped lidocaine base system, as described in section (one). Lidocaine hydrochloride was purchased from microphone with 99% purity, abbreviated as LIDO-HCl.

The sciatic nerve blocking effect of the G2YQ4YG2 entrapped lidocaine base system was prolonged compared to free lidocaine hydrochloride and no significant toxic reaction was observed (table 18).

TABLE 18 G2YQ4YG2 Lidocaine base System formulation and SNB Effect

Long-acting local anesthesia analgesic effect of GQ5YG entrapped bupivacaine alkali system

The SNB model was used to evaluate the narcotic analgesic effect of GQ5 YG-entrapped bupivacaine base system in the same manner as described in section (one).

TABLE 19 GQ5YG entrapped bupivacaine base System formulation and SNB Effect

The analgesic effect of the GQ5YG entrapped bupivacaine base system in the sciatic nerve block model was prolonged to 11-14 hours and no significant toxic response was observed (table 19).

(seven) long-acting local anesthesia analgesic effect of T4Y4 entrapped bupivacaine alkali system

The SNB model was used to evaluate the narcotic analgesic effect of the T4Y 4-entrapped bupivacaine base system, as described in section (I).

TABLE 20 formula of TY 4-coated bupivacaine alkali system and SNB effect

The analgesic effect of the T4Y4 entrapped bupivacaine base system in the sciatic nerve block model was prolonged to 12-16 hours and no significant toxic response was observed (table 20).

Long-acting local anesthesia analgesic effect of TYQ entrapped ropivacaine alkali system

The SNB model was used to evaluate the narcotic analgesic effect of TYQ-entrapped ropivacaine base system as described in section (one).

The analgesic effect of TYQ-entrapped ropivacaine base system in the sciatic nerve block model was prolonged to 16-24 hours and no significant toxic response was observed (Table 21).

Table 21. Formulation of TYQ-entrapped ropivacaine base system and SNB effect

Long-acting local anesthesia analgesic effect of YQT entrapped ropivacaine alkali system

The SNB model was used to evaluate the narcotic analgesic effect of the YQT-entrapped ropivacaine base system as described in part (one).

Table 22. Formulation of YQT-entrapped ropivacaine base system and SNB effect

The analgesic effect of YQT-entrapped ropivacaine base system in the sciatic nerve block model was prolonged to 16-24 hours and no significant toxic response was observed (table 22).

The experiment shows that the medicine preparation prepared by taking the short peptide of the invention as a carrier and coating the local anesthetic has excellent nerve blocking effect, obviously prolongs the effective action time, and can be used for preparing long-acting analgesic or long-acting local anesthetic medicines.

Experimental example 6: influence of pH on drug-loading rate, encapsulation efficiency and local anesthesia analgesic effect of sustained-release preparation

(one) Effect of pH on BUP-GQY formulations

The BG060 preparation prepared in example 3 was taken and the pH of BG060 was adjusted to 6.0, 6.4, 7.0, 7.4 and 8.4 using 1-6M HCl and NaOH, respectively. The concentration of bupivacaine in the supernatant was measured by HPLC, and the measurement method was the same as in example 3.

The SNB model was used to evaluate the analgesic effect of BG060 formulations at different pH values, and the experimental method and the administration volume were the same as those of experimental example 5.

The results show that as pH increases, the drug loading and encapsulation of bupivacaine base by GQY correspondingly increases (table 23).

In addition, the BG060 preparation has quick effect on sensory and motor nerve block under different pH values; in the BG060 formulations at pH 6.0-8.4, nerve blocking time was different with pH change, but the blocking time was still significantly prolonged compared with comparative examples 1 and 2 in experimental example 5, indicating that BG060 formulations at different pH all had significant effect of prolonging analgesic time (table 23).

TABLE 23 drug delivery parameters and SNB Effect of BG060 formulations at different pH values

(II) influence of pH on ROP-GQY preparation

Supernatant concentrations of RG06060, RG06070 and RG06085 were measured by HPLC in the same manner as in Experimental example 3. And the SNB model was used to investigate the local analgesic effect of ROP-GQY formulations of different pH in example 3. Healthy male SD rats were screened for 32, weighing 250-300g, and were randomly divided into 4 groups of 8 animals each, each group being as follows: RG06060, RG06070, RG06085 and comparative example 6. Ropivacaine hydrochloride is purchased from microphone (purity 99%, abbreviated as ROP-HCl).

TABLE 24 formulation of comparative example 6

TABLE 25 drug delivery parameters and SNB Effect of RG060 formulations at different pH values

The results show that GQY can effectively encapsulate ropivacaine alkali to form a uniform preparation. In the ROP-GQY preparation with pH of 6.0-8.5, nerve blocking time slightly changed with pH change, but compared with the free ropivacaine hydrochloride, the sciatic nerve blocking time of the ROP-GQY system with different pH values is obviously prolonged (Table 25).

The experimental results show that under different pH conditions, the pharmaceutical preparation prepared by taking the short peptide of the invention as a carrier can obviously prolong the time of nerve block.

Experimental example 7: stability investigation of BG06064 freeze-dried preparation

1. Experimental method

The experiment examined the stability of BUP-GQY formulations after lyophilization for long term storage. Freeze-drying the quantitative BG06064 preparation for 6 months; then adding ultra-pure water which is the same volume as before freeze-drying for re-dissolution, and using an SNB model to examine the anesthesia and analgesia effects of the freeze-dried preparation. Experimental procedure and dosing volumes were the same as in Experimental example 5.

2. Experimental results

TABLE 26 SNB Effect of lyophilized BG06064 formulations after 6 months of cryopreservation

The results show that BG06064 can be stored for a long time after being prepared into a freeze-dried preparation, and can still obviously prolong the analgesic effect time after being redissolved (table 26).

The experimental result shows that the pharmaceutical preparation prepared by taking the short peptide of the invention as a carrier has good stability and can obviously prolong the analgesic action time after long-time storage.

The above in vivo experiments prove that the pharmaceutical preparation prepared by taking the short peptide of the invention as a carrier has the effects of long-acting analgesia or long-acting local anesthesia, can be used for treating pain before, during or after operation, and can also meet the long-time local anesthesia requirement during operation. On the basis, the invention further tests the whole body safety and the local safety of the pharmaceutical preparation prepared by taking the short peptide as a carrier.

Experimental example 8: systemic safety test of BUP-GQY formulations in rats

1. Experimental method

Referring to experimental example 5, 48 healthy male SD rats (250-300 g) were selected and randomly divided into 6 groups of 8 each, i.e., BG007, BG030, BG060, BG120, control example 1 and control example 2. The rat was placed in the holder to expose the tail, and a 24G venipuncture needle was used to puncture the tail vein and leave the sheath. And then, after the rat is anesthetized, a needle is inserted through the middle point of the connecting line of the femoral trochanter and the ischial tuberosity, and 0.2mL of each sample is injected at a constant speed after the needle tip contacts the bone. 10min, 30min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 10h, 12h, 24h, 28h, 32h, 48h, 52h, 56h, 72h, 76h, 80h after dosing were collected with 0.15mL venous blood, plasma was extracted, and the concentration of bupivacaine in plasma at different time points was detected using a liquid chromatography-mass spectrometer.

Instrument: agilent 1200 high performance liquid chromatograph; agilent G6460 triple quadrupole Mass spectrometer (Agilent Mass-Hunter workstation); beckman low temperature high speed centrifuge (ale 64R,Beckman Coulter company, usa); one ten thousandth electronic analytical balance (Sartorius ME215 s); vortex mixer (IKA MS3 basic), MILLI-Q ultra-pure water purifier

Reagent: acetonitrile, chromatographic purity, fisher, usa; methanol, chromatographic purity, fisher, USA; ethyl acetate, chromatographic purity, fisher company, usa; formic acid, chromatographic purity (Sigma-Aldrich, usa); ropivacaine hydrochloride (purity 94.2% of the institute of pharmaceutical and biological products, china); ropivacaine-d 7 hydrochloride (canadian TRC).

Chromatographic conditions:

chromatographic column: agilent extension C18 (3.0 x 100mm,3.5 μm), mobile phase: a:0.05% formic acid in water, B: acetonitrile, flow rate: gradient elution was performed at 0.3 mL/min. The initial proportion of B is 21%,2-3min, B is increased from 21% gradient to 40%,3-3.1min, B is increased from 40% gradient to 100%, the initial proportion is maintained to 4.5min,4.6min B is reduced from 100% gradient to 21%, and the operation time is 7min. Column temperature: 35 ℃, sample injection amount: 1 mul. Needle washing liquid: meOH-H ₂ O (1:1, v/v), needle wash for 15s.

Mass spectrometry conditions:

electrospray ionization source (ESI source), multiple Reaction Monitoring (MRM), positive ion detection mode.

Drying gas temperature: 350 ℃, dry Gas flow rate (Gas flow): 5L/min, sprayer pressure 45psi, sheath air temperature: sheath gas flow rate at 350 ℃): 11L/min, capillary voltage 3500V.

For quantifying ion pairs:

bupivacaine, m/z 289.2-m/z 140.1, fragmentation voltage: 110V, collision energy: 16eV;

ropivacaine-d 7 (internal standard), m/z 282.5-m/z 133.3, fragmentation voltage: 92V, collision energy: 16eV.

2. Experimental results

TABLE 27 pharmacokinetic parameters of BUP-GQY formulations

The results are shown in Table 27 and FIG. 14. At the same concentration, the peak time and half-life of the BUP-GQY preparation are longer than those of bupivacaine hydrochloride, which proves that the BUP-GQY preparation still has obvious slow release characteristic in vivo. Further, BG007 had a peak blood concentration of about 64% of that of control 1, as compared to control 1 having the same drug concentration; in contrast, BG030 compares with comparative example 2, which has the same drug concentration, and the peak blood concentration is about one third of comparative example 2; although the peak plasma concentration of the BUP-GQY preparation increased with the increase of bupivacaine concentration, there was no significant difference from the peak plasma concentration of comparative example 1, indicating a slightly wider poisoning dose range for the BUP-GQY preparation.

The experimental results show that BUP-GQY preparations with different bupivacaine concentrations have excellent slow release effect and systemic safety.

Experimental example 9: evaluation of local pathological injury of BUP-GQY preparation in sciatic nerve block model

1. Experimental method

Experimental method referring to experimental example 5, 56 healthy male rats were screened and randomly divided into 7 groups, i.e., BG007, BG030, BG060, BG120, control example 1, control example 3 and control example 4, each group of 8 rats was administered with 0.2mL of the aforementioned formulation, and tissue sampling was performed at 4 days and 14 days (n=4/group) after administration, respectively.

Rats were euthanized by tail vein injection of propofol, dissected to expose sciatic nerve, sectioned from the vicinity of the point of administration for 2 cm long sciatic nerve and muscle nearby, and fixed with 10% neutral formaldehyde for 48 hours. The tissue was paraffin-embedded after dehydration and transparency to a slice thickness of 5 μm, dewaxed, hydrated, HE stained, observed under an optical microscope, and assessed for inflammatory cell infiltration and muscle cytotoxicity of the tissue by a pathologist unaware of the experimental grouping. Tables 28 and 29 show the inflammatory cell infiltration and the muscle cytotoxicity scoring criteria, respectively.

2. Experimental results

TABLE 28 inflammatory cell infiltration scoring criteria

Microscope lower view	Scoring of
		Non-inflammatory cell infiltration	0
Inflammatory cell infiltration of surrounding area	1
		Inflammation in deep zone	2
Semimyofascitis cell infiltration	3
		Infiltration of panmyofascitis cells	4

TABLE 29 muscle cytotoxicity scoring criteria

Microscope lower view	Scoring of
		No abnormality	0
Perimuscular nucleus internalization	1
		>5 cell layer nuclear internalization	2
Regeneration of the periphery of the muscle bundles	3
		Deep regeneration	4
Semifascicular regeneration	5
		Regeneration of the full muscle bundle	6

TABLE 30 pathological lesion score 4 days after SNB administration of BUP-GQY slow-release system

* Compared with comparative example 1.

TABLE 31 pathological lesion score 14 days after SNB administration of BUP-GQY sustained release System

* Compared with comparative example 1.

The experimental result shows that compared with 0.9% physiological saline, local administration of 5mM GQY causes mild inflammation 4 days after administration, and recovery is carried out 14 days after administration, so that the GQY short peptide serving as a carrier material has good biocompatibility and no pathological damage to muscle cells and sciatic nerves. The positive preparations are administered for 4 days to generate certain inflammatory reaction and have certain injury effect on muscle cells and nerves; however, the inflammatory response of BG030 group was lighter than that of comparative example 1, and there was no difference in other pathological lesions; similarly, BG007, BG060 and BG120 were similar to comparative example 1 in pathological damage degree, and were not statistically different (table 30 and fig. 15). The above-mentioned pathological damage evaluation index was recovered 14 days after administration, and the pathological damage of BG007, BG030, BG060 and BG120 groups were not different from that of the control example 1 (table 31 and fig. 16).

The experimental results show that the BUP-GQY preparation with different bupivacaine concentrations has small pathological damage and excellent local safety.

The above in vivo experimental data prove that the pharmaceutical preparation prepared by taking the short peptide of the invention as a carrier has the effects of long-acting analgesia or long-acting local anesthesia, and simultaneously has excellent whole body safety and local safety.

In summary, the invention provides a short peptide and a slow release preparation using the same as a carrier material and having long-acting analgesic or/and long-acting local anesthetic effects. The short peptide can be used as a carrier of local anesthetic, and the local anesthetic drug slow-release preparation taking the short peptide as the carrier has the advantages of large drug loading, high encapsulation efficiency, good stability, excellent nerve blocking effect, capability of remarkably prolonging the effective acting time, excellent systemic safety and local safety, and wide application prospect in preparing drugs with long-acting analgesic or/and long-acting local anesthetic effect.

SEQUENCE LISTING

<110> Huaxi Hospital at university of Sichuan

<120> a short peptide and a sustained release having a long-acting analgesic or/and a long-acting local anesthetic effect using the same as a carrier material

Formulations

<130> GYKH1533-2022P0114776CC

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 7

<212> PRT

<213> artificial sequence

<400> 1

Gly Gln Gln Gln Gln Gln Tyr

1 5

<210> 2

<211> 7

<212> PRT

<213> artificial sequence

<400> 2

Ac-Gly Gln Gln Gln Gln Gln Tyr

1 5

<210> 3

<211> 7

<212> PRT

<213> artificial sequence

<400> 3

Tyr Gln Gln Gln Gln Gln Thr

1 5

<210> 4

<211> 10

<212> PRT

<213> artificial sequence

<400> 4

Ac-Gly Gly Tyr Gln Gln Gln Gln Tyr Gly Gly -NH2

1 5 10

<210> 5

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<212> PRT

<213> artificial sequence

<400> 5

Gly Gln Gln Gln Gln Gln Tyr Gly

1 5

<210> 6

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<212> PRT

<213> artificial sequence

<400> 6

Ac-Thr Tyr Thr Tyr Thr Tyr Thr Tyr -NH2

1 5

<210> 7

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<212> PRT

<213> artificial sequence

<400> 7

Ac-Thr Tyr Gln Thr Tyr Gln Thr Tyr -NH2

1 5

<210> 8

<211> 8

<212> PRT

<213> artificial sequence

<400> 8

Ac-Tyr Gln Thr Tyr Gln Thr Tyr Gln -NH2

1 5

Claims

1. A local anesthetic drug sustained release preparation, which is characterized in that: the preparation is prepared by taking a local anesthetic as an active ingredient and taking a short peptide or a nano-structure carrier as a drug carrier, wherein the nano-structure carrier is prepared by self-assembling the short peptide in an aqueous solution, and the amino acid sequence of the short peptide is shown as SEQ ID NO.1 or SEQ ID NO. 2; the local anesthetic is bupivacaine alkali, ropivacaine alkali, bupivacaine salt or more than two of ropivacaine salts;

the preparation is a liquid preparation, and the pH value of the liquid preparation is 6.0-7.0.

2. The local anesthetic drug delivery-release preparation according to claim 1, wherein: the salt is hydrochloride.

3. The local anesthetic drug delivery-release preparation according to claim 1, wherein: in the liquid preparation, the concentration of the local anesthetic is 1.0-200mg/mL, and the concentration of the drug carrier is 1mM-10mM.

4. A local anesthetic drug delivery-release preparation as claimed in claim 3, wherein: in the liquid preparation, the concentration of the local anesthetic is 7.5-120mg/mL, and the concentration of the drug carrier is 5mM.

5. A method for preparing a local anesthetic drug sustained-release preparation as claimed in any one of claims 1 to 4, characterized in that: the method comprises the following steps:

6. The method according to claim 5, wherein: the aqueous solution is water or a buffer solution.