Polypeptide in-situ gel long-acting injection as well as preparation method and application thereof

Abstract

The invention relates to the technical field of pharmaceutical preparations, and particularly discloses a polypeptide in-situ gel long-acting injection as well as a preparation method and application thereof, the injection is prepared from the following components: a polypeptide substance, a gel matrix and a biocompatible solvent, the polypeptide substance is tetrapeptide H-Tyr-D-Arg-Ph-Gly-NH2, and the mass volume percent of the polypeptide substance is 0.1%-4% (w / v); the gel matrix is an amphiphilic compound and natural phospholipid, and the mass ratio of the amphiphilic compound to the natural phospholipid is (0.7-7): 1; the biocompatible solvent is 1, 2-propylene glycol, and the mass fraction of the 1, 2-propylene glycol is 15%-30%. The injection has good fluidity and low viscosity, meets the viscosity requirement of a pharmacopoeia injection, is in contact with body fluid after being injected, is subjected to a solvent exchange reaction and is converted into hexagonal phase liquid crystal in-situ gel to form a medicine storage cavern, so that the release speed of tetrapeptide from the in-situ gel can meet the long-acting pain relieving requirement.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a long-acting polypeptide in situ gel injection, its preparation method, and its application. Background Technology

[0002] H-Tyr-D-Arg-Phe-Gly-NH2 is a Russian-innovative chemically synthesized tetrapeptide drug. According to Taphalgin, "Taphalgin is a Russian innovative tetrapeptide pharmaceutical for subcutaneous injection: review of the results of phase I and II clinical trials," the clinical efficacy and safety of this tetrapeptide have been investigated in phase I, II, and III trials. In the phase I clinical trial with 39 healthy volunteers, the most common adverse events (AEs) were fatigue, dry mouth, decreased sensation, and headache. All observed AEs were mild; they did not require treatment and in most cases resolved / discontinued. According to the clinical investigators, all volunteers reported "good" or "excellent" tolerability. No significant abnormalities were observed in major vital signs, complete blood count, blood biochemistry, coagulation function, urinalysis, or electrocardiogram.

[0003] A phase II clinical trial in patients with malignant tumors and accompanying pain syndromes demonstrated that the tetrapeptide exhibited good safety and efficacy at various doses. Multiple subcutaneous injections of the product showed efficacy comparable to morphine (injectable solution). Given that the study included patients with pain syndromes who had previously received opioid therapy, these patients were switched to subcutaneous tetrapeptide injections to maintain adequate pain relief. Pain intensity scores below 3 on the Neurological Rating Scale (NRS) may indicate that the drug has high analgesic potential, comparable to potent opioids.

[0004] No patients taking individually adjusted doses of H-Tyr-D-Arg-Phe-Gly-NH2 reported breakthrough pain, nor were morphine switches required due to poor efficacy or serious adverse events. Patient-reported adverse events included dizziness, hypotension, skin flushing, fever, diarrhea, nausea, vomiting, constipation, fatigue, somnolence, elevated blood pressure, drowsiness, numbness in the extremities, isolated allergic reactions, restlessness, disorientation, and dry mouth. All adverse events were mild to moderate. Notably, opioid-related adverse events observed before the study, such as constipation, dysuria, decreased appetite, and dry mouth, disappeared or lessened after treatment with this tetrapeptide. Furthermore, during follow-up, clinicians noted reduced lethargy and somnolence, and increased daily activities in patients treated with H-Tyr-D-Arg-Phe-Gly-NH2, indicating improved quality of life.

[0005] Although tetrapeptide drugs have analgesic effects comparable to opioids, without addictive or withdrawal symptoms, their analgesic duration is too short (maximum concentration is reached in 30 minutes with subcutaneous administration; half-life is 1.3 to 2.5 hours). This causes considerable inconvenience and suffering for patients; frequent administration leads to poor patient compliance, and the short duration of analgesia is unsuitable for treatments requiring long-term management, such as postoperative pain. Therefore, developing a long-acting peptide injection with long-lasting analgesia has very broad application prospects and enormous commercial value. This paper presents a long-acting peptide in-situ gel injection, its preparation method, and its application. Summary of the Invention

[0006] The purpose of this invention is to address the deficiencies of the prior art by providing a long-acting polypeptide in situ gel injection, its preparation method, and its application, thereby resolving the problems raised in the background section.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a long-acting polypeptide in-situ gel injection, which is composed of the following components: polypeptide substance, gel matrix, and biocompatible solvent;

[0008] The polypeptide is a tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH2, and the mass-volume percentage of the polypeptide is 0.1% to 4% (w / v).

[0009] The gel matrix is ​​composed of an amphiphilic compound and natural phospholipids, with a mass ratio of the amphiphilic compound to natural phospholipids of 0.7 to 7:1.

[0010] The biocompatible solvent is 1,2-propanediol, and the mass fraction of 1,2-propanediol is 15% to 30%.

[0011] As a preferred embodiment of the present invention, the natural phospholipid is selected from soybean phospholipids and / or egg yolk lecithin, and the amphiphilic compound is selected from one or more of monooleic glycerides, monostearate glycerides, and trioleic glycerides.

[0012] As a preferred embodiment of the present invention, the natural phospholipid in the gel matrix is ​​egg yolk lecithin, and the amphiphilic compound is monooleic glycerol.

[0013] As a preferred embodiment of the present invention, the tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH2 has a mass-volume percentage of 0.5% to 2% (w / v).

[0014] As a preferred embodiment of the present invention, the mass ratio of monooleic glyceride to egg yolk lecithin in the gel matrix is ​​4:5.

[0015] As a preferred embodiment of the present invention, the 1,2-propanediol has a mass fraction of 25%.

[0016] A method for preparing a long-acting peptide in situ gel injection as described above includes the following steps: adding a biocompatible solvent to the gel matrix, adding excess anhydrous ethanol and ultrasonically stirring, and removing excess ethanol by solvent evaporation; then adding the peptide substance to the prepared solution and vortexing until completely dissolved; thus obtaining the long-acting peptide in situ gel injection.

[0017] The application of the above-described polypeptide in-situ gel long-acting injection in the preparation of a pain medication, wherein the pain is for the treatment of acute or chronic postoperative pain; the medication is an in-situ gel long-acting injection containing the tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH2.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] This invention, through long-term experimental research, has discovered that a long-acting peptide in-situ gel injection can prolong the analgesic effect compared to direct peptide injection, providing a novel long-acting formulation for postoperative pain management. This invention, through extensive and innovative experimental research, selected specific types of liquid crystal materials (monoleic glyceryl monoester) and specific types of organic solvents in a specific ratio with tetrapeptides.

[0020] H-Tyr-D-Arg-Phe-Gly-NH2 was used to prepare a long-acting peptide in-situ gel injection. This injection exhibits good fluidity and low viscosity, meeting the viscosity requirements of pharmacopoeia injections. After injection, it comes into contact with body fluids and undergoes a solvent exchange reaction, transforming into a hexagonal phase liquid crystal in-situ gel, forming a drug reservoir. This allows the tetrapeptide to be released from the in-situ gel at a rate sufficient for long-acting analgesia. In other words, the long-acting peptide in-situ gel injection of this invention has excellent long-acting sustained-release effects, reduces the frequency of administration, and has few adverse reactions.

[0021] The long-acting polypeptide in situ gel of the present invention is a flowing solution at room temperature, which transforms into a gel at body temperature, making it injectable and eliminating the need for surgical implantation, thus avoiding surgical trauma.

[0022] The long-acting polypeptide in situ gel injection of the present invention can be directly mixed with drugs. Drugs, genes, proteins and cells can be easily mixed into the solution, especially providing a reservoir-like carrier for some drugs with poor in vivo stability.

[0023] This invention utilizes a lyotropic liquid crystal reservoir system to effectively control the initial burst release effect of the active pharmaceutical ingredient, avoiding the risks caused by a sudden increase in blood drug concentration. At the same time, it can reduce the total dosage without affecting product performance and lower drug manufacturing costs.

[0024] The long-acting polypeptide in-situ gel injection of this invention exhibits excellent biocompatibility and biodegradability, and is unlikely to cause irritation or other adverse immune responses in in vivo tissues. It utilizes glyceryl monooleate, a biodegradable and biocompatible material, FDA-recognized as Generally Recognized as Safe (GRAS), which is eliminated after subcutaneous and intramuscular injection via lipolysis by lipases. Phospholipids are fundamental substances of life and are safe natural phospholipid complexes that play a crucial role in maintaining the physiological activity of biological membranes and in vivo metabolism. It does not cause related toxicity or inflammation and possesses good biocompatibility and biodegradability.

[0025] The long-acting polypeptide in-situ gel injection of this invention can effectively provide acute analgesia for moderate to severe surgeries, such as orthopedic joint replacement, open tumor resection, and thoracic surgery, while avoiding frequent dosing and improving patient compliance. It can also be applied to postoperative chronic pain, day surgery, and ERAS scenarios. Attached Figure Description

[0026] Figure 1 The image shows the gel state formed after the long-acting polypeptide in situ gel injection prepared in Example 1 undergoes a solvent exchange reaction.

[0027] Figure 2A polarized light microscope image of the long-acting polypeptide in situ gel injection prepared in Example 2;

[0028] Figure 3 The in vitro release curves of the long-acting polypeptide in situ gel injection containing different proportions of liquid crystal materials and organic solvents prepared in Example 3 are shown.

[0029] Figure 4 The rheological analysis diagram of the long-acting polypeptide in-situ gel prepared in Example 4 is shown in Figure A, where A is the shear rate scan and B is the shear stress scan.

[0030] Figure 5 The graph shows the change in the mechanical withdrawal threshold of incision pain in rats after preparing the long-acting polypeptide in situ gel injection obtained in Example 5 (n=6).

[0031] Figure 6 The graph shows the change in the number of acetic acid writhing movements in mice after the long-acting polypeptide in situ gel injection prepared in Example 6 (n=6).

[0032] Figure 7 The graph shows the change in maximum analgesic effect (%MPE) of the long-acting polypeptide in situ gel injection prepared in Example 7 in mice (n=6).

[0033] Figure 8 The plasma concentration-time curves of 5.5 mg / kg long-acting peptide in situ gel injection and tetrapeptide original preparation were obtained by subcutaneous injection of the long-acting peptide in situ gel injection and the long-acting peptide in situ gel injection prepared in Example 8 in New Zealand rabbits (n=3). Detailed Implementation

[0034] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0035] Example 1: A method for preparing a long-acting polypeptide in situ gel injection, the injection being composed of the following components: a pharmaceutically active polypeptide substance, a gel matrix, and a biocompatible solvent.

[0036] The polypeptide is a tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH2, and the mass-volume percentage of the polypeptide is 0.1% to 4% (w / v).

[0037] The synthesis method of the tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH2 is as shown in the patent publication CN 108697758 A, which describes a drug for effectively controlling acute and / or chronic pain and its administration method.

[0038] On an automated peptide synthesizer, peptides were synthesized using a solid-phase method on a Rink polystyrene-based polymer support via the Fmoc protocol, employing the DEPCDI / HOBt amino acid activation method. The peptides were cleaved from the resin and unblocked using a mixture of TFA and m-cresol. The peptides were purified by reversed-phase HPLC with a gradient of acetonitrile. The peptides were then identified by mass spectrometry.

[0039] The gel matrix is ​​composed of an amphiphilic compound and natural phospholipids, wherein the mass ratio of the amphiphilic compound to the natural phospholipids is 0.7 to 7:1; the natural phospholipids are selected from soybean phospholipids and / or egg yolk lecithin, and the amphiphilic compound is selected from one or more of monooleic glycerides, monostearate glycerides, and trioleic glycerides.

[0040] The biocompatible solvent is 1,2-propanediol, with a mass fraction of 15% to 30%.

[0041] The steps include the following:

[0042] Weigh out a certain mass ratio of glyceryl monooleate, egg yolk lecithin, and a certain amount of 1,2-propanediol, place them in a round-bottom flask, add excess anhydrous ethanol, and sonicate to fully dissolve all components. Then, use a rotary evaporator under reduced pressure (45℃, 80 r·min) to evaporate the mixture. -1 Remove anhydrous ethanol (30 min). Add the tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH2 to the prepared solution and vortex until completely dissolved to obtain a long-acting peptide in-situ gel injection. Inject the injection into excess PBS (pH=7.4), where solvent exchange will occur, transforming it into a gel form. Figure 1 ).

[0043] The tetrapeptide was synthesized as follows: Using an automated peptide synthesizer, the Fmoc protocol was employed on a Rink polystyrene-based polymer support. A solid-phase synthesis method using DEPCDI / HOBt amino acid activation was used to cleave the peptide from the resin, followed by deblocking with a mixture of TFA and m-cresol. The peptide was purified by reversed-phase HPLC with a specific gradient of acetonitrile. Mass spectrometry was used to determine the peptide's identity.

[0044] Example 2:

[0045] The long-acting polypeptide in-situ gel injection prepared in Example 1 was observed under a polarized light microscope. To examine the internal morphology of the injection, a small sample was placed on a glass slide and covered with a coverslip, then observed under a polarized light microscope at 100x magnification at room temperature. The results are as follows. Figure 2 As shown, the injectable liquid crystal is a hexagonal phase liquid crystal and exhibits a fan-shaped texture pattern.

[0046] Example 3:

[0047] An in vitro drug dissolution experiment was conducted on the long-acting polypeptide in situ gel injection prepared in Example 1.

[0048] The method is as follows: Weigh 1g of the long-acting polypeptide in-situ gel prepared in Example 1 and add it dropwise to centrifuge tubes containing 50ml of PBS buffer. Place the centrifuge tubes in a shaker at 40rpm and 37℃. Release media were collected at 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 hours for measurement, and an equal amount of fresh release media was added. The drug concentration at different time points was measured using high-performance liquid chromatography (HPLC). The in vitro release curve of the drug was obtained, the cumulative release percentage was calculated, and the release curve was plotted. The results are as follows: Figure 3 As shown.

[0049] The results showed that the cumulative release rate at 192 h was F3>F4>F2>F1. No significant burst was observed from the release curves, and the cumulative release rate of the tetrapeptide under in vitro conditions could reach over 80%. The long-acting in-situ gel injection of this polypeptide exhibited good sustained-release characteristics in in vitro drug release experiments. Furthermore, formulation F3 (a mass ratio of monooleate glycerol to egg yolk lecithin of 4:5; a mass fraction of 1,2-propanediol of 25%; and a tetrapeptide mass concentration of 4 w / w%) was selected for characterization and further in-depth pharmacodynamic studies.

[0050] The chromatographic conditions for high performance liquid chromatography (HPLC) were as follows: mobile phase: A phase: water + 0.1% TFA, B phase: 60% acetonitrile + 40% water, gradient elution: 0–5 min, A:B = 80:10; 5–10 min, A:B = 70:20; 10–15 min, A:B = 60:30; detection wavelength: 280 nm; flow rate: 8 mL / min; injection volume: 10 μL; column temperature: 30 ℃.

[0051] Among them, F1, F2, F3, and F4 are formulations of long-acting polypeptide in-situ gel injections containing 15%, 20%, 25%, and 30% propylene glycol, respectively.

[0052] Example 4:

[0053] The rheological properties of the optimal peptide in situ gel long-acting injection formulation prepared in Example 1 were analyzed.

[0054] The method is as follows: Take an appropriate amount of the blank precursor solution, drug-loaded precursor solution, and their corresponding gel of the optimal formulation, and spread them evenly on the test platform, ensuring that the gap between the parallel plate clamps is completely filled and air bubbles are eliminated. Start the test after the instrument has stabilized. Use a 40mm aluminum alloy flat plate system, and set the shear rate scan range to 0.01–100 s. -1Real-time data acquisition of shear rate, shear stress, viscosity, and torque was performed to analyze flow characteristics. The experimental temperature was controlled at 25℃, and the sample was equilibrated for 10 minutes before measurement. After the test, the fixture surface was wiped with alcohol and a new sample was used. In oscillation mode, the angular frequency range was set to 0.1–100 rad / s, the shear strain was fixed at 1%, and the angular frequency, shear stress, storage modulus (G′), and loss modulus (G″) were recorded simultaneously.

[0055] Experimental results showed that both the blank precursor solution and the drug-containing precursor solution exhibited shear-thinning behavior, meaning that the viscosity decreased significantly with increasing shear rate, consistent with the characteristics of pseudoplastic fluids. Figure 4 A). The shear stress in both systems increased linearly with increasing shear rate. The increase in shear stress was more significant in the drug-containing prodrug solution, and the value was consistently higher than that in the control group at the same shear rate. This further illustrates that the addition of the drug enhances the viscous behavior of the system, thereby increasing its resistance to flow deformation. Figure 4 B).

[0056] Example 5:

[0057] The optimal peptide in-situ gel long-acting injection formulation prepared in Example 1 was administered subcutaneously, and the changes in postoperative incision pain threshold in rats were detected using a plantar dynamometer. Healthy male SD rats, weighing approximately 200–300 g (within ±15% of body weight), were selected; three rats were used for each formulation, with two spare rats. All rats were fed a standard diet at 20–25°C and 40–70% RH, with free access to food and water. Before measurement, rats successfully modeled according to the Brennan method were placed in the experimental apparatus for 30 minutes to acclimatize. When mechanical stimulation was applied, the rats exhibited positive reactions such as rapid paw withdrawal and paw licking. The pressure was stopped when the rats showed positive reactions such as rapid paw withdrawal or paw licking. The average of the three measurements was taken as the mechanical paw withdrawal threshold. The mechanical paw withdrawal threshold (MWTs) of rats was measured at 0, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, and 192 hours after administration. Baseline MWTs were measured for each rat before and after surgery.

[0058] The Brennan method involves using a No. 11 scalpel blade to make a longitudinal, layer-by-layer incision in the skin and subcutaneous tissue from 0.5 cm proximal to the rat's toe. After incising the skin, ophthalmic forceps are used to lift and expose the foot muscle. The muscle is then longitudinally incised down to the periosteum, while maintaining the integrity of the muscle's origin and insertion as much as possible. A longitudinal incision of approximately 1.0 cm in length is formed. Gauze is applied to compress and stop the bleeding. The wound is then sutured with 5-0 sutures, disinfected again with povidone-iodine, and penicillin ointment is applied to the wound to prevent infection.

[0059] Experimental results show that ( Figure 5Postoperatively, the mean squared incision length (MWTs) decreased by 51.5% compared to preoperative values. The baseline MWTs after modeling were significantly lower than the baseline before modeling, indicating the successful establishment of the rat incision pain model. Pain thresholds in all treatment groups increased from baseline within one week, and the immediate-release peptide formulation reduced the pain threshold to the preoperative pain threshold 8 hours after administration. Within 192 hours, the pain thresholds of the long-acting peptide in-situ gel injection group (low, medium, and high dose groups) were significantly different from those of the blank incision group and the direct peptide injection group. The onset time of the long-acting peptide in-situ gel injection group was not significantly delayed compared to the direct peptide injection group. The experimental results indicate that the pain threshold can be maintained for up to 7 days, and the long-acting peptide in-situ gel injection allows for the slow release of the tetrapeptide in vivo, maintaining an effective blood drug concentration and achieving a long-lasting analgesic effect.

[0060] Example 6:

[0061] Healthy female ICR mice, weighing approximately 20–23 g (with a weight deviation within ±15%), were selected; 6 mice were used for each prescription, with 2 spares. All mice were fed standard feed at 20–25℃ and 40–70% RH, with free access to food and water. Twenty-four mice were weighed and randomly divided into three groups: a blank control group, a long-acting polypeptide in-situ gel injection group (low, medium, and high dose groups), and the remaining groups received the corresponding drugs via subcutaneous injection of equal volumes. 180 minutes later, each group of mice was intraperitoneally injected with 1% glacial acetic acid (0.1 ml / 10 g). This administration of glacial acetic acid was repeated for seven consecutive days. The time to the first writhing response and the number of writhing movements within 45 minutes were observed.

[0062] Experimental results show that ( Figure 6 All doses of the long-acting peptide in-situ gel injection group showed significant analgesic effects in the early stages of the experiment, with a maximum analgesic effect percentage (MPE%) approaching 100%. However, the analgesic effect gradually weakened over time, reaching its lowest level on day 10. The high-dose group maintained a high MPE% (MPE% = 29.55%) throughout the experiment, with a slower attenuation of analgesia compared to the low-dose group (6.5 mg / kg). The analgesic effect of the medium-dose group (12.5 mg / kg) was between the two, and the number of writhing movements in the mice in the medium-dose group was further reduced, showing a better inhibitory effect than the low-dose group (p<0.05). The experimental results indicate that the long-acting peptide in-situ gel injection has a significant inhibitory effect on writhing behavior in mice within 7 days, demonstrating that the long-acting peptide in-situ gel injection of the present invention has a long-lasting analgesic effect.

[0063] Example 7:

[0064] Mice were allowed to acclimatize to the experimental environment for 30 minutes before the experiment to reduce the interference of environmental factors on the experimental results. Female ICR strain mice were selected as research subjects. Qualified individuals with a stable pain threshold of 5–30 seconds were screened through a preliminary experiment: mice were placed on a hot plate at a constant temperature of 55±0.5℃, and the latency of the first licking of the hind paw was recorded. Mice with a single pain threshold <5 seconds (hypersensitivity) or >30 seconds (hypoesthesia) were excluded. The baseline pain threshold of qualified mice was measured before drug administration: the pain threshold was measured twice at 10-minute intervals (T01, T02), and the arithmetic mean was taken as the baseline pain threshold (T0). The experiment lasted for ten days, and the pain threshold (T1) was measured daily under the same conditions after drug administration. If there was no response within 60 seconds, the mice were immediately removed to avoid tissue damage, and the result was recorded as 60 seconds. Finally, the changes in pain threshold before and after drug administration were statistically analyzed to evaluate the duration and effectiveness of the analgesic effect of the drug.

[0065] Experimental results show that ( Figure 7 The long-acting analgesic effect of the polypeptide in-situ gel long-acting injection was demonstrated by the change in %MPE (percentage of maximum possible effect) over time in different dose groups (6.5, 12.5, 25 mg / kg) and the vehicle (control group). All dose groups significantly inhibited hot plate-induced paw pain in mice and prolonged the pain threshold. The low, medium, and high dose groups showed statistically significant differences compared to the blank control group. The high-dose group exhibited a significant analgesic effect after administration (higher initial %MPE) and the longest duration of effect, maintaining a certain level of %MPE even on days 7-10, indicating its potent and sustained analgesic effect. Therefore, the polypeptide in-situ gel long-acting injection can prolong the analgesic effect, and the prolonged drug metabolic half-life at high doses maintains an effective blood drug concentration for a longer period.

[0066] Example 8:

[0067] Pharmacokinetic studies were conducted in New Zealand rabbits, and an LC-MS / MS method was established to determine the concentration of the original compound in New Zealand rabbit plasma. Before administration, 20.658 mL of physiological saline was added to a glass vial containing 6.26 mg of the tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH2, vortexed, and sonicated to obtain a clear, transparent solution with a concentration of 0.3 mg / mL. The administration volume was 1 mL / kg. Blood samples were collected at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h after administration.

[0068] Experimental results showed that an LC-MS / MS method for determining the concentration of the original compound in New Zealand rabbit plasma was successfully established. Linear regression analysis using the ratio of the area of ​​the tetrapeptide to the internal standard yielded a standard curve for the tetrapeptide concentration in plasma: y = 8.07·10. -5 x+1.07·10-5 , (R 2 =0.9978), the results showed good linearity within the concentration range of 1 ng / mL to 2000 ng / mL. The lower limit of quantification of the tetrapeptide in plasma was 1 ng / mL. Figure 8 A pharmacokinetic comparison of the long-acting peptide in situ gel injection (5.5 mg / kg) and the original tetrapeptide formulation is presented. The long-acting peptide in situ gel injection showed a rapid increase in plasma drug concentration at early time points after administration (15 min, 30 min, 1 h, 2 h), reaching a high level rapidly in the initial stage (0–24 h), followed by a slow decline, with detectable drug concentration still observed at 240 h. This indicates that the formulation has a significant sustained-release effect, with a long duration of drug release in vivo, maintaining effective plasma drug concentrations for a prolonged period. The original formulation's C... max and AUC 0-t The C values ​​for the long-acting peptide in situ gel injection were 223.67±24.63 ng / mL and 472.06±82.29 h·ng / mL, respectively. max和 AUC 0-t The concentrations were 339.4 ± 55.37 ng / mL and 35975.91 ± 4578 h·ng / mL, respectively. The results indicate that, compared with the original formulation, the Cg of the peptide in-situ gel long-acting injection is significantly higher. max It increased by 1.52 times, maintaining effective blood drug concentrations for a longer period. The half-life (t) of the long-acting polypeptide in-situ gel injection... 1 / 2 =12.04±1.17h) compared to the original formulation half-life (t) 1 / 2 The clearance rate (2.64±0.39 mL / min / kg) was significantly prolonged, increasing by approximately 16.05 times. This result reflects the sustained-release characteristics of the drug-loaded formulation, which significantly reduces the rate of drug elimination in vivo and thus significantly prolongs the duration of drug action. The clearance rate of the long-acting polypeptide in-situ gel injection (2.64±0.39 mL / min / kg) was lower than that of the original formulation (11.32±2.18 mL / min / kg), further confirming its slow elimination in vivo.

[0069] The above embodiments merely illustrate implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A long-acting polypeptide in-situ gel injection, characterized in that: This injection consists of the following components: polypeptide substance, gel matrix, and biocompatible solvent; The polypeptide is a tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH2, and the mass-volume percentage of the polypeptide is 0.1% to 4% (w / v). The gel matrix is ​​composed of an amphiphilic compound and natural phospholipids, with a mass ratio of the amphiphilic compound to natural phospholipids of 0.7 to 7:

1. The biocompatible solvent is 1,2-propanediol, and the mass fraction of 1,2-propanediol is 15% to 30%.

2. The long-acting polypeptide in-situ gel injection according to claim 1, characterized in that: The natural phospholipids are selected from soybean phospholipids and / or egg yolk lecithin, and the amphiphilic compounds are selected from one or more of monooleic glycerides, monostearate glycerides, and trioleic glycerides.

3. The long-acting polypeptide in-situ gel injection according to claim 2, characterized in that: The gel matrix contains egg yolk lecithin as the natural phospholipid and monooleoglyceride as the amphiphilic compound.

4. The long-acting polypeptide in-situ gel injection according to claim 1, characterized in that: The tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH2 has a mass-volume percentage of 0.5% to 2% (w / v).

5. The long-acting polypeptide in-situ gel injection according to claim 2, characterized in that: The mass ratio of monooleic glyceride to egg yolk lecithin in the gel matrix is ​​4:

5.

6. The long-acting polypeptide in-situ gel injection according to claim 2, characterized in that: The mass fraction of the 1,2-propanediol is 25%.

7. A method for preparing a long-acting polypeptide in-situ gel injection as described in any one of claims 1 to 6, characterized in that: Includes the following steps: After adding a biocompatible solvent to the gel matrix, an excess of anhydrous ethanol was added and ultrasonically stirred. The excess ethanol was then removed by solvent evaporation. The polypeptide substance is then added to the prepared solution and vortexed until completely dissolved; thus, a long-acting polypeptide in-situ gel injection is obtained.

8. The use of a long-acting polypeptide in-situ gel injection as described in any one of claims 1 to 6 in the preparation of a pain-relieving drug, characterized in that: The pain is for the treatment of acute or chronic postoperative pain; the drug is an in-situ gel long-acting injection containing the tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH2.

Images