CN114344299A - Lipid drug delivery system with long-acting sustained release effect and preparation method thereof - Google Patents

Abstract

The invention discloses a lipid drug release system with long-acting slow release effect and a preparation method thereof, wherein the lipid drug release system is transparent yellowish to yellow clear liquid and comprises the following components: active ingredients, namely phospholipid serving as a core raw material of a lipid drug release system, organic solvent and medicinal oil which are mutually soluble with water and serve as dispersion media for dissolving the phospholipid and the active ingredients, water and cholesterol which are used for regulating the drug release behavior, and an antioxidant with an antioxidant effect; wherein the active ingredient is ropivacaine free base. The preparation method comprises the steps of weighing the active ingredients, the phospholipid, the cholesterol, the medicinal oil and the antioxidant according to the prescription, shearing at a high speed or stirring at a high speed, heating to 40-70 ℃ to completely dissolve the system, replacing air in the system with nitrogen in the process to prevent oxidation, cooling to 20-40 ℃, adding the organic solvent, stirring uniformly, adding a proper amount of water for injection, stirring rapidly until the system is clear, filtering through a filter membrane to sterilize, subpackaging, filling with nitrogen, plugging and sealing to obtain the composition.

Description

Lipid drug delivery system with long-acting sustained release effect and preparation method thereof

Technical Field

The invention belongs to the field of pharmaceutics, and mainly relates to a lipid release system with long-acting slow release effect and a preparation method thereof.

Background

Pain is an unpleasant sensation and emotion caused by existing or impending tissue damage, is a relatively common warning signal in the clinic that may be accompanied by anxiety in the patient, and is an emotional experience for the patient. The term "pain" means, for the patient, pain and/or disease that may be suffered, and, for the physician, a clinical condition; the concept of pain has been used to date and has not changed since the work written by merck in 1964 was recognized and widely used, and was subsequently published in a form published in 1979. Melzack et al, in journal of science, proposed a new theory of pain, driving human understanding of pain mechanisms and pain management. In 2016, Williams et al reviewed the previous definition of pain, and in order to better grasp the nature of pain now understood, proposed the following new definitions: pain is a painful experience that is associated with a patient's sensory, emotional, cognitive, and social factors or with its own underlying tissue damage.

Diseases with "pain" as the main symptom are called "painful diseases" clinically, and are called "painful diseases" for short. According to the duration and nature of pain, acute pain and chronic pain can be distinguished. Acute and chronic pain is common clinical symptoms of orthopedic patients, and slight pain can cause mental pain of the patients and reduce the life quality of the patients; severe pain can cause various system dysfunction and low immunity of the human body to induce various complications, even cause pain disability or pain shock, and threaten the life of patients. Therefore, the research on scientific and effective clinical diagnosis and treatment means is vital to relieve the clinical symptoms of patients with acute and chronic pains and improve the life quality of the patients.

Background problem one: the deficiency of anesthetic drugs and the solution of the invention

Local anesthetics (abbreviated as local anesthetics) are drugs which can eliminate the pain sensation of local tissues under the condition that people are conscious. Wherein, the amide local anesthetic comprises lidocaine, ropivacaine, bupivacaine and the like. Although they have similar parent-nucleus chemical structures, the substituent groups have great influence on the spatial structure, physicochemical properties, pharmacological efficacy and drug toxicity of the drug, particularly on formulation formation. For example, ropivacaine and bupivacaine are structurally similar, but the former is used for surgical anesthesia, labor and post-operative analgesia, the latter is used for local infiltration anesthesia, peripheral nerve block and intraspinal block; for another example, the prior literature adequately demonstrates that ropivacaine and bupivacaine also exhibit dramatic differences in toxicity. Therefore, the selection of reasonable analgesic elements for development has always eluded the problem in the art.

Through extensive research, the inventors found that the amide local anesthetics have great differences in the formation of new dosage forms. For example, although there are reports on the documents of lipid drug delivery systems for amide local anesthetics, no ropivacaine sustained release preparation prepared by using the lipid drug delivery system is successfully marketed at home and abroad. The reason for this is that lidocaine, ropivacaine, bupivacaine and the like are all long-acting amide local anesthetic drugs, and free alkali is insoluble in water, so that the products on the market at present are all hydrochloride or mesylate, and the solubility problem can be effectively solved, so that the injection prepared is convenient for clinical use. This is the most common, basic method of use for such drugs. However, there are obvious problems in that the salt rapidly enters blood circulation after local injection due to its good solubility, the half-life is short, and the irritation is strong due to the low pH. In order to realize long-acting effect, a continuous administration method is generally adopted clinically, which brings great inconvenience to pain patients. Therefore, for researchers of drug scientists for treating pain, the problem is to find a solution to the long-acting pain-relieving problem among a plurality of drugs for treating pain, possible structural modification or new dosage form treatment schemes. The idea of the inventor is to select ropivacaine and to select the free base of ropivacaine and to use a new formulation. The free alkali has indissolvability, is different from a salt form and is rapidly transported in vivo, so that the half-life period is short, and the novel dosage form is matched with the chemical structure and the physicochemical property of the medicament, so that the sustained medicament release is realized, the long-acting effect is achieved, and the side effect is reduced to the greatest extent.

Ropivacaine is a novel long-acting amide local anesthetic drug, first marketed in the netherlands since 1996, and introduced for clinical anesthesia in 1999 in China. The medicine on the market is prepared into a clear and transparent sterilized aqueous solution by hydrochloride or mesylate and is used for anesthesia of surgical operation or acute pain control, and the administration modes comprise lumbar epidural administration, thoracic epidural administration and subarachnoid cavity administration. The onset time is about 10-20 minutes, t1/2 is 1.8 hours, and anesthesia or analgesia lasts 2-6 hours. A large number of clinical researches prove that ropivacaine has definite curative effect on obstetrical anesthesia and painless childbirth, has light motor nerve block, does not influence the childbirth process and newborn, and is a common and ideal analgesic medicament at present. In order to achieve the purpose of analgesia, a continuous intravenous drip mode is generally adopted in clinic, no long-acting ropivacaine preparation is on the market at present, and other means are needed for postpartum analgesia.

As a similar drug which can be referred to, bupivacaine is also an amide local anesthetic, and the hydrochloride thereof, namely bupivacaine hydrochloride, is commonly used for treating peripheral nerve block, brachial plexus block, epidural anesthesia, postoperative pain and the like, and has the characteristics of stronger anesthesia efficiency, obvious separation of motor and sensory blocks and the like. Bupivacaine is mainly used and has the advantages of quick response, long action duration and the like, but the bupivacaine has a certain drug toxicity effect on the central nervous system and the heart and can cause adverse anesthesia reactions such as hypotension, respiratory depression, bradycardia and the like, but a common injection (with the concentration of 0.5%) only has the analgesic effect of 5-7 h, the postoperative pain usually lasts for 48-72h, and the time period is the most difficult to control, so that the bupivacaine liposome Exparel adopting Depofoam is sold abroad, has the slow release function, and the long-acting analgesic time reaches 72 h. However, the clinical application of bupivacaine lipid preparations has been hampered by various reasons including the drug itself and the components of the preparation.

The inventor believes that the properties of the drug itself should be sufficiently removed from the pharmaceutical preparation, particularly the novel preparation, and thus a reasonable formulation and formulation should be designed. For example, ropivacaine and bupivacaine do not look large in chemical structure, but differ enough to result in a large difference in physicochemical properties, ropivacaine having a pka8.1, log p of 2.9; the pKa of bupivacaine is 14.85, the LogP is 3.9, so that the acidity of ropivacaine is higher than that of bupivacaine, the fat solubility of ropivacaine is lower than that of bupivacaine, and the fat solubility of ropivacaine is small, so that the time for the ropivacaine to reach the coarse motor nerve is delayed, and the unique action characteristic of separating the motor from the sensory block of the ropivacaine is formed, and the characteristic enables the ropivacaine to be more favorable for the recovery of early motor function after operation in clinic. It can be seen that under the premise of great difference of drug properties, ropivacaine and bupivacaine are applied in a complex preparation system, and the effect is different. The reasonable method is to design a specific and specific ropivacaine free base preparation scheme, thereby improving the clinical application value of the medicament.

The second background problem: deficiencies of phospholipid formulations and solutions of the present invention

The phospholipid-based drug delivery systems include liposomes, fat emulsions, microemulsions, and the like, and are widely used, but the types of preparations are particularly complex, and vary from drug to drug and from system to system. Wherein, the used auxiliary material phospholipid has the self-assembly characteristic, so a drug delivery system can be formed under the appropriate condition. Self-emulsifying drug delivery systems are liquid formulations consisting of an oil phase, a non-ionic surfactant, and a co-surfactant. However, it is not easy to solve the problem of phospholipids as carriers to form specific novel formulations, and not a simple additive effect. Moreover, the new dosage form of the medicine is different from the traditional preparation, and the preparation components and the dosage form system are very complicated.

The inventors have surprisingly found that by taking advantage of the essential feature that under suitable conditions a phase transition can occur, thereby forming a drug reservoir, the drug is slowly released, thereby achieving a long-lasting sustained release effect. The phospholipid is a basic substance for forming cell membranes and also a main component for forming a phospholipid reservoir, has the characteristic of self-assembly in water, and can spontaneously form liposome, micelle and gel with a spatial structure to become a drug carrier; in addition, phospholipids have a surface active effect and can effectively solubilize poorly soluble molecules. Meanwhile, as the phospholipid is an important component substance of the cell membrane, the phospholipid has strong affinity with the cell membrane and good biocompatibility. At present, various preparation technologies and dosage forms using phospholipid as an auxiliary material, such as fat emulsion and liposome, are available on the market. Therefore, the preparation prepared by using the phospholipid as the auxiliary material has excellent safety and biocompatibility, and provides basic guarantee for the safety of medication. In the present invention, the role of the phospholipid is to form a drug reservoir.

Cholesterol and phospholipids are the basic materials that co-constitute membranes and liposomes. Cholesterol is an amphoteric substance and can regulate the fluidity and permeability of phospholipid bilayers. Free radicals are generated in the phospholipid bilayer to accelerate the oxidation of phospholipid, and cholesterol is added into the bilayer to solidify the membrane, so that the generation of free radicals is reduced, and the oxidation level is reduced. Phospholipid can self-assemble into a bilayer structure in water, and the structural integrity of the phospholipid bilayer can be changed by adding a proper amount of cholesterol into the phospholipid bilayer structure, so that the stability of the bilayer is greatly increased, the phenomenon that a burst release effect is generated due to the quick release of a medicament is avoided, and the toxic reaction caused by the overhigh instantaneous medicament concentration is avoided. In other words, in the present invention, the dual role of constructing a drug reservoir and preventing burst release of the drug is achieved using phospholipids and cholesterol.

Further, when the ambient temperature is higher than the phase transition temperature, the bilayer stability of the phospholipid self-assembly system is changed, so that the drug is released. The phase transition temperature of the natural phospholipid is about-20 ℃, if a drug reservoir is constructed by only one natural phospholipid, the phase transition temperature of a system formed by self-assembly is consistent, and the phase transition temperature is lower, so that the reservoir has poor stability after injection, and the burst effect is easily caused. A system formed by mixing a plurality of phospholipids with different phase transition temperatures is selected, and the storage stability formed after the phase transition temperature is increased.

Furthermore, the addition of cholesterol can effectively change the phase transition temperature of the system. Above the phase transition temperature, it can inhibit the rotational isomerization movement of fatty acyl chain in phospholipid molecule, and reduce the fluidity of membrane; below the phase transition temperature, the membrane lipids are in crystalline alignment, which in turn induces the formation of a distorted conformation of the fatty acyl chains, preventing the appearance of the crystalline state. Hydroxyl in cholesterol molecules and carbonyl in phospholipid molecules can form a compound through hydrogen bonds, free movement of fatty acyl chains is reduced, membrane compression is caused, the area is reduced, combination is tight, and the mobility is reduced, so that the leakage rate of the medicine is reduced, and the burst effect is avoided. In short, cholesterol itself exerts two roles in bidirectional regulation.

The dispersion medium is one of the essential components of injection, and water for injection, medicinal oil or water-miscible organic solvent are often used as the dispersion medium, aiming at solving the problem of solubility of the active ingredients. In the present invention, the choice of the dispersion medium is peculiar.

Medicinal oil is commonly used as solvent, and has good safety, soybean oil has been used as medicinal oil for injection for more than 60 years, and its rat intravenous injection LD₅₀It was 22.1 g/kg. Because of its good safety, it is used in many marketed drugs.

Medium Chain Triglycerides (MCT) are derived from coconut oil and purified to medium chain fatty acids, sinceThis has good hydrophilicity and oxidation stability. Can be used as solvent for dissolving fat soluble components such as phospholipid, ropivacaine and cholesterol. The good safety of the compound has been used as a pharmaceutical raw material and auxiliary material for a plurality of injection drugs, such as medium/long-chain fat emulsion injection, mixed oil injection (SMOF) and the like. Acute toxicity tests of rabbits show that oral LD₅₀Greater than 5g/kg, subcutaneous LD₅₀More than 2 g/kg.

Organic solvents miscible with water, such as ethanol, propylene glycol, glycerol, tert-butanol, etc., are also used as common solvents for changing system properties, stability and related parameters of injection.

Propylene glycol is mainly used as preservative, bactericide, wetting agent, plasticizer, solvent, stabilizer and cosolvent in the pharmaceutical field. Can be administered by intramuscular injection, intravenous injection, nasal administration, oral administration, ophthalmic administration, otic administration, topical administration, etc.

Ethanol is used primarily as a solvent and is also commonly used as an antimicrobial preservative for solutions. Topical ethanol solutions are also used as permeation enhancers and disinfectants. Ethanol is an inhibitor of the central nervous system and the intake of small to moderate amounts of ethanol can lead to intoxication symptoms. The ingestion of high-concentration ethanol can cause spinal reaction reduction, lethargy, amnesia, hypothermia, hypoglycemia, coma, numbness, respiratory depression and cardiovascular failure, and the lethal concentration of human blood ethanol is 400-500 mg/100 mL. Topical application of ethanol above 50% v/v may be irritating to the skin.

Although organic solvents such as ethanol and propylene glycol are often used as solvents of injections, the organic solvents still have certain irritation to injection sites, so that the dosage of the organic solvents needs to be reduced as much as possible, and adverse reactions during injection are reduced.

Dispersion medium animal toxicity data are as follows:

selection of the solvent (organic solvent) for the dispersing medium

	LD50(subcutaneous injection)	LD50(Abdominal injection)	LD50(intravenous injection)	LD50(oral administration)
					Species of	g/kg	g/kg	g/kg	g/kg
Ethanol	8.29	0.93	1.97	3.45
					Propylene glycol	17.34	9.72	6.63	22.0

From animal toxicity data, propylene glycol is safer than ethanol for various routes of administration.

In order to prepare a drug reservoir with slow release effect and low irritation and prolong the action time of active ingredients in vivo so as to achieve the slow release effect, the technical scheme adopted by the invention is to dissolve phospholipid and active ingredients by taking an organic solvent and oil which are mutually soluble with water as solvents; cholesterol and water are used as auxiliary materials for controlling the release behavior of the drug delivery system, and a proper amount of antioxidant is added at the same time, so that the stability of the preparation system is ensured.

The invention utilizes the phospholipid to form a self-assembly system under certain conditions so as to form a drug reservoir, embeds the active ingredient in the drug reservoir, has slow release effect, and utilizes the phospholipid as a nonionic surfactant with solubilization effect. In addition, the excellent biocompatibility of the liposome enables the liposome to be widely used in the pharmaceutical field, and a plurality of fat emulsion, liposome and phospholipid mixed micelle products all use phospholipid as main auxiliary materials at present. The invention utilizes the characteristics of phospholipid to prepare a lipid drug release system with long-acting slow release effect. The outstanding characteristic of the lipid drug delivery system is that the lipid drug delivery system is designed for ropivacaine free alkali.

The third background problem: the disadvantages of the existing lipid preparation

Chinese patent 201380036669 "depot formulation of hydrophobic active ingredient and method of preparation thereof" provides a non-aqueous, proliposome depot formulation for hydrophobic APIs and a method of manufacture thereof, wherein the step of emulsification is not included, and wherein the composition is not exposed to an aqueous phase at any stage prior to entering the patient's body. In addition, the present invention provides a depot formulation using only GRAS excipients, substantially free of synthetic phospholipids. A pharmaceutical composition comprising: a hydrophobic Active Pharmaceutical Ingredient (API); from about 40% to about 60% by weight of Phosphatidylcholine (PC) or a pharmaceutically acceptable salt thereof; from about 30% to about 50% by weight castor oil; and about 2% to about 10% by weight ethanol, wherein the composition is in the form of a clear solution, free of particles greater than 100nm in size, stable at ambient temperature, and substantially free of water. Chinese patent 201380036700 "depot formulation of local anesthetic and method of preparation thereof" provides a non-aqueous, proliposomal depot local anesthetic formulation that beneficially generates liposome or other lipid vesicle structures in situ upon contact with body fluids. The invention also provides a method for preparing the depot formulation of the invention, wherein the composition is not exposed to an aqueous phase at any stage of the preparation process. The composition is free of water, except for residual moisture that may be present in the excipients used to make the composition. Both of these patents are non-aqueous systems and the main component is a slow release carrier containing castor oil, which is reported to be low to moderate hazardous in MSDS, with some irritation and allergic effects on the skin and eyes. Moreover, both patents are used for hydrophobic drugs in a general way, and are not specific drug delivery systems, and the scenes used in the literature are far from the practical application.

Chinese patent 201810592597 improves 201380036669 and 201380036700 systems, provides a formulation and a preparation method of a phospholipid-cosolvent-oil sustained-release drug delivery system of local anesthetics, and soybean oil, medium chain triglyceride and corn oil are used in the preparation scheme to replace castor oil in the above patents, so that skin irritation is effectively avoided. But benzyl alcohol and benzyl benzoate were used as solvents. Also, it is a non-aqueous delivery system, and when it is injected subcutaneously, drug diffusion depends on solvent diffusion, and the more water-soluble solvent, the faster the diffusion speed. The phospholipid self-assembly is dependent on water, when enough tissue fluid in subcutaneous tissues interacts with the phospholipid, a system can generate phase change, the phospholipid self-assembly can be formed, and the sustained release effect is achieved, so that the long-acting effect is realized. Therefore, there are cases where the release of the drug is too fast in the former stage of the system. Meanwhile, in the patent technology, the dosage of oil and solvent is relatively large, and the sum of the solvent and the oil reaches 40-90% of the total amount of the system, so that the drug release is mainly based on solvent diffusion.

Among them, ingestion or inhalation of benzyl alcohol may cause dizziness, vertigo, nausea, vomiting and diarrhea. A large number of inhalations can lead to central nervous system depression and dyspnea. The adverse reactions include: intravenous toxicity, intrathecal neurotoxicity, hypersensitivity and infant toxicity syndrome. The FDA recommends that benzyl alcohol should not be used in the rinse. LD₅₀(mouse, intravenous injection) 0.32g/kg, more toxic than ethanol. Benzyl benzoate can be rapidly hydrolyzed in vivo into benzoic acid and benzyl alcohol, which are further metabolized into hippuric acid, which is excreted via urine. Benzyl benzoate is used as adjuvant for topical administration for treating scabies and intramuscular injection, and the concentration of oral preparation is 25% (V/V). Adverse reactions include skin irritation and allergic reactions. LD₅₀(mouse, oral) 1.4g/kg, also more toxic than ethanol.

Therefore, the toxicity of benzyl alcohol and benzyl alcohol used in the patent is stronger than that of ethanol, so that strong irritation can be generated when injection is performed, and adverse reactions such as local red swelling, pain and the like can be caused. Further, the composition of ropivacaine described in this patent document does not solve the technical problems of the present invention.

Chinese patent 201310403977 "in situ lipid gel pharmaceutical formulation and its preparation method and use" provides an in situ lipid gel pharmaceutical formulation, which is characterized in that the active substance is a local anesthetic, preferably an amide local anesthetic, more preferably bupivacaine, levobupivacaine, ropivacaine, lidocaine or their pharmaceutically acceptable salts, and particularly preferably bupivacaine, levobupivacaine or their pharmaceutically acceptable salts. Among them, water is introduced into lipid formulations and is thought to be able to regulate the viscosity of the formulation. The technical scheme of the invention does not contain oil and cholesterol.

The patent mainly uses phospholipid, ethanol and water as auxiliary materials, and the essence of the preparation is that the phospholipid forms a multi-layer liposome with a bilayer structure by self-assembly after meeting water, so that the preparation has a slow release effect. However, since the phase transition temperature of the soybean lecithin or egg yolk lecithin used in the technical scheme is about-20 ℃, when the ambient temperature is below the phase transition temperature of the liposome, the lipid bilayers are tightly arranged, the fluidity is small, the drug leakage is small, and the lipid membrane undergoes phase transition with the rise of the temperature, the cross section of the membrane is increased, the permeability and the fluidity are increased, and the drug is inevitably accelerated to leak from the liposome, so the phase transition temperature is an important parameter for measuring the stability of the lipid system. Therefore, when the temperature is higher than the phase transition temperature, the permeability of the membrane of the lipid bilayer is increased, the drug is released, and the temperature of a human body is far higher than the phase transition temperature, so the phase transition is easy to occur after subcutaneous injection, and the diffusivity of the used solvent ethanol is strong, so that the drug release speed is accelerated, the burst release is caused, the use safety of the drug is influenced, and the prescription still has no practical use.

A well flowing clear transparent system with an ethanol content of up to about 33% was obtained with a preferred phospholipid/ethanol ratio of 2:1 in example 1 as described in the patent. Local stimulation caused by ethanol has been studied, and it is shown that gel is prepared and local stimulation of injection site is studied by using Ting Zhang (Zhang, Ting, ethyl. A high-efficiency, low-sensitivity, phosphopeptides-based phase separation gel for long-term delivery of peptides, 45(2015), 1-9.) and the local stimulation is directly related to the amount of ethanol, the more ethanol is used, the more severe local stimulation is, and connective tissue necrosis and epidermal ulcer are generated at the injection site when the amount of ethanol reaches 30-45%, so that the amount of ethanol is strictly controlled.

In addition, the heating temperature of 30-80 ℃, preferably 30-50 ℃ and the stirring time of more than 30 minutes, which are described in the document, can cause the ethanol in the system to volatilize when stirring and heating are carried out simultaneously, thereby changing the proportion of the ethanol in the composition, and because the proportion of the water is 20% -80% of the sum of the phospholipid and the organic solvent, the amount of the water depends on the organic solvent, when the organic solvent is volatilized, particularly at the critical point of the system, the original prescription amount of the water is still added, the phase change of the system can be caused, and the turbidity and even the drug precipitation can occur in the process of storage, transportation and storage of the preparation. 201310403977 therefore does not solve the technical problem addressed by the present invention.

Chinese patent 202110046374.7 "a depot ropivacaine pharmaceutical composition and its preparation method and use" seems to make a new improvement to the ropivacaine drug depot by providing a depot ropivacaine pharmaceutical composition comprising, in weight percent, 0.5% to 10% of ropivacaine based on the total weight of the composition; 2% to 25% of a pharmaceutical solvent; 8% to 55% of a pharmaceutically acceptable phospholipid; 15% to 89% of medicinal oil; 0.5% to 10% of a pharmacodynamic enhancer; about 0% to 1% of an antioxidant; about 0% to 8% of an acid base modifier. However, this document returns to the way of non-aqueous preparations and fails to overcome various drawbacks of non-aqueous preparations, including failure to achieve self-assembly of phospholipids to achieve drug depot in a short time and failure to avoid strong solvent diffusion effects due to the use of large amounts of solvents. The inventor also carries out long-term follow-up research on a large number of documents relating to ropivacaine lipid preparations, and finds that the existing documents are roughly divided into a nonaqueous system and a water-containing system, and the preparation forms and the in vivo action mechanisms are various and complicated. For another example, chinese patent 201410067859 provides a ropivacaine nano lipid carrier temperature-sensitive in-situ gel and a preparation method thereof, the main components of which are raw material drugs of ropivacaine, a solid lipid material, a liquid lipid material, a surfactant, a cosurfactant, a gel matrix and water for injection. The technical scheme is that ropivacaine is firstly prepared into an emulsion, and then the emulsion is added into a gel matrix prepared from poloxamer, so that the ropivacaine is far away from a phospholipid storage system. Therefore, there is currently a lack of a truly effective, targeted solution to the long-acting ropivacaine drug depot formulation, and none of the solutions makes the long-acting ropivacaine drug depot formulation a drug on the market. This stems from the special properties of ropivacaine and the technical complexity of new dosage forms for lipid drug depots.

In the invention, based on the specific properties of ropivacaine, phospholipid is adopted as a main raw material for forming a drug reservoir, medicinal oil and an organic solvent which is mutually soluble with water are used as dispersing solvents to dissolve active ingredients, so that the using amount of the organic solvent is effectively reduced, meanwhile, the addition of cholesterol improves the phase transition temperature of a system and the stability of a bilayer structure after phospholipid self-assembly, increases the stability of the formed reservoir in vivo and controls the drug release speed, thereby effectively avoiding burst effect, controlling the phase transition critical point by water content, and promoting the rapid formation of the drug reservoir after injection, thereby avoiding burst release. The technical scheme adopted by the invention is essentially different from the above patent documents.

Disclosure of Invention

The invention adopts phospholipid as a key material for forming a lipid drug release system, medicinal oil and an organic solvent which is mutually soluble with water as solvents to dissolve the phospholipid, active ingredients and cholesterol, simultaneously adopts the phase change temperature of a cholesterol regulation system to enhance the in vivo stability of the system after phase change and avoid burst effect, and in addition, water is used for regulating the phase change critical point to promote the rapid formation of a drug reservoir in vivo so as to realize the purpose of slow release. The components and the dosage thereof are matched with each other to prepare the ropivacaine lipid drug release system with long-acting slow release effect.

The invention aims to provide a lipid drug release system with long-acting sustained release effect and a preparation method thereof, which are used for transferring active ingredients of drugs, thereby realizing the long-acting effect that the release time of the active ingredient ropivacaine reaches 48 to 72 hours. The lipid sustained-release preparation is a novel high-safety lipid sustained-release system with long-acting sustained-release effect, has wide clinical application prospect, must become powerful supplement of relevant preparations of clinical sustained-release medicines, and increases the selectivity of clinical medication.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

the invention discloses a lipid drug release system product with long-acting sustained release effect; the lipid drug release system is a clear and transparent uniform liquid consisting of active ingredients, phospholipid, drug release behavior regulating substances, a dispersion medium and an antioxidant.

In a preferred embodiment of the present invention, the active ingredient is a fat-soluble active ingredient. The active ingredient is free alkali of ropivacaine which is a local anesthetic.

The ropivacaine has definite curative effect on obstetrical anesthesia and painless delivery, has light motor nerve block, does not influence the delivery process and newborn, comprehensively considers the solubility of the active ingredients and the clinical use safety, and is further preferably the free base of the ropivacaine.

The phospholipid is used as a key component of a lipid slow release system, is a main carrier of a medicinal active component, and is preferably phospholipid with good biocompatibility and high safety in order to further ensure the safety of medication. In addition, it is also necessary to select phospholipids having excellent solubility in a dispersion medium to reduce the amount of a solvent used to secure a drug concentration, while minimizing the administration volume upon local injection, facilitating clinical procedures and reducing adverse reactions. At the same time, the selected phospholipid needs to be compatible with other formulation components.

In yet another preferred embodiment of the present invention, the phospholipid comprises a natural phospholipid or a synthetic phospholipid. The natural phospholipid is derived from semen glycines, ovum gallus Domesticus flavus and flos Helianthi, and the commonly used natural phospholipids include semen glycines phospholipid, ovum gallus Domesticus flavus lecithin; synthetic phospholipids include phospholipids hydrogenated using natural phospholipids or obtained using total synthesis methods, including: hydrogenated Soybean Phospholipids (HSPC), Dipalmitoylphosphatidylcholine (DPPC), Dimyristoylphosphatidylcholine (DMPC), Distearoylphosphatidylcholine (DSPC).

The content of phosphatidylcholine in the phospholipid is more than 70%, and preferably the content of phosphatidylcholine is more than 95%. Preferably, soybean lecithin or egg yolk lecithin is used, and the synthetic phospholipid is used in an amount of 0-5%, preferably 1-5% of the natural phospholipid.

The natural phospholipid has a lower phase transition temperature which is generally less than 0 ℃, a multi-layer structure storage with bilayer is formed after injection, when the ambient temperature is below the lipid phase transition temperature, the lipid bilayer is tightly arranged, the fluidity is low, the drug leakage is low, and along with the temperature rise, the lipid membrane undergoes phase transition, the cross section of the membrane is increased, the permeability and the fluidity are increased, and the drug can be accelerated to leak from the liposome. Because the temperature of a human body is far higher than the phase transition temperature of natural phospholipid, the permeability of a bilayer generated by self-assembly after injection is increased, so that the release of the medicine is accelerated, and the medicine is easy to leak so as to cause burst release. Therefore, the first improvement of the invention is that the drug release is improved by increasing the phase transition temperature of the system. The synthetic phospholipid containing choline structure has high phase transition temperature, most of the phase transition temperature of cholesterol and some synthetic phospholipids is higher than 20 ℃, the phase transition temperature of the system can be improved by adding the synthetic phospholipid, for example, the phase transition temperature of soybean phospholipid is about-20 ℃, when a small amount of cholesterol is added, the phase transition temperature of the system is about 40 ℃, and the improvement of the phase transition temperature is beneficial to the improvement of the system stability after the reservoir is formed. This outstanding advantage makes the ropivacaine free base drug reservoir a core guarantee. Accordingly, the prior art does not consider the technical problem of phase transition temperature of ropivacaine lipid reservoir, which inevitably results in burst or over-rapid release of the drug.

As a preferable scheme, the substrate is composed of natural phospholipid (or synthetic phospholipid) and cholesterol, and the phase transition temperature of the system is increased.

In another preferred scheme, natural phospholipid, synthetic phospholipid and cholesterol form a matrix, and the phase transition temperature of the system is increased. Wherein the synthetic phospholipid is 0-5%, preferably 1-5%.

The active pharmaceutical ingredients, phospholipids and cholesterol are all lipid soluble ingredients, and can be dissolved in oil or organic solvents.

In order to solve the problem of dissolving phospholipid, active ingredients of medicaments and cholesterol, the injection is prepared to be easy to inject.

In another preferred embodiment of the present invention, the dispersion medium is a medicinal oil and a water-miscible organic solvent. Wherein the medicinal oil is one or more of soybean oil, medium chain triglyceride, olive oil, tea seed oil, and fish oil, preferably soybean oil. The ratio of medicinal oil to phospholipid is 4:1-1:4, more preferably 1:1-1: 4.

The water-miscible organic solvent is one or more of ethanol, propylene glycol and glycerol. Preferably, ethanol is used as the dispersion medium in combination with propylene glycol or glycerol, preferably in a ratio of from 1:1 to 10:0(w/w), more preferably from 7:3 to 10:0 (w/w).

The phospholipid is important for the system, is not only an important carrier for transferring the active ingredients of the medicine, but also a key component for increasing the solubility of the active ingredients of the medicine due to the property of the surfactant, is beneficial to reducing the solvent, thereby reducing the use volume of a final finished product and improving the safety of clinical use.

Data relating to the solubility of pharmaceutical active ingredients in different dispersion media

From the above data, it can be seen that the solubility of ropivacaine free base in absolute ethanol is optimal, but the solubility drops sharply when water is added; the phospholipids are effective in increasing the solubility of ropivacaine in soybean oil and ethanol-water.

Solubility data for cholesterol in medicinal oils and organic solvents

In the technical implementation process of the invention, the medicinal oil is surprisingly found to improve the stability of the system and effectively avoid the precipitation of the medicinal active ingredients and cholesterol in the system.

The dispersion medium, in addition to its important role as a solvent to dissolve the active ingredient, phospholipids and cholesterol, also affects the release of the drug, generally the more solvents, the faster the diffusion and the faster the release of the drug. Therefore, the technical scheme further researches the release behavior of the medicinal oil and other solvents and the influence of the dosage of the solvents on the release behavior.

The method comprises the following steps: about 1.0g of the sample was taken and placed in a dialysis bag (3000Da) which was placed in phosphate buffer and incubated at 37 ℃ with stirring at 100 rpm, and samples were taken at different time points and assayed.

The data on the effect of different solvents on the release of the active ingredient are as follows:

the results show that the release is faster with ethanol as solvent than with soybean oil as solvent.

In another preferred embodiment of the present invention, the ratio of the medicinal oil to the phospholipid in the dispersion medium is 4:1 to 1: 4. Further preferably 1:1 to 1: 4.

Due to the diffusion effect of the solvent, in addition to the effect of the medicinal oil on the release, the amount of water-miscible organic solvent also affects the release of the drug.

In another preferred embodiment of the present invention, the dispersion medium further comprises an organic solvent miscible with water, and the ratio of the phospholipid to the organic solvent is 1:1-2: 1. Further preferably 1.5:1 to 2: 1.

The dispersion medium contains water-miscible organic solvent (including one or more of ethanol, propylene glycol and glycerol) besides medicinal oil, and preferably ethanol and propylene glycol are used as dispersion medium in combination with medicinal oil. The ratio of ethanol to propylene glycol or glycerol is preferably 1:1 to 9:1(w/w), more preferably 1:1 to 2:1 (w/w).

In a preferred embodiment of the present invention, the system contains water, which is provided in the form of water for injection, and other forms of water, such as water containing sodium chloride and buffer salts, are avoided, so that the mono-or divalent ions contained therein are prevented from causing catalytic oxidation of phospholipids and unsaturated medicinal oils.

The proportion of the organic solvent with water accounts for 10-30% (w/w), preferably 20-30% (w/w).

Cholesterol is also one of the components of cell membranes, belongs to an amphoteric substance and can regulate the fluidity and permeability of phospholipid bilayers. Surprisingly, the addition of the cholesterol can change the drug release behavior of the lipid drug release system, and the drug release is slowed down along with the increase of the dosage of the cholesterol, thereby providing a great effect for realizing the long-acting release of the lipid drug release system.

In yet another preferred embodiment of the present invention, the ratio of cholesterol to phospholipid is 1:5 to 1:20 (w/w).

The cholesterol is a key raw material for changing the release behavior of the drug, the addition of the cholesterol obviously delays the release of the drug, and the action mechanism of the cholesterol is another mechanism which improves the phase change temperature of a system, changes the integrity of a bilayer formed by self-assembly of phospholipid and changes the strength of the bilayer, so that the burst effect caused by drug leakage is not easy to occur.

In another preferred embodiment of the present invention, the antioxidant is one of vitamin E acetate and dl-alpha-tocopherol, and the amount of the antioxidant is 0.1-1% (w/w).

In order to achieve the expected effect of the invention, the preferred scheme of the invention is as follows:

(1) in a preferred embodiment of the present invention, the active ingredient is ropivacaine; the content of active ingredient is 2-8%, preferably 3-6%.

The ratio of the medicinal oil to the phospholipid is 4:1-1:4, and more preferably 1:1-1: 4;

the ratio of the phospholipid to the organic solvent is 1:1-2:1, and the preferable ratio is 1.5:1-2: 1;

the ratio of cholesterol to phospholipid is 1:5-1:20(w/w), more preferably 1:8-1:20 (w/w);

the proportion of the organic solvent to water is 10 to 30% (w/w), and more preferably 20 to 30% (w/w);

the proportion of the antioxidant is 0.1 to 1 percent of the sum of the phospholipid and the oil.

(2) In another preferred embodiment of the present invention, the preparation method comprises the following steps:

weighing active ingredients, phospholipid, cholesterol, medicinal oil and antioxidant according to a formula, shearing at a high speed or stirring at a high speed, heating to 40-70 ℃, preferably heating to 40-50 ℃ to completely dissolve the system, replacing air in a container with nitrogen in the process to prevent oxidation, and cooling to 20-40 ℃.

② adding organic solvent and stirring uniformly.

③ adding a proper amount of water for injection, and rapidly stirring the mixture while adding the water until the system is clear.

Fourthly, filtering the mixture through a filter membrane with the diameter of 0.22 mu m to remove bacteria.

Fifthly, subpackaging, charging nitrogen, plugging and sealing to obtain the product.

(3) In another preferred embodiment of the present invention, in the step (r), the phospholipid, cholesterol and active ingredients in the system are dispersed by high-speed stirring with high-speed shearing, so as to increase the specific surface area and accelerate the dissolution, and the dissolution time is less than 1 hour, more preferably less than 30 minutes. Meanwhile, nitrogen is adopted to replace air in the system, so that the oxidation of phospholipid and oil is reduced as much as possible.

(4) In another preferred embodiment of the present invention, in the step (i), the dissolved system should be cooled, preferably to 20 to 40 ℃, more preferably to 20 to 30 ℃, and then the organic solvent is added according to the step (ii), so that the ratio change of the organic solvent in the system due to volatilization caused by heating can be effectively avoided.

(5) In another preferable scheme of the invention, the injection water is added while stirring rapidly in the third step, so that the precipitation of the active pharmaceutical ingredient or cholesterol caused by phase transition of the system due to high local water content is avoided.

(6) More preferably, the scheme is as follows: the active drug ropivacaine free base is 2-8 parts, preferably 3-6 parts, based on 100 parts by weight.

40-60 parts of phospholipid, preferably 45-55 parts of phospholipid based on 100 parts by weight.

15-35 parts of ethanol, preferably 10-25 parts, based on 100 parts by weight.

Soybean oil 5-20 parts, preferably 8-15 parts, per 100 parts by weight.

Cholesterol is 2-8 parts, preferably 2-5 parts, based on 100 parts by weight.

0.01-1 part of antioxidant, preferably 0.02-0.5 part, based on 100 parts by weight.

1 to 10 parts, preferably 1.5 to 8 parts, more preferably 4 to 8 parts of water for injection based on 100 parts by weight.

The above preferred embodiments may be used singly or in combination of any two or more

Another aspect of the present invention is to provide an application of a combination of water for injection and cholesterol in adjusting the drug release behavior of the lipid drug release system product with a long-acting sustained-release effect, wherein the effects include that cholesterol increases the phase transition temperature of the system, and simultaneously enhances the integrity and stability of phospholipid bilayers, thereby improving the stability of a drug reservoir; the water is used for preparing a reservoir in a phase transition critical state, when a small amount of external aqueous liquid contacts the system, the phase transition of the system is promoted immediately, so that the drug reservoir is formed quickly, and the drug release speed is further controlled by the effective combination of the synergistic effect of the two.

The more preferable scheme of the invention is that the raw materials of the lipid drug release system product with long-acting slow release effect comprise active ingredients, phospholipid, drug release behavior regulating substances, dispersion medium and antioxidant. No further additional components are contained, apart from unavoidable impurity constituents or customary substances for osmotic pressure regulation which have to be added. The lipid drug delivery system generally adopts the administration mode of injection at the periphery of a wound or is prepared into a pharmaceutically acceptable spray to be sprayed on the surface of the wound or the accessible mucosal tissue.

The lipid drug carrier system of the invention is light yellow to yellow clear transparent liquid, after being administrated, the lipid drug carrier interacts with tissue liquid and then undergoes phase transition, the system undergoes self-emulsification and self-assembly to form a drug reservoir, and active ingredients are slowly released.

The advantages of the present invention compared to prior art solutions can be summarized as follows:

(1) the invention has good safety in clinical safety, and the medicinal oil with lower toxicity is used as the solvent, so that the dosage of the organic solvent can be effectively reduced, thereby reducing the local irritation and ensuring the value and the use safety of the preparation in clinical application. Meanwhile, the technical difficulty is overcome, the stability of the preparation is ensured, and the long-acting release effect is achieved.

(2) The phospholipid is a key component for realizing slow release, can effectively improve the content of the local anesthetic in the composition, and can be combined with water in vitro or tissue fluid in vivo to generate phase transition due to the self-assembly characteristic, so that a drug storage with a space structure is formed, and the slow release effect is realized; in addition, the phospholipid is also an important component of cell membranes, and has good safety and biocompatibility. The invention finds that the phase transition temperature is a core element for controlling the drug release of a lipid drug release system, is a core element for constructing a drug reservoir, and is realized by a scheme that a substrate is composed of natural phospholipid (and/or synthetic phospholipid) and cholesterol, more preferably, the substrate is composed of natural phospholipid, synthetic phospholipid and cholesterol, and the phase transition temperature is increased.

(3) The medicinal oil and the organic solvent are contained in the invention, and the medicinal oil has a slower diffusion speed than the organic solvent, so that the condition that the front-stage release is too fast due to too fast diffusion of the medicine caused by the independent use of the organic solvent in a system is avoided, and the medicinal oil and the organic solvent are organically combined to adjust the release speed of the medicine. Meanwhile, the medicinal oil can also increase the solubility of the medicine and cholesterol in the system, thereby improving the medicine-loading rate and maintaining the stability of the system.

(4) Compared with the prior art, the invention reduces the dosage of the organic solvent to the maximum on the premise of ensuring the content of the active ingredients, the clarity of the system and the influence of the viscosity during injection on the injection, so as to reduce the burst release of the medicine caused by the solvent diffusion effect, thereby achieving the effect of stable and slow release and simultaneously reducing the local irritation of the organic solvent during injection to the maximum.

(5) Compared with the prior art, the cholesterol is added into the system, when the system is combined with interstitial fluid to generate phase transition, the cholesterol and phospholipid form a tighter bilayer structure, and the stability of the system after phase transition is more effectively maintained, so that the drug release speed is effectively controlled, and the long-acting effect is maintained.

(6) The water adopted by the invention is water for injection, wherein the content of monovalent and divalent ions is low, and the oxidation of unsaturated fatty acid in phospholipid and medicinal oil caused by ions can be effectively avoided, thereby improving the safety. At the same time, water is also a key element for regulating drug release

(7) The antioxidant and the metal ion complexing agent used in the invention are common pharmaceutic adjuvants, so that the safety is good, and meanwhile, the oxidation of active ingredients and adjuvants, namely phospholipid and medicinal oil, can be effectively reduced by adding the antioxidant, thereby reducing degradation and ensuring the safety of products.

(8) Finally, the design process of the invention is always developed around the ropivacaine free alkali, the components and the dosage are reasonably designed, and the effect of excessively depending on single components and dosage is avoided, so that the ropivacaine lipid drug release system prepared by the invention has the advantages that the drug effect duration time can reach more than 48-72h through testing, the drug release process is stable, the repeated drug administration times are reduced, the compliance of patients in the clinical use process is improved, and the treatment operation is effectively reduced.

Drawings

FIG. 1 is a pre-self-assembly configuration of the lipid delivery system of example 9;

FIG. 2 is the self-assembled configuration of the lipid delivery system of example 9;

FIG. 3 shows the results of the hot plate test of the control group and the test group in example 11;

FIG. 4 is the results of the percentage (%) of the maximal analgesic effect of the test substance given by a single peri-sciatic injection in rats in example 11;

FIG. 5 shows the results of the rate (%) of increase in pain threshold in rats after a single peri-sciatic injection of the test substance in example 11.

Detailed Description

In order to further illustrate the technical solution of the present invention, the following examples are given for the purpose of illustration and not as a limitation of the present invention.

Example 1

Samples containing ropivacaine free base were prepared according to the recipe in table 1, left at room temperature for 24h, and observed for properties.

TABLE 1 prescription composition and State

The water content can change the state of the system, and the system changes phase with the increase of the added amount of water and gradually changes from clear to turbid. In the system, when the proportion of water in the ethanol reaches 35%, the system is not stable any more.

Tests prove that the defects of the mixed solvent (relation between the solvent and the drug loading) are not simple to combine, and the mixed solvent is mixed according to a certain proportion to form a required system. It is illustrated as a complex system. Water is one of the core components in the present invention that affects the release or formation of the drug depot. The more water content, the faster the reservoir is formed, but the more the active pharmaceutical ingredient in the composition is precipitated; the water content is low, the speed of forming a drug storage is slow, the drug release is mainly expressed as the solvent diffusion behavior, and the release speed is high. Therefore, to the extent that there is a reasonable range, the moisture and organic solvent ratios are described in tables 1 and 3 of the examples in the patent. Table 6 also describes the effect of moisture content on release.

Example 2

Samples containing ropivacaine free base were prepared according to the recipe in table 2, left at room temperature for 24h, and observed for properties.

TABLE 2 prescription composition and State

In the above proportions, clear and transparent systems can be obtained, and the liquid obtained from various compositions has good fluidity.

Example 3

Samples containing ropivacaine free base were prepared as prescribed in table 3, left at room temperature and observed for 0h and 24 h.

TABLE 3 prescription composition and State

The solubility of ropivacaine in propylene glycol is much lower than that of ethanol, so when ethanol is reduced, ropivacaine is precipitated.

Example 4

Samples containing ropivacaine free base were prepared as prescribed in table 4, left at room temperature for 24h and then subjected to in vitro release studies.

Release degree study method: about 1.0g of the sample was placed in a dialysis bag (3000Da), and the dialysis bag was placed in 250mL of phosphate buffer (pH7.4), incubated at 37 ℃ with a stirring speed of 100 rpm, and sampled at 0.5, 1, 2, 4, 6, 8, 24, 32, 48, and 72 hours, respectively, for measurement.

Table 4 effect of the amount of medicinal oil on drug release data are as follows:

soybean oil is one of the solvents used as medicinal oil, has a diffusion effect, and the more the dosage of soybean oil is, the faster the drug is released.

Example 5

Samples containing ropivacaine free base were prepared as prescribed in table 5, left at room temperature for 24h and then subjected to in vitro release studies.

Release degree study method: about 1.0g of the sample was placed in a dialysis bag (3000Da), and the dialysis bag was placed in 250mL of phosphate buffer (pH7.4), incubated at 37 ℃ with a stirring speed of 100 rpm, and sampled at 0.5, 1, 2, 4, 6, 8, 10, 24, 32, 48, and 72 hours, respectively, for measurement.

Table 5 influence of organic solvents on drug release data are as follows:

the content of the organic solvent in the system directly influences the release of the medicine, and the higher the content of the organic solvent is, the faster the release is.

Example 6

Samples containing ropivacaine free base were prepared as prescribed in table 6, left at room temperature for 24h and then subjected to in vitro release studies.

Release degree study method: about 1.0g of the sample was placed in a dialysis bag (3000Da), and the dialysis bag was placed in 250mL of phosphate buffer (pH7.4), incubated at 37 ℃ with a stirring speed of 100 rpm, and sampled at 0.5, 1, 2, 4, 6, 8, 10, 24, 32, 48, and 72 hours, respectively, for measurement.

Table 6 the data on the effect of water content on drug release are as follows:

the basic reason is that the addition of water can effectively change the structure of the original system, so that the anhydrous system is quickly changed, the drug reservoir is quickly constructed, and the drug release is changed from simple solvent diffusion into dual functions of solvent diffusion and reservoir slow release, thereby changing the release behavior of the drug.

Example 7

Samples containing ropivacaine free base were prepared as prescribed in table 7, left at room temperature for 24h and then subjected to in vitro release studies.

Release degree study method: about 1.0g of the sample was placed in a dialysis bag (3000Da), and the dialysis bag was placed in 250mL of phosphate buffer (pH7.4), incubated at 37 ℃ with a stirring speed of 100 rpm, and sampled at 0.5, 1, 2, 4, 6, 8, 24, 32, 48, and 72 hours, respectively, for measurement.

Table 7 influence of cholesterol on the drug release of the system the data are as follows:

the cholesterol can effectively change the phase transition temperature of the system and the integrity of the bilayer after the system forms a reservoir, thereby effectively reducing the burst release of the drug caused by low phase transition temperature of the system.

Example 8

Samples containing ropivacaine free base were prepared as prescribed in table 8, left at room temperature for 24h and then subjected to in vitro release studies.

Release degree study method: about 1.0g of the sample was placed in a dialysis bag (3000Da), and the dialysis bag was placed in 250mL of phosphate buffer (pH7.4), incubated at 37 ℃ with a stirring speed of 100 rpm, and sampled at 0.5, 1, 2, 4, 6, 8, 24, 32, 48, and 72 hours, respectively, for measurement.

Table 8 the data on the effect of synthetic phospholipids on the drug release of the system are as follows:

example 9

Samples containing ropivacaine free base were prepared as shown in Table 9, about 1g of the sample was taken and placed in a dialysis bag near to dryness, and the dialysis bag was placed in a phosphate buffer (pH7.4) at 37 ℃ for observation of changes in properties at 0.5, 1, 2, 4 and 8 hours, respectively.

TABLE 9 change of samples during dialysis

The sample prepared by the method can generate phase transition after meeting a small amount of water, so that the morphological change is generated, and the yellow clear transparent liquid is gradually changed into milk white, which is shown in the figure 1 and the figure 2.

Example 10

Based on the above studies, preferred embodiments of the present invention include, but are not limited to, the following:

(1) 1.5g of ropivacaine, 20.0g of soybean phospholipid, 10.0g of ethanol, 5.0g of soybean oil, 1.0g of cholesterol, 0.05g of vitamin E and 3.0g of water for injection;

(2) 1.8g of ropivacaine, 20.0g of soybean phospholipid, 10.0g of ethanol, 5.0g of soybean oil, 1.5g of cholesterol, 0.05g of vitamin E and 2.0g of water for injection;

(3) 2.0g of ropivacaine, 20.0g of soybean phospholipid, 10.0g of ethanol, 7.5g of soybean oil, 2.0g of cholesterol, 0.075g of vitamin and 2.0g of water for injection;

(4) 1.3g of ropivacaine, 18.0g of soybean phospholipid, 2.0g of hydrogenated soybean phospholipid, 10.0g of ethanol, 10.0g of soybean oil, 2.0g of cholesterol, 0.075g of vitamin E and 2.0g of water for injection;

(5) 1.5g of ropivacaine, 19.5g of soybean phospholipid, 0.5g of distearoyl phosphatidylcholine, 15.0g of ethanol, 7.5g of soybean oil, 1.0g of cholesterol, 0.075g of vitamin E and 3.0g of water for injection;

(6) 1.5g of ropivacaine, 19.5g of egg yolk lecithin, 0.5g of distearoyl phosphatidylcholine, 15g of ethanol, 7.5g of soybean oil, 1.0g of cholesterol, 0.075g of vitamin E and 1.5g of water for injection;

(7) 1.4g of ropivacaine, 20.0g of soybean phospholipid, 9.0g of ethanol, 1.0g of propylene glycol, 5.0g of soybean oil, 1.0g of cholesterol, 0.075g of vitamin E and 2.0g of water for injection;

(8) 1.4g of ropivacaine, 20.0g of soybean phospholipid, 9.0g of ethanol, 1.0g of propylene glycol, 5.0g of soybean oil, 1.0g of cholesterol, 0.075g of vitamin E and 2.0g of water for injection;

(9) 1.5g of ropivacaine, 20.0g of soybean phospholipid, 10.0g of ethanol, 5.0g of soybean oil, 2.0g of cholesterol, 0.05g of vitamin E and 2.5g of water for injection;

(10) 2.5g of ropivacaine, 20.0g of soybean phospholipid, 10.0g of ethanol, 20.0g of soybean oil, 4.0g of cholesterol, 0.05g of vitamin E and 1.5g of water for injection;

to further verify that the composition disclosed in the present invention has sustained release effect, samples of ropivacaine lipid sustained release formulations were prepared according to the above-described preferred embodiment, and comparative studies were performed on the in vitro release rate of ropivacaine hydrochloride injection, while pharmacokinetic studies were performed using SD rats, and the results are shown in tables 10, 11, 12 and 13.

Release degree study method: about 1.0g of the sample was placed in a dialysis bag (3000Da), and the dialysis bag was placed in 250mL of phosphate buffer (pH7.4), incubated at 37 ℃ with a stirring speed of 100 rpm, and sampled at 0.5, 1, 2, 4, 6, 8, 24, 32, 48, 72, 96, and 120 hours, respectively, for measurement.

TABLE 10 in vitro Release Rate Studies of ropivacaine injection and samples prepared according to the protocol of the present invention

The pharmacokinetic study method comprises the following steps: taking 6 SD rats, randomly dividing into 2 groups, namely a test group and a control group, wherein the test group is administered with a ropivacaine lipid sustained-release preparation, the control group is a ropivacaine hydrochloride injection, the administration dose is 40mg/kg, the administration mode is subcutaneous injection, and the test group and the control group are respectively subcutaneously injected after administration: blood concentration is measured by taking blood from vein after 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, 48h and 72h after administration. The test group results are as follows:

TABLE 11 rat pharmacokinetic study test group-40 mg/kg

TABLE 12 rat pharmacokinetic study control group 40 mg/kg-rat

Note: BLQ: is lower than the detection limit

TABLE 13 pharmacokinetic parameters of control and test groups

According to the pharmacokinetic research result, T of the test group_1/2Approximately 13.8 times that of the control group, Tmax delayed approximately 1 times relative to the control group, and AUC (0- ∞) data indicate that the test group is 1.6 times that of the control groupThis also demonstrates that the samples used in the test group had a significant sustained release effect. From the result of monitoring the blood concentration, the lipid drug delivery system disclosed by the invention has a sustained release effect, the action time can reach 48-72h, and the result of a control group shows that the action time is only 8h, which is consistent with the action time of 2-6 h in the specification of ropivacaine hydrochloride injection, and further proves that the composition disclosed by the invention has a long-acting sustained release effect.

Example 11

In order to further verify that the lipid release system disclosed in the present invention has sustained release effect, a sample of ropivacaine lipid sustained release preparation was prepared according to the protocol of example 10 and subjected to analgesic test using hot plate method.

The experimental method comprises the following steps:

the SD rat is adapted to the animal room for 2-7 days, the environment of the animal room is kept at 23 +/-2 ℃, the humidity is 40-70%, and the light and the shade are alternated for 12 hours. Animals were housed 5 per cage, 4 per cage after group, and bedding was changed twice per week (corncob bedding, dam Chuan commerce Co., Ltd., Suzhou).

After the acclimation period, all rats were subjected to a pain threshold sensitivity screen the day before dosing. The rats were fixed, the right hind paw of the rat was placed on a 56 ℃ hot plate, the time to withdrawal was observed and recorded, and the mean value of the three measurements was taken and recorded as the basal pain threshold. If the result of the measurement of the time for shortening the feet exceeds 5s, the basal pain threshold of the rat is not satisfactory and should be removed. Then, 24 animals are selected according to the weight of the rat and the basic pain threshold value and are averagely divided into 3 groups, and each group comprises 8 animals: a control group, a ropivacaine lipid sustained-release preparation 20mg/kg group and a ropivacaine lipid sustained-release preparation 40mg/kg group. After the experimental animals were grouped, the next day, the right sciatic nerve was injected with the solvent control or test substance. The specific method comprises the following steps: rats are anesthetized with isoflurane and then in prone position, after local hair is removed, the skin is cut after local disinfection with iodine tincture and 75% alcohol in sequence, muscles are separated in a blunt manner, sciatic nerves are exposed and dissociated, and finally wounds are sutured and disinfected in sequence. Measuring pain threshold of injection side feet at 4h, 6h, 8h, 24h, 32h, 48h and 72h after administration, if the pain threshold exceeds 10s and the foot shrinkage phenomenon still does not occur during hot plate test after administration, the injection side feet should be separated from the hot plate to avoid burn. The percentage of maximal analgesic effect and the rate of increase in threshold pain were calculated and observed for their time-effect relationship. The maximum percentage of analgesic effect and the rate of increase in threshold pain are calculated as follows:

percent maximal analgesic effect (MPE%) (post-dose pain threshold-basal pain threshold)/(10-basal pain threshold) × 100%

The pain threshold increase rate (average pain threshold after administration-average pain threshold before administration)/average pain threshold before administration × 100%

The dosing schedule is shown in table 14, and the hotplate test data is shown in table 15, figure 4, figure 5.

TABLE 14 animal dosing regimens

And (3) test results:

compared with a control group, the hot plate reaction time (p is less than 0.01 or 0.001) of the rat 4h to 24h after the administration can be obviously prolonged by 20mg/kg of the ropivacaine lipid sustained-release preparation which is injected around the sciatic nerve once, a certain prolonging effect is still realized on the hot plate reaction time in 32h to 48h, and the hot plate reaction time is gradually reduced along with the time after the administration. The ropivacaine lipid sustained-release preparation is administered by single peripheral injection of sciatic nerve at a dose of 40mg/kg, so that the hot plate reaction time (p is less than 0.01 or 0.001) of the rat 4-48 h after administration can be remarkably prolonged, the hot plate reaction time is still prolonged to a certain extent within 48-72h, and the hot plate reaction time is gradually reduced along with the time duration after administration. The effect of the test substance ropivacaine lipid sustained release formulation on the prolongation of the rat hot plate response time was dose-wise positively correlated (table 15, figure 3).

Table 15 effect of single periischial injection of test substances on hot plate response time in rats (n-8)

Compared with a control group, the ropivacaine lipid sustained-release preparation is administered by 20mg/kg and 40mg/kg through single periischiadic nerve injection, so that the hot plate reaction time of the rats after administration for 4h-48h can be prolonged remarkably, the hot plate reaction time is prolonged to a certain extent after 48h, but no statistical significant difference exists, and the hot plate reaction time is gradually reduced along with the time after administration.

The maximum analgesic effect percentage and pain threshold increasing rate of the ropivacaine lipid sustained-release preparation given by single peripheral injection of sciatic nerve are highest 4h after administration and then gradually reduced, and the analgesic effect of the ropivacaine lipid sustained-release preparation on rat hot plate induced pain is in positive correlation in dosage.

The maximum percentage of analgesic effect and the pain threshold increase rate of the ropivacaine lipid sustained-release preparation given by 20mg/kg and 40mg/kg in single peri-sciatic nerve injection were highest 4h after administration and then gradually decreased, and the test substance ropivacaine lipid sustained-release preparation showed a positive dose-related analgesic effect on rat hot-plate-induced pain (table 16, table 17, fig. 4, fig. 5).

TABLE 16 Effect of single peri-sciatic injection of test substances on percent (%) maximal analgesic effect in rats (n-8)

TABLE 17 Effect of single peri-sciatic nerve injection administration of test substances on the rate (%) of threshold increase in pain in rats (n ═ 8)

Conclusion of the hot plate test:

the hot plate reaction time of rats can be obviously prolonged by injecting 20mg/kg and 40mg/kg ropivacaine lipid sustained release preparations around the sciatic nerve once, and the analgesic effect is obviously related to the sustained release dosage of the ropivacaine lipid.

Claims (14)

1. A lipid delivery system product having a long-lasting sustained release effect, the lipid delivery system product comprising: active ingredients, phospholipids, drug release behaviour modifying substances, dispersion media, and antioxidants:

wherein the active ingredient is ropivacaine free base;

wherein the phospholipid is natural phospholipid, synthetic phospholipid or a combination thereof;

wherein, the substance for regulating the drug release behavior of the drug release system is a composition of water for injection and cholesterol;

wherein the dispersion medium is a composition of medicinal oil and water-soluble organic solvent;

wherein the antioxidant is a fat-soluble antioxidant;

the ratio of the phospholipid to the organic solvent is 1:1-2:1,

the ratio of the medicinal oil to the phospholipid is 4:1-1:4,

the proportion of the water for injection in the organic solvent is 10-30% w/w,

the ratio of cholesterol to phospholipid is 1:5-1:20w/w,

the proportion of the antioxidant is 0.1 to 1 percent of the sum of the phospholipid and the oil,

the content of active ingredients is 2-8%.

2. The product of claim 1, wherein the phospholipid is a combination of natural phospholipid and synthetic phospholipid, wherein the synthetic phospholipid accounts for 1-5% of the phospholipid.

3. The product of claim 1, wherein the ropivacaine free base content is 2-8%.

4. A product according to any of claims 1-3, wherein said phospholipid is selected from natural phospholipids and/or synthetic phospholipids; wherein the natural phospholipid is selected from soybean phospholipid and egg yolk lecithin; the synthetic phospholipid is selected from natural phospholipid, hydrogenated soybean phospholipid, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and distearoylphosphatidylcholine, and has phase transition temperature higher than 0 deg.C.

5. A lipid delivery system product with long-acting sustained release according to any of claims 1 to 3, wherein the content of phosphatidylcholine in the phospholipid is greater than 70%.

6. A lipid delivery system product with long-acting sustained release according to any of claims 1 to 3, wherein the dispersion medium comprises a medicinal oil and a water-miscible organic solvent, and the ratio of phospholipid to organic solvent is 1:1-2:1, more preferably 1.5:1-2: 1; the ratio of medicinal oil to phospholipid is 4:1-1:4, more preferably 1:1-1: 4.

7. A lipid delivery system product with long-acting sustained release according to any of claims 1 to 3, wherein the dispersion medium comprises a pharmaceutically acceptable oil and a water-miscible organic solvent, which is effective in dissolving the active ingredient, phospholipids and cholesterol.

8. The product of claim 7, wherein the oil is one or more of soybean oil, medium chain triglycerides, olive oil, tea seed oil, and fish oil.

9. The product of claim 7, wherein the water-miscible organic solvent is one or more of ethanol, propylene glycol, and glycerol; preferably, ethanol is used as the dispersion medium in combination with propylene glycol or glycerol, preferably in a ratio of from 1:1 to 10:0, more preferably from 7:3 to 10: 0.

10. A lipid delivery system product with long-acting sustained release according to any of claims 1 to 3, wherein the water is water for injection, and the ratio of water to organic solvent is 10-30%, preferably 20-30%.

11. A lipid delivery system product with long-acting sustained release according to any of claims 1 to 3, wherein the ratio of cholesterol to phospholipid is 1:5 to 1: 20.

12. A method for preparing a product of a lipid delivery system with a long-acting sustained release effect according to any one of claims 1 to 11, comprising the steps of:

weighing active ingredients, phospholipid, cholesterol, medicinal oil and antioxidant according to the prescription, stirring, heating to 40-70 deg.C to completely dissolve the system, under nitrogen protection, cooling, adding organic solvent, stirring, adding appropriate amount of water for injection, rapidly stirring to clarify the system, filtering with 0.22 μm filter membrane for sterilization, packaging, charging nitrogen gas, adding plug, and sealing.

13. The method for preparing a product of a lipid delivery system with a long-lasting and sustained release effect according to claim 12, comprising the following steps

Weighing active ingredients, phospholipid, cholesterol, medicinal oil and antioxidant according to a formula, shearing at a high speed or stirring at a high speed, heating to 40-70 ℃, preferably to 40-50 ℃, completely dissolving the system, replacing air in a container with nitrogen in the process, preventing oxidation, and cooling to 20-40 ℃;

adding organic solvent and stirring uniformly;

thirdly, adding a proper amount of water for injection while rapidly stirring until the system is clear;

fourthly, filtering the mixture through a filter membrane with the diameter of 0.22 mu m to remove bacteria;

fifthly, subpackaging, charging nitrogen, plugging and sealing to obtain the product.

14. A lipid delivery system with long-acting sustained release according to any of claims 1 to 3, wherein the delivery is by injection around the wound or as a pharmaceutically acceptable spray applied to the wound surface or to the accessible mucosal tissue.

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