Lipid drug release system with long-acting slow release function and preparation method thereofTechnical Field
The invention belongs to the field of pharmacy, and mainly relates to a lipid release system with a long-acting slow release effect and a preparation method thereof.
Background
Pain is an unpleasant sensation and emotion caused by an existing or impending tissue injury, a warning signal that is relatively common in the clinic that may be accompanied by anxiety in the patient, and an emotional experience in the patient. The term "pain" means, for the patient, pain and/or a possible suffering from a disease, which is a clinical symptom for the doctor; the concept of pain has been used until now, as has been described in the publication in 1979, since the start of its work written in the merck of 1964 on pain and its widespread use. Melzack et al, in the journal of science, set forth new theories of pain that motivate an understanding of the mechanisms of pain and management of pain. In 2016, by Williams et al, based on previous review of the definition of pain, in order to better grasp the pain essence as understood at present, the following new definition was proposed: pain is a painful experience that is associated with the patient's sensory, emotional, cognitive, and social factors or with the underlying tissue damage itself.
Diseases with "pain" as a main symptom are called "painful diseases" in clinic, and are abbreviated as "painful diseases". Depending on the duration and nature of pain, acute pain and chronic pain can be classified. Acute and chronic pain is a common clinical symptom of an orthopedics patient, and slight pain can cause mental pain of the patient and reduce the life quality of the patient; severe pain can cause dysfunction and hypoimmunity of various systems of the human body to induce various complications, even painful disability or painful shock, and threaten the life of the patient. Therefore, scientific and effective clinical diagnosis and treatment means are important for relieving clinical symptoms of patients with acute and chronic pain and improving the life quality of the patients.
Background problem one: insufficiency of anesthetic drugs and solution of the invention
Local anesthetics (abbreviated as "local anesthetics") are a class of drugs that can cause the pain of local tissues to disappear under the condition of consciousness. Wherein the amide local anesthetic comprises lidocaine, ropivacaine, bupivacaine, etc. Although they have similar chemical structures of the parent nucleus, the substituent groups have great influence on the spatial structure, physicochemical properties, pharmacological effects and drug toxicity of the drug, especially the formation of the preparation. For example, ropivacaine is structurally similar to bupivacaine, but the former is used for surgical anesthesia, labor and post-operative analgesia, and the latter is used for local infiltration anesthesia, peripheral nerve block and intraspinal block; for another example, the prior literature is well documented that ropivacaine and bupivacaine also exhibit a dramatic difference in toxicity. Therefore, the selection of reasonable analgesic elements for research and development has been a constant dilemma in the art.
Through a great deal of research, the inventor discovers that the amide local anesthetics have great difference in the molding of new dosage forms. For example, although reports are made on the literature about lipid delivery systems for amide local anesthetics, no ropivacaine sustained-release preparation prepared by the lipid delivery systems has been successfully marketed at home and abroad at present. The reason for this is that lidocaine, ropivacaine, bupivacaine and the like are long-acting amide local-anesthetic drugs, and free alkali is insoluble in water, so that the products on the market at present are hydrochloride or mesylate, and the problem of solubility of the products can be effectively solved, so that the injection is prepared and is convenient for clinical use. This is the most common and basic method of use of such drugs. However, there are obvious problems in that the salt thereof has good solubility, so that it can rapidly enter into blood circulation after local injection, has a short half-life period, and has strong irritation due to a low pH. In order to achieve a long-acting effect, a continuous administration method is generally adopted clinically, and great inconvenience is brought to patients with pain. Therefore, the primary problem faced by pharmaceutical researchers for treating pain is to find a solution to the problem of long-acting analgesia among numerous drugs for treating pain, possible structural modifications, or new dosage form treatment schemes. The present inventors have thought to select ropivacaine and to select the free base of ropivacaine and to solve this by means of a new dosage form. Wherein, the free alkali has indissolvable property, is different from the salt form which is rapidly transported in vivo and has short half-life, and the new dosage form is matched with the chemical structure and physicochemical property of the drug to realize continuous drug release, achieve long-acting effect and reduce side effect to the greatest extent.
Ropivacaine is a novel long-acting amide local anesthetic drug that was first marketed in the netherlands since 1996 and was introduced for clinical anesthesia in 1999 in our country. The medicines on the market are prepared into clear and transparent sterilized aqueous solution by hydrochloride or mesylate for anesthesia in surgical operation or acute pain control, and the administration modes are lumbar epidural administration, thoracic epidural administration and subarachnoid administration. The onset time is about 10-20 minutes, t is 1.8 hours, and the anesthesia or analgesia lasts for 2-6 hours. Clinically, a large number of researches prove that the ropivacaine has definite curative effect on obstetrical anesthesia and painless delivery, has light motor nerve block, does not influence the labor and the neonate, and is a commonly used and ideal analgesic at present. In order to achieve the analgesic effect, a continuous intravenous drip mode is generally adopted in clinic to achieve the analgesic purpose, no long-acting ropivacaine preparation is marketed at present, and other means are needed for postpartum analgesia.
The bupivacaine is also an amide local anesthetic drug, and the hydrochloride, 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 strong anesthesia efficacy, obvious separation of motor and sensory block and the like. The bupivacaine has the advantages of quick response, long action duration and the like because of the main effect, but the bupivacaine has certain drug toxicity to the central nervous system and the heart, can cause anesthesia adverse reactions such as hypotension, respiratory depression, bradycardia and the like, but the common injection (with the concentration of 0.5%) only has the analgesic effect of 5-7 h, and the postoperative pain usually lasts for 48-72 h, and is most difficult to control in the time period, so that the bupivacaine liposome Exparel adopting Depofoam is marketed abroad, has a slow release function, and has long-acting analgesic time as long as 72h. However, bupivacaine lipid formulations have been found to be a clinical obstacle for various reasons, such as the drug itself and the formulation components.
The inventors believe that pharmaceutical formulations, particularly novel formulations, are sufficiently devoid of the properties of the drug itself to design reasonable dosage forms and formulations. For example, ropivacaine and bupivacaine appear to be of a chemical structure that is not large enough to result in a large difference in physicochemical properties, having a pka of 8.1 and a LogP of 2.9; the bupivacaine has a pKa of 14.85 and a LogP of 3.9, which makes the acidity of the ropivacaine stronger than that of the bupivacaine and the fat solubility of the ropivacaine is Yu Bubi, and the ropivacaine has small fat solubility and the time for reaching the thick motor nerves is prolonged, so that the unique action characteristic of separating the motor from sensory retardation of the drug is formed, and the characteristic makes the ropivacaine more favorable for the recovery of early motor functions after operation in clinic. It is easy to see that the ropivacaine and bupivacaine are applied in a complex preparation system on the premise of great difference of pharmaceutical properties, and the necessary effects are quite different. The reasonable method is to design a specific ropivacaine free base preparation scheme, so as to improve the clinical application value of the medicine.
Background problem two: deficiency of the Phospholipids formulations and solutions of the present invention
Phospholipid-based drug delivery systems include liposomes, fat emulsions, microemulsions, etc., which are very widely used, but the type of formulation is particularly complex and varies from drug to drug and from system to system. Wherein, the used auxiliary material phospholipid has self-assembly property, so that a drug carrying system can be formed under proper conditions. The self-emulsifying drug delivery system is a liquid formulation consisting of an oil phase, a nonionic surfactant, and a cosurfactant. However, it is not easy to solve phospholipids as carriers to form specific new formulations and is not a simple additive effect. Moreover, the new formulation of the medicine is different from the traditional formulation, and the formulation components and the formulation system are very complex.
The inventors have surprisingly found that by virtue of its essential feature, a phase transition can occur under suitable conditions, thereby forming a drug depot, slowly releasing the drug, and thus achieving a long-acting sustained release effect. Phospholipids are basic substances forming cell membranes and also are main components forming phospholipid reservoirs, have self-assembly property in water, and can spontaneously form liposome, micelle and gel with space structures, so that the liposome, micelle and gel become drug carriers; in addition, phospholipids have a surface active effect and can effectively solubilize insoluble molecules. Meanwhile, because 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 techniques and dosage forms which adopt phospholipids as auxiliary materials are marketed, such as fat emulsion and liposome. Therefore, the preparation prepared by using the phospholipid as the auxiliary material has excellent safety and biocompatibility, and provides basic guarantee for medication safety. In the present invention, the phospholipid functions to form a drug depot.
Cholesterol and phospholipids are the basic substances that together constitute membranes and liposomes. Cholesterol belongs to an ampholytic substance, and can regulate the fluidity and permeability of a phospholipid bilayer. The generation of free radicals in the phospholipid bilayer can accelerate oxidation of phospholipid, and the addition of cholesterol in the bilayer can solidify the membrane, so that the generation of free radicals is reduced, and the oxidation level is reduced. The phospholipid can be self-assembled 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 bilayer structure, so that the stability of the bilayer is greatly improved, the rapid release of the medicament is avoided to generate a burst effect, and the toxic reaction caused by the excessively high concentration of the instantaneous medicament is avoided. In other words, in the present invention, phospholipids and cholesterol are utilized to both build up a drug depot and prevent drug burst.
Further, when the ambient temperature is higher than the phase transition temperature, the stability of the bilayer of the phospholipid self-assembled system is changed, so that the drug is released. The phase transition temperature of the natural phospholipid is about-20 ℃, for example, a drug depot is constructed by only one natural phospholipid, the phase transition temperature of a system formed after self-assembly is consistent, and the phase transition temperature is lower, so that the stability of the depot is poor after injection, and the burst effect is easy to cause. And a system formed by mixing phospholipids with different phase transition temperatures is selected, and the stability of a formed reservoir is increased 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 chains in phospholipid molecules, reducing the fluidity of the membrane; below the phase transition temperature, the membrane lipids are in a crystalline arrangement, which in turn induces the formation of a twisted conformation of fatty acyl chains, preventing the appearance of the crystalline state. The hydroxyl in cholesterol molecule can also form a compound with carbonyl in phospholipid molecule by hydrogen bond, and the free movement of fatty acyl chain is reduced, which causes the compression of membrane, the area reduction, the tight combination and the mobility reduction, thus reducing the leakage rate of medicine, and avoiding the burst effect. In short, cholesterol itself plays two roles in bi-directional regulation.
The dispersion medium is one of the indispensable components of the injection, and water for injection, medicinal oil or water-miscible organic solvent is often used as the dispersion medium, so as to solve the solubility problem of the active ingredient. In the present invention, the choice of dispersion medium is unique.
Medicinal oil is often used as a solvent, has good safety, soybean oil has been used as medicinal oil for injection for about 60 years at the earliest, and LD50 for intravenous injection in rats is 22.1g/kg. Because of its good safety, it is used in a number of marketed drugs.
Medium Chain Triglycerides (MCT) are derived from coconut oil and purified to form medium chain fatty acids, and therefore have good hydrophilicity and oxidative stability. As the solvent, a solvent in which a fat-soluble component such as phospholipid, ropivacaine and cholesterol are dissolved in a certain amount can be used. The high safety of the compound is used as a pharmaceutical raw material and auxiliary material for a plurality of injection medicines, such as medium/long chain fat emulsion injection, mixed oil injection (SMOF) and the like. Acute toxicity tests using rabbits indicate that oral LD50 is greater than 5g/kg and subcutaneous LD50 is greater than 2g/kg.
Organic solvents which are mutually soluble with water, such as ethanol, propylene glycol, glycerol, tertiary butanol and the like, are also used as common solvents for changing the system properties, the stability and the related parameters of the injection.
Propylene glycol is mainly used as a preservative, a bactericide, a wetting agent, a plasticizer, a solvent, a stabilizer and a latent solvent in the pharmaceutical field. Can be administered by intramuscular injection, intravenous injection, nasal administration, oral administration, ocular administration, aural 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 ingestion of small to moderate amounts of ethanol can lead to symptoms of alcohol intoxication. Intake of high concentration ethanol can lead to reduced spinal cord reaction, somnolence, amnesia, reduced body temperature, hypoglycemia, coma, numbness, respiratory depression, cardiovascular failure, and human blood ethanol lethal concentration of 400-500 mg/100mL. 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 for injection, they still have a certain irritation to the injection site, so that the amount of the organic solvent needs to be reduced as much as possible, thereby reducing adverse reactions during injection.
The dispersion medium animal toxicity data are as follows:
solvent (organic solvent) selection of dispersion medium
| LD50 (subcutaneous injection) | LD50 (intraperitoneal injection) | LD50 (intravenous injection) | LD50 (oral administration) |
| Species of type | 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, the safety of propylene glycol is better than that of ethanol in various administration routes, and the injection dosage of the dispersion medium is far lower than that of LD50 according to the dosage conversion of the propylene glycol, so that the dispersion medium has good safety.
In order to prepare a drug reservoir with slow release function and low irritation, and prolong the action time of the active ingredient in the body so as to achieve the slow release function, the invention adopts the technical scheme that organic solvent and oil which are mutually soluble with water are used as solvents to dissolve phospholipid and the active ingredient; cholesterol and water are used as auxiliary materials for controlling the release behavior of a drug release system, and meanwhile, a proper amount of antioxidant is added, so that the stability of the preparation system is ensured.
The invention utilizes phospholipid to form a self-assembly system under certain conditions so as to form a drug reservoir, embeds active ingredients therein and has slow release function, and besides, the phospholipid is also a nonionic surfactant and has solubilization function. In addition, the excellent biocompatibility enables the phospholipid to be widely used in the field of pharmacy, and a plurality of fat emulsion, liposome and phospholipid mixed micelle products all take phospholipid as main auxiliary materials at present. The invention utilizes the characteristic of phospholipid to prepare a lipid drug release system with long-acting slow release function. The lipid delivery system is characterized by being designed for ropivacaine free base.
Background problem three: deficiency of existing lipid formulations
Chinese patent 201380036669 "depot formulations of hydrophobic active ingredients and methods of making same" provides non-aqueous, proliposome depot formulations for hydrophobic APIs and methods of making same, wherein no emulsification step is included, and wherein the composition is not exposed to an aqueous phase at any stage prior to entry into the patient's body. Furthermore, the present invention provides depot formulations substantially free of synthetic phospholipids using only GRAS excipients. A pharmaceutical composition comprising: a hydrophobic Active Pharmaceutical Ingredient (API); about 40% to about 60% by weight of Phosphatidylcholine (PC) or a pharmaceutically acceptable salt thereof; about 30% to about 50% by weight castor oil; and about 2% to about 10% by weight of ethanol, wherein the composition is in the form of a clear solution, free of particles having a size greater than 100nm, stable at ambient temperature, and substantially free of water. Chinese patent 201380036700, "depot formulations of local anesthetics and methods of making same," provides non-aqueous, proliposome depot local anesthetic formulations that beneficially generate liposomes or other lipid vesicle structures in situ upon contact with body fluids. The invention also provides a method for preparing a 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 patents are nonaqueous systems and have the main component of a slow release carrier containing castor oil, which is reported to be low to moderate hazardous in MSDS and has a certain irritating and allergic effect on skin and eyes. Moreover, both patents are broadly applied to hydrophobic drugs, and are not specific drug delivery systems, and the scenes used in the literature are far from practical.
The system of China patent 201810592597 is improved on 201380036669 and 201380036700, and a phospholipid-mixed solvent-oil slow-release drug delivery system formula and a preparation method of the local anesthetic are provided, wherein soybean oil, medium-chain triglyceride, corn oil and the like are used for replacing castor oil in the patent in a preparation scheme, so that skin irritation is effectively avoided. But wherein benzyl alcohol and benzyl benzoate are used as solvents. Moreover, it is a non-aqueous delivery system, where drug diffusion is dependent on solvent diffusion, the more water-soluble solvent, the faster the diffusion rate when it is injected subcutaneously. The self-assembly of the phospholipid is necessary to depend on water, when enough tissue fluid in subcutaneous tissue interacts with the phospholipid, the system can be subjected to phase change, the phospholipid can be formed by self-assembly, and the phospholipid has a slow release effect, so that long-acting is realized. Thus, there are situations where drug release is too fast in the front-end of the system. Meanwhile, the oil and solvent are relatively large in the technology of the patent, and the sum of the solvent and the oil reaches 40-90% of the total system, so that the drug release is mainly solvent diffusion.
Among them, benzyl alcohol intake or inhalation may cause dizziness, nausea, vomiting and diarrhea. Massive inhalation can lead to central nervous system depression and dyspnea. Adverse reactions include: intravenous injection toxicity, intrathecal injection neurotoxicity, hypersensitivity and infantile toxicity syndrome. FDA suggests that benzyl alcohol should not be used in the rinse solution. LD50 (mice, intravenous injection) was 0.32g/kg, toxicity was greater than ethanol. Benzyl benzoate can be rapidly hydrolyzed in vivo to benzoic acid and benzyl alcohol, which is further metabolized to hippuric acid for excretion through urine. Benzyl benzoate is used as an adjuvant for topical administration for treating scabies and intramuscular injection and is usually used in a concentration of 25% (V/V) for oral administration. Adverse reactions include skin irritation and allergic reactions. LD50 (mouse, oral) was 1.4g/kg, also more toxic than ethanol.
Therefore, benzyl alcohol and benzyl ester used in the patent are both more toxic than ethanol, and thus may cause strong irritation when injection is performed, resulting in adverse effects such as local redness and swelling, pain, and the like. Further, the formulation of ropivacaine described in the patent document does not solve the technical problems of the present invention.
Chinese patent 201310403977, in situ lipid gel pharmaceutical formulation, and preparation method and use thereof, 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 pharmaceutically acceptable salts thereof, particularly preferably bupivacaine, levobupivacaine or pharmaceutically acceptable salts thereof. Wherein water is introduced into the lipid formulation and is believed to be capable of adjusting 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 is essentially characterized in that phospholipid is self-assembled to form a multi-layer liposome with a bilayer structure after meeting water, so that the liposome has a slow release effect. However, because the phase transition temperature of the soybean lecithin or the egg yolk lecithin used in the technical scheme is about minus 20 ℃, when the ambient temperature is below the phase transition temperature of the liposome, the lipid bilayer is closely arranged, the fluidity is small, the medicine leakage is small, and as the temperature is increased, the lipid membrane is subjected to phase transition, the cross section of the membrane is increased, the permeability and the fluidity are increased, and the medicine can be naturally accelerated to leak from the liposome, so the phase transition temperature is an important parameter for measuring the stability of a 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 medicine is released, the temperature of a human body is far higher than the phase transition temperature, so that the phase transition is easy to occur after subcutaneous injection, and the solvent ethanol used for the preparation has strong diffusivity, so that the medicine release speed is possibly accelerated, the burst release is caused, and the use safety of the medicine is influenced, so that the prescription still has no practical use.
The preferred phospholipid/ethanol ratio of example 1 described in this patent gives a clear transparent system with good flowability up to about 33% ethanol. Local irritation caused by ethanol has been studied ,Ting Zhang(Zhang,Ting,etal.A high-efficiency,low-toxicity,phospholipids-based phase separation gel for long-term delivery of peptides.Biomaterials,45(2015),1–9.) to prepare a gel and the local irritation at the injection site has been studied, and the results show that the local irritation is directly related to the amount of ethanol, the more the amount of ethanol is, the more serious the local irritation is, and connective tissue necrosis and epidermoid ulcer are generated at the injection site when the amount of ethanol reaches 30-45%, so that strict control of the amount of ethanol is required.
In addition, the heating temperature is 30-80 ℃, preferably 30-50 ℃, and the stirring time is more than 30 minutes, ethanol in the system can volatilize when stirring and heating are carried out simultaneously, so that the proportion of the ethanol in the system is changed, the water accounts for 20-80% of the sum of phospholipid and organic solvent, the water consumption depends on the organic solvent, and after the organic solvent volatilizes, particularly at the critical point of the system, the water with the original prescription amount is still added, so that the system can be subjected to phase change, and turbidity and even drug precipitation can occur in the storage and transportation and preservation processes of the preparation. Therefore, 201310403977 does not solve the technical problem proposed by the present invention.
Chinese patent 202110046374.7, "a depot ropivacaine pharmaceutical composition, a method of preparing the same, and uses thereof," appears to make new improvements to ropivacaine drug depots, providing a depot ropivacaine pharmaceutical composition comprising, by weight percent, 0.5% to 10% 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 a pharmaceutically acceptable oil or fat; 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 nonaqueous formulations, and fails to overcome various drawbacks of nonaqueous formulations, including failure to complete phospholipid self-assembly in a short period of time to achieve drug depots, and failure to avoid the strong solvent diffusion effects resulting from the use of large amounts of solvents. The inventors have also conducted long-term follow-up studies on a large number of documents related to ropivacaine lipid preparations, and found that the existing documents are largely classified into nonaqueous systems and aqueous systems, and the preparation forms and in-vivo action mechanisms are various and complex. In another example, chinese patent 201410067859 provides a "ropivacaine nano-lipid carrier temperature-sensitive in-situ gel and its preparation method", which comprises main components of ropivacaine, solid lipid material, liquid lipid material, surfactant, cosurfactant, gel matrix and water for injection. The technical scheme is that ropivacaine is prepared into emulsion firstly, and then the emulsion is added into gel matrix prepared by poloxamer, which is far from a phospholipid reservoir system. Therefore, there is currently no truly effective and targeted solution for the long-acting formulation of ropivacaine drug depots, and there is no solution that makes the long-acting formulation of ropivacaine drug depots a marketed drug. This results from the special nature of ropivacaine and the technical complexity of new formulations of lipid drug depots.
In the invention, based on the unique property of ropivacaine, phospholipid is adopted as a main raw material for forming a drug reservoir, and drug oil and an organic solvent which is mutually soluble with water are adopted as a dispersion solvent to dissolve active ingredients, so that the dosage 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, and the stability of the formed reservoir in vivo and the drug release speed are improved, thereby effectively avoiding the burst effect, controlling the critical point of phase transition by using the water content, promoting the rapid formation of the drug reservoir after injection, and avoiding burst. The technical scheme adopted by the invention is essentially different from the patent documents.
Disclosure of Invention
The invention adopts phospholipid as a key material for forming a lipid drug release system, adopts medicinal oil and a water-miscible organic solvent as solvents to dissolve phospholipid, active ingredients and cholesterol, adopts the phase transition temperature of a cholesterol regulation system to enhance the in-vivo stability of the system after phase transition, avoids the burst effect, and adopts water to regulate the phase transition critical point to promote rapid formation of a drug depot in vivo so as to realize the slow release purpose. The ropivacaine lipid drug release system with long-acting slow release function is prepared by mutually matching various components and the dosage thereof.
The invention aims to provide a lipid release system with a long-acting slow-release effect and a preparation method thereof, which are used for transferring active ingredients of medicines, so as to realize the long-acting effect of the active ingredients of ropivacaine with the release time reaching 48-72 hours. The novel high-safety lipid drug release system with the long-acting slow release function has wide clinical application prospect, and can be effectively supplemented for related preparations of clinical slow release drugs, so that the selectivity of clinical drug application is improved.
In order to achieve the above object, the present invention adopts the following technical scheme:
the invention discloses a lipid release system product with a long-acting slow release effect; the lipid drug release system is a clear and transparent uniform liquid composed of active ingredients, phospholipids, drug release behavior regulating substances, a dispersion medium and an antioxidant.
The active ingredient in the preferred embodiment of the present invention is a fat-soluble active ingredient. The active ingredient is the free base of the local anesthetic ropivacaine.
Ropivacaine is used for obstetrical anesthesia and painless delivery, has definite curative effect, is light in motor nerve block, does not affect the labor and newborns, comprehensively considers the solubility of active ingredients and clinical use safety, and is more preferably the free base of ropivacaine.
The phospholipid is used as a key component of a lipid slow-release system and is a main carrier of a medicine active component, and in order to further ensure the safety of medicine application, the phospholipid with good biocompatibility and excellent safety is preferably selected. In addition, it is also desirable to select phospholipids with excellent solubility in the dispersion medium to reduce the amount of solvent used to ensure drug concentration while minimizing the volume of administration upon local injection, facilitating clinical procedures and reducing adverse reactions. At the same time, the phospholipid selected needs to be compatible with the other formulation components.
In a further preferred embodiment of the present invention, the phospholipid comprises a natural phospholipid or a synthetic phospholipid. The natural phospholipids are derived from soybean, egg yolk and sunflower, and commonly used natural phospholipids comprise soybean phospholipids and egg yolk lecithin; synthetic phospholipids include phospholipids obtained by hydrogenation using natural phospholipids or by total synthesis, including: hydrogenated Soybean Phospholipids (HSPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), distearoyl phosphatidylcholine (DSPC).
The content of phosphatidylcholine in the phospholipid is more than 70%, preferably the content of phosphatidylcholine is more than 95%. Preferably, soybean lecithin or egg yolk lecithin is used, and the amount of synthetic phospholipid is 0-5%, preferably 1-5% of that of natural phospholipid.
Natural phospholipids have a low phase transition temperature, generally less than 0 ℃, and form a multi-layer reservoir with a bilayer after injection, when the ambient temperature is below the lipid phase transition temperature, the lipid bilayer is closely arranged, the fluidity is small, the drug leakage is small, and as the temperature increases, the lipid membrane undergoes phase transition, the cross section of the membrane increases, the permeability and fluidity increase, and the drug can accelerate leakage from the liposome. Because the temperature of the human body is far higher than the phase transition temperature of the 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 that burst release is caused. Thus, the first improvement made by the invention is that increasing the phase transition temperature of the system more readily improves drug release. The phase transition temperature of the synthetic phospholipids containing choline structures is higher, the phase transition temperature of cholesterol and some synthetic phospholipids is mostly higher than 20 ℃, the addition of the synthetic phospholipids can improve the phase transition temperature of the system, for example, the phase transition temperature of soybean phospholipids is about-20 ℃, when a small amount of cholesterol is added, the phase transition temperature of the system is increased, and the increase of the phase transition temperature is favorable for improving the stability of the system after the formation of a reservoir. This outstanding advantage provides a central guarantee for ropivacaine free base drug depots. Accordingly, the prior art does not address the problem of the ropivacaine lipid reservoir phase transition temperature, which necessarily results in burst or too fast release of the drug.
As a preferred embodiment, the matrix is composed of natural phospholipids (or synthetic phospholipids) and cholesterol, and the phase transition temperature of the system is raised.
In another preferred scheme, natural phospholipid, synthetic phospholipid and cholesterol form a matrix, and the phase transition temperature of the system is improved. Wherein the synthetic phospholipid is used in an amount (w/w) of 0-5%, preferably 1-5%.
The active ingredients of the medicine, the phospholipid and the cholesterol are all fat-soluble ingredients and can be dissolved in oil or organic solvent.
In order to solve the problems of dissolving phospholipid, active pharmaceutical ingredients and cholesterol, the preparation method is used for preparing the injection which is easy to inject.
In a further preferred embodiment of the present invention, the dispersion medium is a pharmaceutical 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, a combination of ethanol and propylene glycol or glycerin is used as the dispersion medium, and the ratio of ethanol to propylene glycol or glycerin is preferably 1:1 to 10:0 (w/w), more preferably 7:3 to 10:0 (w/w).
The phospholipid is of great importance for the system, is not only an important carrier for transferring the active ingredients of the medicine, but also a key ingredient for increasing the solubility of the active ingredients of the medicine because of the nature of the surfactant, and is beneficial to the reduction of solvents, so that the use volume of a final finished product is reduced, and the safety of clinical use is improved.
Data on the solubility of pharmaceutically active ingredients in different dispersion media
From the above data, it is clear that ropivacaine free base has the best solubility in absolute ethanol, but the solubility drops dramatically when water is added; phospholipids are effective in increasing the solubility of ropivacaine in soybean oil and ethanol-water.
Solubility data of cholesterol in medicinal oils and organic solvents
In the technical implementation process of the invention, the medicinal oil can surprisingly improve the stability of the system and effectively avoid precipitation of the medicinal active ingredients and cholesterol in the system.
In addition to the important role of dissolving the active ingredient, phospholipids and cholesterol as solvents, the dispersion medium also affects the release of the drug, generally the more solvent diffuses faster, the faster the drug is released, and the more water-miscible solvent diffuses than the water-insoluble solvent. Therefore, the technical scheme further researches the influence of the release behavior of the medicinal oil and other solvents and the dosage of the solvents on the release behavior.
The method comprises the following steps: dissolving appropriate amount of active ingredients with absolute ethanol and soybean oil respectively, taking about 1.0g of sample, placing into dialysis bags (3000 Da) respectively, placing the dialysis bags into phosphate buffer solution, maintaining the temperature at 37deg.C, stirring at 100 rpm, sampling at different time points, and measuring.
The effect data of the different solvents on the release of the active ingredient are as follows:
The results show that ethanol is released faster than soybean oil is released with ethanol as solvent. The dosage of ethanol is reduced, and the dosage of medicinal oil is increased, so that the initial release speed can be effectively reduced, and the situation that animals are in an anesthetic state rather than analgesic after administration due to massive burst release is avoided.
In a further 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, besides the solvent type, the amount of the solvent used has an influence on the drug release, and the amount of the water-miscible organic solvent in the formulation also has an influence on the drug release.
In a further preferred embodiment of the present invention, the dispersion medium further comprises an organic solvent miscible with water, and the ratio of phospholipid to organic solvent is 1:1 to 2:1. Further preferably 1.5:1 to 2:1.
Wherein the dispersion medium contains water-miscible organic solvents, including one or more of ethanol, propylene glycol, and glycerin, in addition to the medicinal oil, and preferably ethanol and propylene glycol are used in combination with the medicinal oil as the dispersion medium. 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 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 catalytic oxidation of phospholipids and unsaturated pharmaceutical oils caused by monovalent or divalent ions contained therein can be avoided.
The proportion of the water to the organic solvent is 10-30% (w/w), and is preferably 20-30% (w/w).
Cholesterol is also one of the constituent components of cell membranes, is an amphoteric substance, and can regulate the fluidity and permeability of phospholipid bilayer. Surprisingly, the addition of 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 cholesterol dosage, thereby providing a great effect for realizing long-acting release of the lipid drug release system.
In a further preferred embodiment of the 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 medicine, the addition of the cholesterol obviously delays the release of the medicine, and the action mechanism is that the integrity of a bilayer formed by phospholipid after self-assembly is changed while the phase transition temperature of a system is improved, and the strength of the bilayer is changed, so that the mechanism of the burst effect caused by medicine leakage is not easy to cause.
In a further preferred embodiment of the present invention, the antioxidant is one of vitamin E acetate and dl-alpha-tocopherol, and the dosage thereof 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 invention, the active ingredient is ropivacaine; the content of the 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 phospholipid to organic solvent is 1:1-2:1, and more preferably 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 water in the organic solvent is 10-30% (w/w), and more preferably 20-30% (w/w);
The proportion of the antioxidant is 0.1% -1% of the sum of the phospholipid and the oil.
(2) In a further preferred embodiment of the present invention, the preparation method comprises the steps of:
① Weighing active ingredients, phospholipid, cholesterol, medicinal oil and antioxidant according to the prescription, stirring at high speed or high speed, heating to 40-70deg.C, preferably 40-50deg.C to dissolve the system completely, replacing air in the container with nitrogen to prevent oxidation, and cooling to 20-40deg.C.
② Adding the organic solvent and stirring uniformly.
③ Adding proper amount of water for injection, and rapidly stirring while adding until the system is clear.
④ The sterilized fraction was then filtered through a 0.22 μm filter.
⑤ Packaging, charging nitrogen, adding plug, and sealing.
(3) In a further preferred embodiment of the present invention, the phospholipid, cholesterol and active ingredient in the system are dispersed by high-speed stirring in step ① by using high-speed shearing, so as to increase the specific surface area and accelerate dissolution, and the dissolution time is less than 1 hour, more preferably less than 30 minutes. Simultaneously, the nitrogen is adopted to replace the 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 step ①, the dissolved system should be cooled, preferably to 20-40 ℃, more preferably 20-30 ℃, and then the organic solvent is added according to step ②, so that the ratio of the organic solvent in the system is effectively prevented from being changed due to volatilization caused by heating.
(5) In a further preferred embodiment of the present invention, the injection water is added in step ③ with rapid stirring to avoid phase inversion of the system due to high local water content, thereby leading to precipitation of pharmaceutically active ingredient or cholesterol.
(6) The more preferable scheme is as follows: the active drug ropivacaine free base is 2-8 parts, preferably 3-6 parts, per 100 parts by weight.
The phospholipid is 40-60 parts by weight, preferably 45-55 parts by weight, based on 100 parts by weight.
15-35 Parts, preferably 10-25 parts, of ethanol per 100 parts by weight.
The soybean oil is 5 to 20 parts by weight, preferably 8 to 15 parts by weight per 100 parts by weight of soybean oil.
Cholesterol 2-8 parts, preferably 2-5 parts, per 100 parts by weight.
The antioxidant is 0.01-1 parts, preferably 0.02-0.5 parts, 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, per 100 parts by weight.
The above preferred embodiments can be used singly or in combination of any two or more
In another aspect of the invention, an application of a combination of water for injection and cholesterol in regulating the drug release behavior of a lipid drug release system product with a long-acting slow release effect is provided, wherein the actions comprise increasing the phase transition temperature of the system by cholesterol, and enhancing the integrity and stability of a phospholipid bilayer, thereby improving the stability of a drug reservoir; the water is used for preparing a reservoir in a phase transition critical state, and when a small amount of external water-containing liquid is contacted with the system, the phase transition of the system is promoted, so that a drug reservoir is rapidly formed, and the effective combination of the two synergistic effects further controls the drug release speed.
The more preferable scheme of the invention is that the raw materials of the lipid drug release system product with the long-acting slow release effect consist of active ingredients, phospholipids, drug release behavior regulating substances, dispersion medium and antioxidants. No additional components are contained other than the unavoidable presence of impurity constituents or osmotic pressure regulating conventional substances that must be added. The lipid delivery system of the present invention is generally administered by injection around the wound, or by spraying it as a pharmaceutically acceptable spray onto the wound surface or accessible mucosal tissue.
The lipid drug carrier system of the invention is light yellow to yellow clear transparent liquid, phase transition occurs after interaction with tissue liquid after administration, self-emulsifying self-assembly of the system occurs 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 proved by the application of components such as phospholipid, medicinal oil and the like in various injection varieties in clinical safety consideration, and can effectively reduce the dosage of organic solvents by using medicinal oil with lower toxicity as a solvent, thereby reducing local irritation and ensuring the value of clinical application and the safety of use of the preparation. Meanwhile, the technical difficulty is overcome, the stability of the preparation is ensured, and the sustained release effect is realized.
(2) The phospholipid is a key component for realizing slow release in the invention, not only can effectively improve the content of local anesthetics in the composition, but also can generate phase transition after being combined with water in vitro or tissue fluid in vivo due to the characteristic of self-assembly after meeting water, thereby forming a drug reservoir with a space structure and realizing slow release; in addition, because the phospholipid is also an important component of cell membranes, the phospholipid has good safety and biocompatibility. The invention discovers 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 matrix is composed of natural phospholipid (and/or synthetic phospholipid) and cholesterol, more preferably, the matrix is composed of natural phospholipid, synthetic phospholipid and cholesterol, and the phase transition temperature is improved.
(3) The invention contains medicinal oil and organic solvent, the diffusion speed of the medicinal oil is slower than that of the organic solvent, the problem that the release speed of the front section is too fast due to too fast diffusion of the medicine caused by singly using the organic solvent in the system is avoided, and the medicinal oil and the organic solvent are organically combined to regulate 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 carrying capacity 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 extent on the premise of ensuring the content of the active ingredients, the clarity of the system and the influence of viscosity during injection on injection, so as to reduce the drug burst caused by the solvent diffusion effect, thereby achieving the effect of stable slow release and simultaneously reducing the local irritation of the organic solvent during injection to the maximum extent.
(5) Compared with the prior art, cholesterol is added into the system, when the system is combined with tissue fluid to generate phase transition, the cholesterol and the phospholipid form a tighter double-molecular layer structure, so that the stability of the system after phase transition is maintained more effectively, the speed of drug release is controlled effectively, and the long-acting effect is maintained.
(6) The water adopted by the invention is water for injection, wherein the monovalent and divalent ion content 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 pharmaceutical excipients, so that the safety is good, and meanwhile, the addition of the antioxidant can effectively reduce the oxidation of active ingredients, the auxiliary materials, namely phospholipid and pharmaceutical oil, so that the degradation is reduced, and the safety of the product is ensured.
(8) Finally, the design process of the invention is always developed around the ropivacaine free alkali, the component and the dosage are rationalized, and the excessive dependence on the action of single component and dosage is avoided, so that the ropivacaine lipid drug release system is prepared, the duration of the drug effect can reach more than 48-72 hours after test, the drug release process is stable, the repeated 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 self-assembled pre-morphology of a lipid delivery system of example 9;
FIG. 2 is a self-assembled version of the lipid delivery system of example 9;
FIG. 3 is the results of the hot plate test of the control and test groups of example 11;
FIG. 4 is a graph showing the results of example 11 as percent (%) of maximum analgesic effect of a single perisciatic injection administration of the test substance to rats;
FIG. 5 shows the results of the pain threshold improvement (%) in rats obtained by administering the test substance to the rats by a single sciatic nerve injection in example 11.
Detailed Description
The following examples are provided to further illustrate the technical aspects of the present invention, but are not to be construed as limiting the present invention.
Example 1
Samples containing ropivacaine free base were prepared according to the recipe in table 1, left at room temperature for 24 hours, and observed for behavior.
Table 1 prescription composition and State
The water content can change the state of the system, and the system changes phase from clarification to turbidity gradually along with the increase of the adding amount of water. In the system of the invention, when the water accounts for 35 percent of ethanol, the system is not stable.
Tests prove that the defects of the mixed solvent (the relationship between the solvent and the drug loading) are not the simple combination, and the mixed solvent must be mixed according to a certain proportion to form a required system. It is described as a complex system. Water is one of the core components in the present invention that affects release or forms a drug depot. The more water content, the faster the reservoir is formed, but the more pharmaceutically active ingredients in the composition will precipitate; the water content is low, the speed of forming a drug reservoir is low, the drug release is mainly represented by solvent diffusion behavior, and the release speed is high. There is a reasonable range, and tables 1 and 3 in the embodiments of the patent already describe the morphology of the mixture ratio of water and organic solvent. 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 behavior.
Table 2 prescription composition and State
In the above ratios, a clear and transparent system was obtained, and the fluidity of the liquid obtained by the various compositions was excellent.
Example 3
Samples containing ropivacaine free base were prepared as prescribed in table 3, left at room temperature, and observed for 0h and 24h behavior.
Table 3 prescription composition and State
Ropivacaine has a much lower solubility in propylene glycol than in ethanol, so that ropivacaine precipitates when ethanol is reduced.
Example 4
Samples containing ropivacaine free base were prepared as prescribed in table 4, left at room temperature for 24 hours, and then subjected to in vitro release studies.
The release degree research method comprises the following steps: about 1.0g of the sample was taken, placed in a dialysis bag (3000 Da), the dialysis bag was placed in 250mL of phosphate buffer (pH 7.4), incubated at 37℃and sampled at a stirring rate of 100 revolutions per minute at 0.5, 1,2,4, 6, 8, 24, 32, 48, 72 hours, respectively, and measured.
Table 4 the effect data of the amount of medicinal oil on drug release is as follows:
soybean oil is one of solvents as medicinal oil, and has diffusion effect, and the more the amount 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 24 hours, and then subjected to in vitro release studies.
The release degree research method comprises the following steps: about 1.0g of the sample was taken, placed in a dialysis bag (3000 Da), the dialysis bag was placed in 250mL of phosphate buffer (pH 7.4), incubated at 37℃and sampled at stirring speed of 100 rpm for 0.5, 1, 2,4, 6, 8, 10, 24, 32, 48, 72 hours, respectively, and measured.
Table 5 the effect of organic solvents on drug release is as follows:
the content of the organic solvent in the system directly influences the drug release, 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 24 hours, and then subjected to in vitro release studies.
The release degree research method comprises the following steps: about 1.0g of the sample was taken, placed in a dialysis bag (3000 Da), the dialysis bag was placed in 250mL of phosphate buffer (pH 7.4), incubated at 37℃and sampled at stirring speed of 100 rpm for 0.5, 1, 2,4, 6, 8, 10, 24, 32, 48, 72 hours, respectively, and measured.
Table 6 the effect data of water content on drug release is as follows:
The system uses a small amount of water to change the release behavior of the drug, and the root cause is that the addition of water can effectively change the original system structure, so that the anhydrous system is quickly transformed, a drug reservoir is quickly constructed, and the drug release is changed from pure solvent diffusion to the dual effects 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 24 hours, and then subjected to in vitro release studies.
The release degree research method comprises the following steps: about 1.0g of the sample was taken, placed in a dialysis bag (3000 Da), the dialysis bag was placed in 250mL of phosphate buffer (pH 7.4), incubated at 37℃and sampled at a stirring rate of 100 revolutions per minute at 0.5, 1,2,4, 6, 8, 24, 32, 48, 72 hours, respectively, and measured.
Table 7 the effect of cholesterol on the drug release from the system is as follows:
cholesterol can effectively change the phase transition temperature of the system and the integrity of a bilayer after the system forms a reservoir, so that the burst release of the drug caused by low phase transition temperature of the system can be effectively reduced.
Example 8
Samples containing ropivacaine free base were prepared as prescribed in table 8, left at room temperature for 24 hours, and then subjected to in vitro release studies.
The release degree research method comprises the following steps: about 1.0g of the sample was taken, placed in a dialysis bag (3000 Da), the dialysis bag was placed in 250mL of phosphate buffer (pH 7.4), incubated at 37℃and sampled at a stirring rate of 100 revolutions per minute at 0.5, 1,2,4, 6, 8, 24, 32, 48, 72 hours, respectively, and measured.
Table 8 the effect data of synthetic phospholipids on the drug release profile of the system is as follows:
example 9
Samples containing ropivacaine free base were prepared as prescribed in Table 9, about 1g of samples were taken, placed in near-dry dialysis bags, and the bags were placed in phosphate buffer (pH 7.4) at 37℃for 0.5,1, 2,4, 8h, respectively, to observe changes in their properties.
Table 9 sample variation during dialysis
The samples prepared by the method can undergo phase transition after encountering a small amount of water, so that morphological change occurs, and the yellow clear transparent liquid is gradually changed into milky white, as shown in figures 1 and 2.
Example 10
Based on the above study, preferred embodiments of the present invention include, but are not limited to, the following:
(1) Ropivacaine 1.5g, soybean lecithin 20.0g, ethanol 10.0g, soybean oil 5.0g, cholesterol 1.0g, vitamin E0.05g, and water for injection 3.0g;
(2) Ropivacaine 1.8g, soybean lecithin 20.0g, ethanol 10.0g, soybean oil 5.0g, cholesterol 1.5g, vitamin E0.05g and water for injection 2.0g;
(3) 2.0g of ropivacaine, 20.0g of soybean lecithin, 10.0g of ethanol, 7.5g of soybean oil, 2.0g of cholesterol, 0.075g of vitamin E and 2.0g of water for injection;
(4) Ropivacaine 1.3g, soybean lecithin 18.0g, hydrogenated soybean lecithin 2.0g, ethanol 10.0g, soybean oil 10.0g, cholesterol 2.0g, vitamin E0.075g, and water for injection 2.0g;
(5) Ropivacaine 1.5g, soybean lecithin 19.5g, distearoyl phosphatidylcholine 0.5g, ethanol 15.0g, soybean oil 7.5g, cholesterol 1.0g, vitamin E0.075g, and water for injection 3.0g;
(6) Ropivacaine 1.5g, egg yolk lecithin 19.5g, distearoyl phosphatidylcholine 0.5g, ethanol 15g, soybean oil 7.5g, cholesterol 1.0g, vitamin E0.075g, and water for injection 1.5g;
(7) Ropivacaine 1.4g, soybean lecithin 20.0g, ethanol 9.0g, propylene glycol 1.0g, soybean oil 5.0g, cholesterol 1.0g, vitamin E0.075g, and water for injection 2.0g;
(8) Ropivacaine 1.4g, soybean lecithin 20.0g, ethanol 9.0g, propylene glycol 1.0g, soybean oil 5.0g, cholesterol 1.25g, vitamin E0.075g, and water for injection 2.0g;
(9) Ropivacaine 1.5g, soybean lecithin 20.0g, ethanol 10.0g, soybean oil 5.0g, cholesterol 2.0g, vitamin E0.05g, and water for injection 2.5g;
(10) 2.5g of ropivacaine, 20.0g of soybean lecithin, 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 a sustained release effect, ropivacaine lipid sustained release preparation samples were prepared according to the above-described optimal embodiment, and comparative studies were performed with the in vitro release profile of ropivacaine hydrochloride injection, and pharmacokinetic studies were performed using SD rats, and the results are shown in tables 10, 11, 12 and 13.
The release degree research method comprises the following steps: about 1.0g of the sample was taken, placed in a dialysis bag (3000 Da), the dialysis bag was placed in 250mL of phosphate buffer (pH 7.4), incubated at 37℃and sampled at a stirring speed of 100 rpm for 0.5, 1,2, 4, 6, 8, 24, 32, 48, 72, 96, 120 hours, respectively, and measured.
Table 10 in vitro Release study of ropivacaine injection and samples prepared according to the technical scheme of the invention
Pharmacokinetic study method: 6 SD rats are randomly divided into 2 groups, namely a test group and a control group, wherein the test group is used for dosing ropivacaine lipid sustained release preparation, the control group is ropivacaine hydrochloride injection, the dosing amount is 40mg/kg, the dosing mode is subcutaneous injection, and the groups are respectively subcutaneously injected after the dosing: blood concentration was measured by venous blood sampling after 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, 48h, and 72h post administration. The test group results were as follows:
table 12 pharmacokinetic study of rats control 40 mg/kg-rat
Note that: BLQ: below the detection limit
TABLE 13 pharmacokinetic parameters of control and test groups
According to the results of the pharmacokinetic study, the T1/2 of the test group is about 13.8 times that of the control group, the Tmax is delayed by about 1 time relative to the control group, the data for AUC (0- ≡) indicated that the test group was 1.6 times that of the control group, which also indicated that the samples used in the test group had significant sustained release effect. The blood concentration monitoring result shows that the lipid drug release system disclosed by the invention has a slow release effect, the action time can reach 48-72h, and the comparison group result shows that the action time is only 8h, which is matched with the action time of 2-6 h in the ropivacaine hydrochloride injection specification, and further proves that the composition has a long-acting slow release effect.
Example 11
To further verify that the lipid delivery system disclosed in the invention has a sustained release effect, ropivacaine lipid sustained release preparation samples were prepared according to the protocol of example 10 and analgesic tests were performed using a hot plate method.
The experimental method comprises the following steps:
SD rats are taken and adapted to animal houses for 2-7 days, the environment of the animal houses is kept at 23+/-2 ℃ and the humidity is 40-70%, and the brightness and the darkness are alternated for 12 hours. Animals were kept 5 per cage, 4 per cage after grouping, and the litter was replaced twice a week (corncob litter, Chuan commerce, inc., suzhou).
After the adaptation period, all rats were screened for pain threshold sensitivity one day prior to dosing. The rats were fixed and the right hind feet of the rats were placed on a hot plate at 56 ℃, the time of their withdrawal was observed and recorded, the average value was determined three times and recorded as the basal pain threshold. If the measurement result of the footage time 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 equally divided into 3 groups of 8 animals each, wherein the three groups are respectively: control group, ropivacaine lipid slow-release preparation 20mg/kg group and ropivacaine lipid slow-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, respectively. The specific method comprises the following steps: after the rat is anesthetized by isoflurane, in a prone position, local hair is removed, the rat is sequentially disinfected by iodine tincture and 75% alcohol, then skin is cut, muscles are passively separated, sciatic nerves are exposed and dissociated, and finally wounds are sequentially sutured and disinfected. The injection side foot pain threshold values of 4h,6h,8h,24h,32h,48h and 72h after administration are measured, and if the pain threshold value exceeds 10s after administration and the foot shrinkage phenomenon still does not occur in a hot plate test, the hot plate should be separated from the hot plate so as to avoid burn. The percentage of maximum analgesic effect and the rate of increase in pain threshold were calculated and observed for time-dependent effects. The calculation formula of the maximum analgesic effect percentage and pain threshold improvement rate is as follows:
percentage of maximum analgesic effect (MPE%) = (post-dose pain threshold-basal pain threshold)/(10-basal pain threshold) ×100%
Pain threshold improvement rate = (average pain threshold after administration-average pain threshold before administration)/average pain threshold before administration x 100%
Dosing regimen is shown in table 14, hot plate test data is shown in table 15, fig. 4, fig. 5.
Table 14 animal dosing regimen
Test results:
Compared with a control group, the 20mg/kg of ropivacaine lipid slow-release preparation administered by single sciatic nerve peripheral injection can obviously prolong the hot plate reaction time (p <0.01 or 0.001) of the rats at 4h-24h after administration, and has a certain extension effect on the hot plate reaction time at 32h-48h, and the hot plate reaction time gradually decreases along with the extension of the time after administration. The 40mg/kg ropivacaine lipid slow-release preparation administered by single sciatic nerve peripheral injection can obviously prolong the hot plate reaction time (p <0.01 or 0.001) of the rats at 4h-48h after administration, and has a certain prolonging effect on the hot plate reaction time at 48h-72h, and the hot plate reaction time gradually decreases with the time after administration. The prolonged effect of the subject ropivacaine lipid sustained release formulation on rat hotplate response time was dose-positively correlated (table 15, fig. 3).
Table 15 effect of single perisciatic injection administration of test substance on hot plate response time in rats (n=8,)
Compared with a control group, the 20mg/kg and 40mg/kg of ropivacaine lipid slow-release preparation administered by single sciatic nerve peripheral injection can obviously prolong the hot plate reaction time of the rats at 4-48 h after administration, and has a certain prolongation effect on the hot plate reaction time after 48h, but has no statistically significant difference, and the hot plate reaction time gradually decreases along with the duration of the time after administration.
The maximum analgesic effect percentage and pain threshold improvement rate of the ropivacaine lipid slow-release preparation which is given by single sciatic nerve peripheral injection are highest in 4 hours after the administration, and then gradually decrease, and the analgesic effect of the ropivacaine lipid slow-release preparation of the tested substance on the pain caused by a rat hot plate is positively correlated in dosage.
The maximum analgesic effect percentage and pain threshold improvement rate of ropivacaine lipid sustained release preparation administered by single sciatic nerve peripheral injection were highest at 4h after administration, and then gradually decreased, and the analgesic effect of ropivacaine lipid sustained release preparation as a test substance on rat hotplate pain was positively correlated in dose (table 16, table 17, fig. 4, fig. 5).
Table 16 effect of single sciatic nerve peripheral injection administration of test substance on the percentage (%) of maximum analgesic effect in rats (n=8)
Table 17 effect of single sciatic nerve peripheral injection administration of test substance on pain threshold improvement rate (%) in rats (n=8)
Conclusion of hot plate test:
The single injection of 20mg/kg and 40mg/kg of ropivacaine lipid slow-release preparation around the sciatic nerve can obviously prolong the hot plate reaction time of the rats, and has obvious analgesic effect, and the analgesic effect is positively correlated with the administration dosage of the ropivacaine lipid slow-release.
Example 12
In order to further test the physical and chemical stability of the sample, the sample was prepared according to the preferred scheme in example 10, and after being sealed by filling nitrogen, the sample was placed in an opaque external packaging box for stability study under accelerated stability test conditions of 30 ℃ ± 2 ℃,60% ± 5% RH, 25+ -2 ℃ long-term stability test, 60% ± 10% RH, and simultaneously stability at low temperature conditions of 2-8 ℃ was measured for the properties, particle size, zeta potential, and other indicators, respectively. The results are shown in tables 18-22.
The particle size measurement method comprises the following steps: the sample is placed in a dry measuring cup, placed in a Markov Nano-ZS90 particle size analyzer, the measuring temperature is set to 25 ℃, the measuring time is 100s, the parallel measurement is carried out for 2 times, and the average value is obtained.
The potential measurement method comprises the following steps: the sample is placed in a dry measuring cup, placed in a Markov Nano-ZS90 type particle size analyzer, the measuring temperature is set to 25 ℃, the cycle times are set to 15 times, the parallel measurement is carried out for 2 times, and the average value is obtained.
And (3) establishing a content measurement method:
selection of absorption wavelength:
Instrument: ultraviolet visible spectrophotometer, beijing general analysis general Instrument Co., ltd., new century of T6
The method comprises the following steps: precisely weighing 12.5mg of ropivacaine reference substance, placing in a 50mL measuring flask, and dissolving with methanol to obtain reference substance stock solution; 1mL of control stock solution is precisely measured, diluted to 100mL with methanol, and spectral scanning is performed at 200-400 nm.
Results: ropivacaine has absorption in the range of 200-250nm, and the maximum absorption peak is 202nm, which is close to methanol and the terminal absorption, so that the content measurement needs to avoid 202nm in order to reduce the measurement interference to the greatest extent.
The method for measuring ropivacaine by high performance liquid chromatography is established by the following steps:
Detection instrument: high performance liquid chromatograph LC-2030, shimadzu.
Chromatographic conditions: c18 column (4.6 mm. Times.250 mm,5 μm, shimadzu corporation); mobile phase: acetonitrile-phosphate buffer (triethylamine to adjust the pH to 8.0) (60:40) is used as a mobile phase; the detection wavelength is 225nm; the flow rate is 1.0mL/min; the sample volume was 20. Mu.L.
Preparing a reference substance solution: precisely weighing 12.5mg of ropivacaine reference substance solution, placing in a 50mL measuring flask, and diluting with methanol to scale to obtain reference substance stock solution; precisely measuring 1mL of reference substance stock solution, placing in a 10mL measuring flask, and adding methanol to the scale to obtain reference substance solution.
Preparing a test solution: precisely measuring a proper amount of a sample to be measured, placing the sample into a measuring flask, adding methanol to the scale, and shaking uniformly to obtain a clear and transparent solution.
TABLE 18 accelerated stability study (Property and content)
TABLE 19 accelerated stability study (particle size and zeta potential)
The accelerated stability result shows that under the storage condition, the particle size, the content, the zeta potential and the properties of the sample are not changed obviously.
TABLE 20 Long-term stability study (Property and content)
TABLE 21 Long-term stability study (particle size and potential)
The long-term stability results show that under the storage condition, the particle size, the content, the zeta potential and the properties of the sample are not changed significantly.
In order to further study the stability at low temperature, the samples were placed at 2-8℃to observe the properties, and after the observation was completed, the content was determined by taking a liquid, filtering the liquid through a 0.45 μm filter, and then sampling the liquid to be diluted according to the sample preparation method. The results are shown in Table 22
TABLE 22 Low temperature stability study (2-8deg.C)
As a result of the low-temperature test, the samples of the respective formulations are stored at low temperature, crystals are precipitated, the amount of crystals is related to the ratio of the organic solvent to water in the system, and the larger the ratio of water is, the more crystals are precipitated.
Example 13
To further verify the variability of the analgesic test results and pharmacokinetics of the different animals, ropivacaine lipid sustained release formulation samples were prepared according to the protocol of example 10, using beagle pharmacodynamics and pharmacokinetic studies.
The experimental method comprises the following steps:
8 Beagle dogs, each half of which is male and female, have a mass of 13-15kg, are purchased from the common biomedical Co., ltd. Of An Dile, and are subjected to animal quarantine and adaptive feeding for 1 week, and are fasted for 12 hours before administration, and are free to drink water.
The 8 dogs were divided into two groups according to the administration mode, i.e., sciatic nerve administration group and subcutaneous injection administration group.
4 Test dogs, male and female half, were selected from sciatic nerve administration group, fasted for 12h before administration, and were free to drink water. Numbering and weighing animals; intravenous injection of propofol emulsion (8.00 mg/kg) was induced for anaesthesia, followed by administration of dexmedetomidine (0.03 ml/kg) and butorphanol (0.06 ml/kg) and maintenance of anaesthesia using 0.6% isoflurane. After anesthesia, the skin was shaved locally, disinfected, the skin was surgically cut to isolate the muscles, the sciatic nerve was exposed, and ropivacaine lipid sustained release formulation (6.00 mg/kg) was injected around the sciatic nerve. Sequentially suturing muscle and skin, and sterilizing the operation incision. After the operation is completed, the isoflurane administration is stopped, and the monitor is removed after the animal is awake.
The subcutaneous injection administration group selects 4 test dogs, the male and female dogs are half, and the dogs are fasted for 12 hours before administration and drink water freely. Animal numbering, weighing and recording; ropivacaine lipid sustained release formulation (6.00 mg/kg) was injected subcutaneously into the animal's neck.
For the sciatic nerve administration group, the respiratory times and heart rate of animals are respectively measured before anesthesia of the animals; after anesthesia, pulse oximetry is monitored by using a MINDRAY IMEC monitor, and the respiration frequency and the heart rate are recorded; surgery was performed and administration was performed, and respiratory rate and heart rate were recorded. After the operation is completed, the isoflurane administration is stopped, and the monitor is removed after the animal is awake. Scoring and recording analgesic effects at 0.5h, 2h, 4h, 6h, 8h, 12h, 24h, 32h, 48h, 72h jaw lip, paw, nose, ear, tail, etc. after administration; recording the breathing times and heart rate after administration; the general symptoms of the animals are observed, and the skin at the injection site is changed, and the animals are sleepy and calm.
For the subcutaneous injection administration group, the analgesic effect is scored and recorded at the positions of 0.5h, 2h, 4h, 6h, 8h, 12h, 24h, 32h, 48h, 72h, jaw lip, claw, nose, ear, tail and the like before and after administration; recording the breathing times and heart rate after administration; the general symptoms of the animals are observed, and the skin at the injection site is changed, and the animals are sleepy and calm.
The analgesic effect was digitized by the scoring method in this experiment. The positions of the jaw lip, the claw, the nose, the ear, the tail and the like are used as 5 scoring standards for judging the analgesic effect. The specific scoring scheme is as follows:
"0" score: pain is very severe; when a certain part of the dog is tested by using the hemostatic forceps, the test dog immediately reacts or develops systemic reaction, and the reaction is not obviously different from the reaction before the analgesic is injected.
"1" Score: when a certain part of the hemostatic forceps is clamped, the tested dogs only weakly move limbs to try to avoid flashing, but have slow response and weak action.
"2" Score: when clamping a certain part with a hemostatic forceps, the test dogs only respond to the local stimulation; the systemic reaction is mild.
"3" Score: with a hemostatic clamp on a site, the test dogs did not respond at all.
Summing the scores measured at the 5 parts, and judging that the total score is highly analgesic within 13-15 points; 10 to 12 is divided into moderate analgesia; 5 to 9 are classified as mild analgesia.
Data are expressed as mean ± standard deviation. The SPSS 22 software was used to perform a one-factor anova with multiple sample mean comparisons and a two-to-two comparison t-test. P < 0.05 indicates a significant difference.
Test results
1. Influence of different administration modes on analgesic effect, respiration and heart rate
The sciatic nerve administration group was completely awake 20-40 min after surgery, and the effect of administration time on analgesic effect, respiratory rate and heart rate is shown in table 23. The effect of the timing of subcutaneous administration on analgesic effect, respiratory rate and heart rate is shown in Table 24.
Table 23 effects of sciatic nerve administration on analgesic effect, respiratory rate and heart rate (n=4,)
| Time (h) | Analgesic effect (fen) | Respiration times (times/min) | Heart rate (times/min) |
| 0 (Before anesthesia) | 0.00±0.00 | 24±11.2 | 128±16.0 |
| 0.5 | 2.50±2.65 | 26±5.2 | 116±12.7 |
| 2 | 8.50±6.45 | 26±7.2 | 121±3.8 |
| 4 | 8.75±3.77 | 25±3.8 | 108±3.3 |
| 6 | 8.25±5.56 | 27±8.3 | 110±12.0 |
| 8 | 10.25±6.85 | 22±2.3 | 101±14.4 |
| 12 | 11.25±2.06 | 22±4.0 | 109±11.9 |
| 24 | 9.75±2.06 | 25±5.0 | 109±15.1 |
| 32 | 6.75±2.63 | 27±8.3 | 102±9.5 |
| 48 | 4.25±0.50 | 31±8.5 | 109±9.5 |
| 72 | 1.50±0.58 | 36±6.2 | 113±19.2 |
From the results, after the sciatic nerve is dosed, the medicine shows time-dependent analgesic effect, the analgesic effect is quite obvious in 2-24 hours, and the analgesic effect scores in 8 hours and 12 hours can reach 10.25+/-6.85 minutes and 11.25+/-2.06 minutes (P is less than 0.01); the analgesic effect is obviously reduced after 48 hours, but the analgesic effect is still achieved after 48 hours, and no obvious difference exists between 72 hours and normal time. The respiration times change with the action time of the medicine, but there is no statistical difference yet; at 8h and 12h, the time is reduced to the minimum, and compared with the normal time of 24+/-11.2 times/min, the time is 22+/-2.3 times/min and the time is 22+/-4.0 times/min respectively; recovery at 24 hours, but slightly increased 48 and 72 hours later, may be due to reduced analgesic effect. The heart rate also changed with the duration of the drug action, 8h was minimized to 101+ -14.4 beats/min, followed by some increase, but no statistical difference. The animals have no somnolence phenomenon in the whole test course.
Table 24 effects of subcutaneous administration on analgesic effect, respiratory rate and heart rate ((n=4,))
From the results, after the medicine is subcutaneously injected, the medicine also shows time-dependent analgesic effect, the analgesic effect is obvious in 0.5-24 h, the analgesic effect is highest in 6h, and the score can reach 13.75+/-1.89 minutes; the analgesic effect is obviously reduced at 48 hours compared with 24 hours, but still has analgesic effect. The respiration frequency and heart rate fluctuate in a small range along with the action time of the medicine, but have no obvious change rule. The phenomenon of sleepiness of animals is not seen in the whole course.
The effect of the sciatic nerve administration is similar to that of subcutaneous injection, but the sciatic nerve administration has less fluctuation of the analgesic effect and is more gentle to recover.
At a dose of 6mg/kg, the animals were awake post-operatively and were free of somnolence, indicating that analgesic effect was maintained at this dose, but not anesthetized. Compared with two groups of different administration modes, the analgesic effect of more than 48 hours can be realized.