RU2791481C2 - Anesthetic compositions with delayed release and methods for their preparation - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: group of inventions relates to a method for obtaining an anesthetic composition with delayed release and to a composition for local administration of an anesthetic to a subject in need obtained by this method. A method for obtaining a delayed-release anesthetic composition involves: dissolving a lipid mixture and at least one amide-type anesthetic in a solvent system to obtain a liquid structure; removal of a solvent system from a liquid structure where the solvent system contains tert-butanol or tert-butanol/water as a co-solvent, to obtain a lipid structure with a high degree of capture (HELS); hydration of HELS using an aqueous buffer solution at a pH of 5.5 to 8.0. Moreover, as a result of hydration of HELS, multilayer vesicles (MLV) with a captured amide-type anesthetic are formed; moreover, the median diameter of MLV vesicles with a captured amide-type anesthetic is at least 1 mcm; and the molar ratio of an amide-type anesthetic and phospholipid in MLV vesicles with a captured amide-type anesthetic is at least 0.5:1. The anesthetic composition obtained by the above method contains multilayer vesicles with a captured amide-type anesthetic, the median diameter of which is at least 1 mcm, and the molar ratio of an amide-type anesthetic and phospholipid in the specified vesicles is at least 0.5:1.

EFFECT: use of the composition obtained by the above method provides a rapid onset of anesthesia and a prolonged duration of local anesthesia with minimal toxicity.

12 cl, 5 dwg, 2 tbl, 5 ex

Description

Link to related application

[0001] The present application claims priority under U.S. Provisional Application No. 62/550,983, filed August 28, 2017, and U.S. Provisional Application No. 62/621,730, filed January 25, 2018, each of which is included herein by reference in its entirety.

The field of technology to which the present invention relates

Scope of the present invention

[0002] The present invention relates to a drug delivery system for delivering a sustained release anesthetic composition. The present invention relates to a method for preparing a drug delivery system. The present invention also relates to a sustained release pharmaceutical composition adapted to a drug delivery system that has a prolonged duration of action.

State of the art information

[0003] Several approaches have been described for the development of local anesthetics with sustained release, including: 1) obtaining local anesthetics in the form of multilayer liposomes using the dehydration-rehydration method (US patent No. 6926905), 2) obtaining local anesthetics in the form of giant multivesicular (GMV ) liposomes using the ammonium sulfate gradient loading method (US Pat. No. 7,357,944); and 3) obtaining local anesthetics in the form of multivesicular liposomes (MVL) using the water-in-oil method (US Pat. No. 8,182,835).

[0004] To obtain local anesthetics in the form of multilamellar liposomes by the dehydration-rehydration method, phospholipid and cholesterol dissolved in tert-butanol were lyophilized and then hydrated to obtain multilamellar vesicles (MLV) and the MLV vesicles were homogenized to obtain small monolamellar vesicles (SUV). The local anesthetic, eg bupivacaine, was then dissolved in the SUV solution, followed by lyophilization, hydration and washing with hyperosmotic saline in order to remove free bupivacaine.

[0005] To obtain local anesthetics in the form of GMV liposomes, a thin lipid film was obtained by dissolving the lipid in a solvent, removing the solvent, and hydrating using an ammonium sulfate solution to obtain MLV vesicles. The MLV vesicles were then homogenized to give SUV vesicles, which were freeze-thawed to give GMV liposomes. The external liposomal medium was replaced to create a gradient, an anesthetic such as bupivacaine was actively loaded into GMV liposomes, and unencapsulated bupivacaine was removed.

[0006] To obtain local anesthetics in the form of MVL, bupivacaine, for example, was converted into a suitable salt form so that it could be easily dissolved in an aqueous solution, and then an aqueous solution of bupivacaine was mixed with a lipid component in an organic solvent by mechanical turbulence to obtain an emulsion type water-in-oil. The water-in-oil emulsion was then dispersed in a second aqueous phase to form spherules in the solvent. Finally, after removal of the organic solvent, a local anesthetic was obtained in the form of MVL.

[0007] In 1991, Legros et al. (US patent No. 5244678) disclosed the preparation of liposomal bupivacaine in the form of MLV containing L-a-phosphatidylcholine (EPC) and cholesterol in a molar ratio of 4:3. This group then disclosed the preparation of anesthetics in liposomal form by preparing a lipid film composed of EPC and a non-polar anesthetic, followed by hydration of the lipid film using a pH controlled buffer in which the non-polar anesthetic remains in unloaded form (US Pat. No. 6,149,937). For example, when preparing liposomal bupivacaine, the lipid film is preferably hydrated using a pH 8.1 buffer (pKa value for bupivacaine is 8.1) in which 50% of bupivacaine remains in unloaded form. Legros et al. also disclosed a method for preparing lyophilized compositions of an amphiphilic drug encapsulated in liposomes (WO1997042936), which were obtained by preparing a thin film containing lipid components and an amphiphilic drug composition, in particular bupivacaine, hydrating the thin film using a buffer solution with a pH of 8.1 s obtaining bupivacaine encapsulated in liposomes, lyophilization of bupivacaine encapsulated in liposomes, together with sorbitol as a membrane stabilizer, and then rehydration before use to obtain liposomal bupivacaine in the form of MLV.

[0008] Some of the aforementioned prior art examples fail to achieve high drug uptake, i. e. high drug to lipid ratio. While some of the prior art examples illustrate formulations with expected high drug to lipid ratios, the manufacture of these formulations requires laborious procedures and high manufacturing costs. Therefore, there is an unmet need for improved and simplified manufacturing methods for the preparation of sustained release liposomal local anesthetics.

Brief summary of the present invention

[0009] The present invention relates to a method for preparing a sustained release anesthetic composition comprising one-step lyophilization to form a high uptake lipid structure (HELS) containing a local anesthetic and a lipid mixture containing one or more phospholipids and/or cholesterol and then hydration HELS using a pH-adjusted buffer to produce multilamellar vesicles (MLVs) with trapped local anesthetic and optionally non-captured local anesthetic. This sustained release anesthetic composition provides rapid onset of anesthesia and prolonged duration of action of local anesthesia with minimal toxicity. In some embodiments, the local anesthetic is an amide type anesthetic.

[0010] An exemplary local anesthetic of the present invention is an amide type anesthetic such as ropivacaine. Other local anesthetics that may be used include lidocaine, bupivacaine, and levobupivacaine. In some embodiments, the HELS of the present invention is prepared by dissolving the non-polar ropivacaine, phospholipid, and cholesterol in a solvent system, e.g., t-butshall alone or t-butanol/water as a co-solvent, followed by removal of the solvent system using a lyophilization technique. In some embodiments, the ropivacaine composition is prepared by hydration of HELS using a pharmaceutically acceptable buffer at a pH greater than 5.5. Theoretically, unloaded ropivacaine is 0.8% of available ropivacaine at pH 6.0 based on pKa calculation (ropivacaine pKa value is 8.1). However, when buffer pH 6.0 is selected as the hydration solution, the binding efficiency (AE) of the resulting anesthetic composition is over 64%, demonstrating that the percentage of unloaded amide-type anesthetic does not significantly contribute to AE.

[0011] However, to adjust the ratio of entrapped anesthetic to non-entrapped anesthetic in the MLV vesicles of the anesthetic composition, the pH of a pharmaceutically acceptable buffer solution can be adjusted. In certain embodiments, the molar ratio of amide-type anesthetic to phospholipid (moldrug:molphospholipid) in the MLV with the captured amide-type anesthetic of the anesthetic composition is at least 0.5:1 and can provide a subject in need thereof with a sufficient amount of amide-type anesthetic to in order to prolong the duration of anesthesia after topical administration in vivo. In addition, limiting the amount of uncaptured amide-type anesthetic can result in a rapid onset of anesthesia with a minimized impact on maximum plasma concentration (C _max ).

[0012] Other objects, advantages and new features of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings.

Brief description of the drawings

[0013] FIG. 1 is a graph showing the plasma concentration of ropivacaine in rats following subcutaneous (SC) injection of a ropivacaine composition (hydrated with pH 5.5 histidine solution, solid square; hydrated with pH 6.0 histidine solution, solid triangle; and hydrated with pH 6.5 histidine solution, solid circle) or after SC injection of non-formulated ropivacaine (open diamond) (all data presented as mean ± standard deviation (SD));

[0014] in FIG. 2A and 2B are a series of graphs showing the effect after SC administration of the formulation of ropivacaine (circle), ropivacaine alone (square) and vehicle (triangle) on mouse paw withdrawal thresholds when exposed to mechanical stimuli (all data shown as mean ± standard error (SEM)); in fig. 2A is a plot of paw withdrawal threshold versus time (g); in fig. 2B is a graph of mechanical pain threshold (%) versus time; And

[0015] in FIG. 3A and 3B are a series of graphs showing the anesthetic effect over time after a single intradermal (1C) injection of a ropivacaine formulation compared to the same dose of ropivacaine (all data are presented as mean±SEM); in fig. 3A, the anesthetic effect is illustrated in a cohort of guinea pigs that received a dose of 3.0 mg per papule 1C of an injection of ropivacaine composition (solid square) or ropivacaine alone (open square); in fig. 3B, the anesthetic effect is illustrated in a cohort of guinea pigs that received a dose of 1.5 mg per papule 1C of an injection of either ropivacaine formulation (solid triangle) or ropivacaine alone (open triangle).

Detailed disclosure of the present invention

[0016] The following terms used above and throughout this disclosure, unless otherwise indicated, should be understood as having the following meanings.

[0017] In the context of the present invention, the singular includes the plural, unless the context clearly indicates otherwise.

[0018] All numbers herein can be understood as modified by "approximately", which, when referring to a measured value such as amount, duration of time, and the like, is meant to cover deviations of ±10%, preferably ±5%, more preferably ± 1% and even more preferably ± 0.1% of the indicated value, since such deviations are suitable for obtaining the desired amount of drug, unless otherwise indicated.

[0019] "Binding Efficiency" (AE) is the amount of drug entrapped in multilamellar vesicles (MLVs) in the anesthetic composition and is calculated from the ratio of the amount of drug entrapped in the MLV to the amount of drug in the original anesthetic composition. Captured drug MLVs can be prepared by methods known in the art, in accordance with the physical properties of MLVs and general knowledge in the art. Preferably, drug-entrapped MLV can be obtained by separating the non-capturing drug from the anesthetic composition using centrifugation techniques, e.g., conventional centrifugation, density gradient centrifugation, differential centrifugation, or using filtration techniques, e.g., diafiltration, gel filtration, membrane filtration.

Multilayer vesicle

[0020] In the context of the present invention, the term “multilayer vesicle (MLV)” or “multilayer vesicle (MLV)” refers to a particle characterized in that it has an internal water space separated from the external environment by a membrane of one or more bilayers forming a vesicle . Bilayer membranes of multilamellar vesicles are usually formed by lipids, i.e. amphiphilic molecules of synthetic or natural origin, which contain spatially separated hydrophobic and hydrophilic domains. According to certain embodiments of the present invention, the multilamellar vesicle is formed by more than one layer of a lipid bilayer membrane.

[0021] In general, MLV bilayer membranes comprise a lipid mixture typically containing dialiphatic chain lipids such as phospholipids, diglycerides, dialiphatic glycolipids, single lipids such as sphingomyelin and glycosphingolipid, steroids such as cholesterol and its derivatives, and their combinations. Examples of phospholipids of the present invention include, without limitation, 1,2-dilauroyl-from-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-from-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1.2 -distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dimyristoyl-sn-glycero-3- phospho-(1'-rac-glycerol) (sodium salt) (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DPPG), 1- palmitoyl-2-stearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (PSPG), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac- glycerol) (sodium salt) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dimyristoyl-sn-glycero-3-phospho- L-serine (sodium salt) (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS), 1,2-di stearoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DSPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dimyristoyl-sn- glycero-3-phosphate (sodium salt) (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA), 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt ) (DSPA), 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl -sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dipalmitoyl-sn-glycero-3-phospho-(1'-myoinositol) (ammonium salt) (DPPI), 1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (DSPI), 1,2-dioleoyl -sn-glycero-3-phospho-(1'-myoinositol) (ammonium salt) (DOPI), cardiolipin, L-α-phosphatidylcholine (EPC) and L-α-phosphatidylethanolamine (EPE).

Local anesthetics

[0022] The term "local anesthetics" refers to one or more groups of substances that cause loss of sensation in a limited area in a subject, caused by a decrease in excitation in nerve endings or inhibition of the transmission of nerve excitation in peripheral nerves. In some embodiments, local anesthetics are amide type anesthetics. The typical structure of an amide type anesthetic contains a lipophilic moiety and a hydrophilic moiety, which are connected via a -NHCO- bond near the center of the molecule. Suitable amide-type anesthetics include, without limitation, lidocaine, bupivacaine, levobupivacaine, ropivacaine, mepivacaine, pyrrocaine, articaine, and prilocaine. In certain embodiments, the amide type anesthetic is a ropivacaine base.

High capture lipid structure

[0023] The term "high entrapment lipid structure" (HELS) refers to a solid phase lyophilized lozenge, a non-film solid mass, or a dried powder containing a lipid mixture and one or more amide-type anesthetics that can be formulated, stored for a long time. time to extend the shelf life of the composition and hydrate immediately before clinical use. The lipid mixture described above may contain one or more phospholipids without cholesterol, or may contain one or more phospholipids with a cholesterol content in mole percent of not more than 50% relative to the amount of the entire lipid mixture. Alternatively, the mole percent cholesterol content, based on the lipid blend, is from about 0% to about 50%, and optionally from about 33% to about 40%. According to some variants of implementation of the present invention, the phospholipid(s) and cholesterol are contained in a molar ratio of from 1:1 to 3:1.

[0024] HELS can be prepared by 1) dissolving a lipid mixture and one or more amide-type anesthetics in a solvent system to form a liquid structure containing one or more solvents to form a homogeneous solution, and 2) removing the solvent(s) to solidify the lipid formulation. mixtures and anesthetic(s) of the amide type. The removal of the solvent can be carried out by known methods such as freeze drying (lyophilization) or spray drying. Examples of solvent systems suitable for freeze drying include, but are not limited to, t-butanol and t-butanol/water co-solvent systems with other non-aqueous solvents such as acetone, acetonitrile, ethanol, n-propanol, isopropanol, n-butanol, methanol, dichloromethane, dimethyl sulfoxide and carbon tetrachloride, or without them. Examples of solvent systems suitable for spray drying include, but are not limited to, water, ethanol, methanol, chloroform, dichloromethane, diethyl ether, carbon tetrachloride, ethyl acetate, and dioxane.

Anesthetic composition

[0025] The term "anesthetic composition" refers to a multilamellar vesicle (MLV) product suitable for topical administration. In certain embodiments, the anesthetic composition comprises an amide-type anesthetic captured by MLV as well as a non-captured amide-type anesthetic. The term "capture" or "trapping" refers to an MLV bilayer membrane that encapsulates, encapsulates, or binds to the target drug. Captured amide-type anesthetic MLV vesicles can be prepared by methods known in the art, preferably by separating the uncaptured amide-type anesthetic from the anesthetic composition using centrifugation methods, e.g., conventional centrifugation, density gradient centrifugation, differential centrifugation, or by filtration methods, e.g. diafiltration, gel filtration, membrane filtration. The size distribution of MLV vesicles with an entrapped amide-type anesthetic of the present invention can be determined by methods known in the art. An exemplary particle size of an MLV with an entrapped amide type anesthetic is at least 1 µm and optionally greater than 5 µm, such as in the range of 5 µm to 50 µm or 10 µm to 25 µm. Alternatively, the median diameter (D50) of MLV vesicles with captured amide-type anesthetic composition of the anesthetic composition is at least 1 µm and optionally greater than 5 µm, such as in the range of 5 to 50 µm or 10 to 25 µm.

[0026] In order to obtain an anesthetic composition for use, the HELS structure is hydrated with an aqueous buffer solution at a pH value greater than 5.5. In some embodiments, the aqueous buffer solution has a pH value in the range of 5.5 to 8.0 and optionally 6.0 to 7.5.

[0027] Suitable aqueous buffer solutions of the present invention include, without limitation, citrate, acetate, malate, piperazine, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), histidine, bis-tris, phosphate, ethanolamine, N-(2 -acetamido)iminodiacetic acid (ADA), carbonate, N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 1,4-piperazinediethanesulfonic acid (PIPES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), imidazole , N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), triethanolamine, lysine, tris, and glycylglycine. The amount of uncaptured amide-type anesthetic in the composition can be controlled based on the partition coefficient of the anesthetic by selecting an appropriate pH value for the aqueous buffer solution based on the clinical indication and the total injection dose.

[0028] In some embodiments, the aqueous buffer contains histidine at a concentration ranging from 1 mM to 200 mM, 10 mM to 150 mM, or 40 mM to 120 mM.

[0029] The amount of uncaptured amide-type anesthetic depends on the binding efficiency (AE) of the anesthetic composition, which is determined by the centrifugation method. Mathematically, the amount of uncaptured amide-type anesthetic is expressed as follows:

[0030]

[0031] where A _uncaptured is the amount of uncaptured amide type anesthetic; And _total is the total amount of amide-type anesthetic in the anesthetic composition; and AE is obtained by dividing the amount of amide-type anesthetic entrapped in MLV vesicles by the total amount of amide-type anesthetic in the anesthetic composition. The AE according to the present invention is at least 60% and optionally from 70% to 95%.

[0032] The molar ratio of amide-type anesthetic to phospholipid (mol of drug:mol _{of phospholipid} , D:PL) of MLV vesicles with captured amide-type anesthetic is preferably at least 0.5:1, including without limitation 0.7:1, 0, 9:1, 1.2:1, or 1.4:1, and the median diameter (D50) of MLV vesicles with captured amide-type anesthetic is preferably at least 1 µm, such as at least 5 µm, and optionally ranges from 5 µm to 20 µm or 5 µm to 15 µm.

[0033] The concentration of the amide-type anesthetic in the anesthetic composition must be above 2 mg/ml to achieve a clinical therapeutic effect. Suitable concentrations of the amide type anesthetic include, without limitation, 2 mg/ml to 30 mg/ml and 10 mg/ml to 20 mg/ml. The limited amount of uncaptured anesthetic in the anesthetic compositions of the present invention may provide the advantage of achieving a higher maximum tolerated dose (depending on the plasma concentration of anesthetic that causes central nervous system and cardiovascular toxicity) and can be used to ensure fast action. In some embodiments, the C _max after administration of the ropivacaine composition is 16.7% of the C _max after administration of ropivacaine not in formulation form, indicating that 6 times the permitted clinical dose can be used within the safety window of this anesthetic.

[0034] For clinical use, AE according to certain embodiments of the present invention is in the range of 70% to 95%. The remaining MLVs with the captured amide-type anesthetic act as a depot for the gradual release of the amide-type anesthetic into the local environment so as to maintain a therapeutically effective dose at the local site. In some embodiments, the half-life of ropivacaine produced by a single SC administration of a ropivacaine composition of the present invention is extended by at least 10-fold compared to the half-life of non-composition ropivacaine. The duration of the anesthetic effect after administration of the ropivacaine composition of the present invention is significantly longer than the duration of the anesthetic effect of ropivacaine not in the form of the composition.

[0035] The present disclosure will be further described based on the following specific non-limiting examples.

Examples

[0036] The following examples illustrate the preparation and properties of certain embodiments of the present invention.

Example 1

Preparation of Ropivacaine Compositions

[0037] HSPC and DMPC were purchased from NOF Corporation. Cholesterol was purchased from Sigma-Aldrich and ropivacaine was purchased from Apollo Scientific or Dishman. All other chemicals were purchased from Sigma-Aldrich.

[0038] To obtain several HELS, various lipid mixtures with ropivacaine were used in the following molar ratios: HSPC: cholesterol: ropivacaine = 1.5: 1: 2.2, HSPC: cholesterol: ropivacaine = 2: 1: 2.9, OMPC: cholesterol:ropivacaine = 2:1:2.9; and OMRS:OPPO:cholesterol:ropivacaine = 1.85:0.15:1:2.9. The lipids and ropivacaine were mixed and then dissolved in t-butanol or mpem-butanol/water as a co-solvent (1/1, v/v) to form liquid structures. Each sample of liquid structures was frozen for 30 to 60 minutes and then lyophilized overnight to obtain HELS in the form of a lyophilized cake.

[0039] To obtain lipid structures for use as a filler control, a lipid mixture in a molar ratio of OMPC:cholesterol = 2:1 was weighed and then dissolved in tert-butanol. The resulting sample was frozen for 60 minutes and then lyophilized overnight to obtain a filler in the form of a lyophilized cake.

[0040] Lyophilized cakes were hydrated with various buffers at various pH values at suitable temperatures (e.g., above 25°C/ambient temperature (AT) for DMPC and above 60°C for HSPC) for 2 to 10 minutes to obtain respectively compositions ropivacaine and excipient compositions.

Example 2

Study of characteristics of ropivacaine formulations

[0041] The binding efficiency (AE) of each of the above preparations was determined as follows. Two hundred microliters of each formulation of ropivacaine was transferred to a centrifuge and spun for 5 minutes at 3000×g at 4°C. After separation of the supernatant, MLV vesicles with captured ropivacaine were obtained and resuspended to a final volume of 200 μl. A reference optical density standard was established for each drug substance (eg, ropivacaine) based on known concentration solutions of the drug substance to be tested. The drug amounts of both the original formulation of ropivacaine and the MLV vesicles with captured ropivacaine were measured using an ultraviolet/visible (UV/visible) spectrophotometer. AE is the ratio of the amount of drug in the MLV with captured ropivacaine to the amount of drug in the ropivacaine formulation. The D:PL of MLV vesicles with captured ropivacaine was calculated by multiplying the D:PL of the HELS structure by AE. A summary of the results is given in Table 1.

[0042] The particle size of each composition of ropivacaine was measured using a laser diffraction analyzer (LA-950V2, Horiba). The median diameter (D50) of ropivacaine-entrapped MLV vesicles formed by hydration of HELS (OMPC:cholesterol = 2:1) using 50 mM histidine buffer (pH 6.5) was 11.1 ± 0.3 µm (n=3) .

[0043]

Example 3

Pharmacokinetic study of ropivacaine formulations

[0044] For the pharmacokinetic (PK) study, female Sprague-Dawley cannulated jugular vein (JVC) rats were used. The rats were kept in a holding chamber that operated on a daily cycle of 12 hours light/12 hours dark and which provided free access to water and food. Ropivacaine formulations were prepared according to Example 1 where OMPC:cholesterol:ropivacaine = 2:1:2.9 HELS structures were hydrated using 50 mM histidine buffers at pH 5.5, 6.0 and 6.5, respectively. Ropivacaine not in the form of a composition was prepared by dissolving ropivacaine hydrochloride monohydrate in 0.9% NaCl to 24.0 mg/ml. The in vivo PK profiles of the respective liposomal formulations of ropivacaine and non-formulated ropivacaine administered to groups of rats (n=3 or 4 per group) were compared after subcutaneous (SC) injection at a dose of 20.0 mg/kg ropivacaine. Blood samples were collected at 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours and 72 hours after injection. Plasma samples were obtained by centrifugation and stored frozen at −80° C. until analysis. The PK data obtained from the samples were analyzed using a non-compartmental model (WinNonlin® software). The RK parameters derived from this model are shown in Table 2.

[0045]

[0046] The C _max of the ropivacaine composition was lower when the pH of the hydration solution was more alkaline. Compared to the non-formulated ropivacaine group, C _max was 55.5% for the ropivacaine formulation hydrated with a pH 5.5 histidine solution, 35.5% for the ropivacaine formulation hydrated with a pH 6 histidine solution. 0, and 16.7% for a ropivacaine formulation hydrated using a histidine solution pH 6.5. The half-life (T _1/2 ) for all three formulations of ropivacaine was significantly prolonged compared to the T _1/2 of ropivacaine not in the form of a composition. Based on the area under the curve (AUC _0-t ), from 84.6% to 90.8% of ropivacaine was released within 72 hours after injection of the ropivacaine composition. The results of the PK study are shown in FIG. 1. After administration of the same dose, plasma ropivacaine could be detected up to 72 hours in all ropivacaine formulation groups, however ropivacaine could not be detected in plasma after 24 hours in the non-formulation ropivacaine group.

Example 4

Anesthetic effect in a paw dissection mouse model

[0047] Wild-type male C57/BL6 mice (8 weeks old, Envigo) were used to evaluate anesthetic efficacy after paw dissection as described in Anesthesiology. 2003 Oct; 99(4): 1023-7 and J Nemo sci Methods. 1994 Jul; 53(1): 55-63. The mouse chamber was operated on a 12-hour light/12-hour dark cycle, ensuring lights were turned off and preventing researchers and technicians from entering the mouse chamber during the dark cycle. A ropivacaine composition and vehicle were prepared according to Example 1, where both the HELS structure with a ratio of OMPC:cholesterol:ropivacaine = 2:1:2.9 and the lipid structure of the vehicle with a ratio of DMPC:cholesterol = 2:1 were hydrated using 50 mM histidine buffer at pH 6.0. Ropivacaine not in the form of a composition was obtained by dissolving ropivacaine in a 9.4% sucrose solution containing 0.1 Η HCl, in an amount of 18.3 mg/ml. An in vivo formulation efficacy study of ropivacaine, non-formulated ropivacaine and vehicle (n=8 per group) was compared after SC injection after paw dissection at a dose of 0.18 mg ropivacaine per wound.

[0048] Before the operation, the initial (T=-2 hours) thresholds of sensitivity to pain caused by mechanical action (von Frey) were set for 32 mice; baseline sensitivity thresholds were measured on the left hind paw of mice. All 32 mice had a foot incision (5 mm long and 5 mm deep) on their left hind paw. Two hours after surgery (T=0 hours), the mechanical pain threshold of each mouse was assessed again, and mechanical allodynia was confirmed in each mouse. Thirty-two mice were randomized into 4 groups (8 mice per group). When anesthetized with 2.5% isoflurane anesthesia, each mouse received a SC injection of vehicle (10 µl), ropivacaine formulation (10 µl at 18.3 mg/ml), or unformulated ropivacaine (10 µl at 18.3 mg /ml). The 50% paw withdrawal threshold of each mouse was obtained using the up and down method at baseline (-2) and at target times (0, 1, 2, 3, 4, 5, 6, 8, and 24 hours). ) after treatment with SC injection.

[0049] The anesthetic efficacy of a formulation of ropivacaine (circle), ropivacaine not in formulation form (square) and vehicle (triangle) after paw dissection is shown in FIG. 2A and 2B. The average 50% paw withdrawal threshold for each treatment group was plotted; data, represented as paw withdrawal threshold (g), was plotted against time (FIG. 2A). In order to account for baseline mechanical pain thresholds in individual mice, the 50% paw withdrawal thresholds of each mouse after surgery and treatment were normalized to its baseline 50% paw withdrawal threshold (T=-2 hours) . The mean normalized 50% paw withdrawal threshold for each treatment group was plotted; data, presented as % change in mechanical pain threshold relative to baseline thresholds, was plotted against time (FIG. 2B). The onset of the anesthetic effect of the ropivacaine formulation and non-formulated ropivacaine formulation after administration was similar, with the withdrawal threshold increasing from 0.04 g to 0.26 g and 0.22 g, respectively, at the first time point (T=1 hour). The ropivacaine formulation group had the largest (~88%) and longest (at least 5 hours) anesthetic effect, and the unformulated ropivacaine group also experienced some degree of pain relief compared to the vehicle group. .

Example 5

Anesthetic effect in guinea pigs using a modified pinprick model in the papule from 1C injection

[0050] Male guinea pigs (8 weeks old, about 500 g, Charles River Laboratories) were used to evaluate anesthetic efficacy as described in J Pharmacol Exp Ther. 1945; 85:78-84. All guinea pigs were housed in group cages at 2 animals per cage with unlimited access to guinea pig food (Healthy Pet®) and water to ensure proper care and nutrition. Conditions were maintained at 65-75°F (~18-23°C) with a daily cycle of 12 hours light/12 hours dark. After an initial acclimation period to laboratory conditions of 12 days, the guinea pigs were randomly assigned #1 - #8. The composition of ropivacaine was prepared according to Example 1, where HELS with a ratio of OMPC:cholesterol:ropivacaine = 2:1:2.9 was hydrated using 50 mm histidine buffer at pH 6.0. Ropivacaine not in the form of a composition was obtained by dissolving ropivacaine hydrochloride monohydrate in water of the highest purity to 20.5 mg/ml.

[0051] The results of an in vivo efficacy study in guinea pigs (n=4 or 6 per group) were compared for the composition of ropivacaine and ropivacaine not in the form of a composition after intradermal (1C) injection at a dose of 3.0 mg of ropivacaine per papule from 1C injection and 1 .5 mg ropivacaine per papule from 1C injection, respectively. The backs of the guinea pigs were shaved one day before the experiment. On the day of the experiment, four areas were drawn on the back before drug administration, and the sensitivity of these areas was determined by pinprick. Each animal received four designated formulations per back, which created 4 papules, respectively. The reaction to pinpricks at the injection site was tested at time points 0 min, 15 min, 1 hour, 2 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours and 23 hours after injection of the composition. Pin pricks were made first in the control area outside the papule at each time point. After observing the animal's normal response to a pinprick outside the papule, six pricks were made inside the papule, and the pricks to which the guinea pig did not respond were recorded as no response. Animals that showed 100% response to all injections were not monitored at other time points.

[0052] The anesthetic effects were determined for the ropivacaine formulation group compared to the ropivacaine non-composition group at the same dose, and the results are shown in FIG. 3A and 3B. The onset of anesthesia for both the ropivacaine formulation and non-formulation ropivacaine, at doses of both 3.0 mg (Fig. 3A) and 1.5 mg ropivacaine (Fig. 3B), was observed at the first time point, within 15 min. In the ropivacaine formulation group, sustained anesthetic effects were observed compared to what was observed in the non-formulation ropivacaine group for both doses. For a dose of 3 mg ropivacaine per papule from 1C injection, a significantly prolonged anesthetic effect was observed at 10 hours and 12 hours post-injection (p<0.05) for the ropivacaine formulation group compared to the non-formulation ropivacaine group. . For a dose of 1.5 mg ropivacaine per papule from 1C injection, the anesthetic effect was also more prolonged for the ropivacaine formulation group compared to the ropivacaine non-formulation group, and a significant difference (p<0.05) was observed at time times 2 hours, 4 hours and 5 hours after injection.

Claims (15)

1. A method for producing a sustained release anesthetic composition, comprising: dissolving the lipid mixture and at least one amide type anesthetic in the solvent system to form a liquid structure; And removing the solvent system from the liquid structure, where the solvent system contains tert-butanol or tert-butanol/water as a co-solvent, to obtain a high capture lipid structure (HELS); And hydration of HELS using an aqueous buffer solution at pH 5.5 to 8.0; moreover, as a result of hydration of HELS, multilamellar vesicles (MLV) are formed with a captured amide-type anesthetic; moreover, the median diameter of the MLV vesicles with the captured amide-type anesthetic is at least 1 μm; and the molar ratio of amide-type anesthetic to phospholipid in the MLV vesicles with the captured amide-type anesthetic is at least 0.5:1. 2. The method of claim 1, wherein the HELS is in the form of a lozenge, a powder, a solid mass not in the form of a film, or a combination thereof. 3. The method of claim 1, wherein the step of removing the solvent system comprises lyophilization or spray drying of the liquid structure. 4. The method according to claim 1, wherein the lipid mixture contains cholesterol. 5. The method according to p. 4, and the content of cholesterol in mole percent in the lipid mixture is not more than 50%. 6. The method according to claim 4, wherein at least one phospholipid and cholesterol are present in a molar ratio of 1:0.01 to 1:1. 7. The method according to any one of paragraphs. 1-6, wherein at least one amide type anesthetic is lidocaine, bupivacaine, levobupivacaine, ropivacaine, mepivacaine, pyrrocaine, articaine, or prilocaine. 8. The method according to any one of paragraphs. 1-6, wherein at least one amide type anesthetic is a ropivacaine base. 9. The method according to any one of paragraphs. 1-6, and the aqueous buffer solution contains histidine in a concentration in the range from 1 to 200 mm. 10. An anesthetic composition with delayed release for the local administration of a local anesthetic to a subject in need of it, and the composition is obtained by the method according to any one of paragraphs. 1-6, wherein the median diameter of MLV vesicles with trapped amide-type anesthetic in the anesthetic composition is at least 1 µm; and the molar ratio of the amide-type anesthetic to the phospholipid in the MLV vesicles with the captured amide-type anesthetic is at least 0.5:1. 11. The sustained release anesthetic composition of claim 10, wherein at least one amide-type anesthetic is a ropivacaine base. 12. The sustained release anesthetic composition of claim 10, wherein the aqueous buffer solution contains histidine at a concentration ranging from 1 to 200 mM.

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