Sustained-release anesthetic compositions and methods of preparation thereof

Abstract

Provided is an anesthetic composition for locally administrating an amide-type anesthetic into a subject in need thereof. The anesthetic composition has multilamellar vesicles with en trapped amide-type anesthetic prepared by hydrating a highly entrapped lipid structure comprising an amide-type anesthetic and a lipid mixture with an aqueous buffer solution at a pH higher than 5.5. Also provided is a method to prepare an anesthetic composition using a simpler and more feasible process for large-scale manufacture and for providing a high molar ratio of amide-type anesthetic to phospholipid content as compared to the prior art. This anesthetic composition has a prolonged duration of efficacy adapted to drug delivery.

Description

[0001] SUSTAINED-RELEASE ANESTHETIC COMPOSITIONS AND

[0002] METHODS OF PREPARATION THEREOF CROSS-REFERENCE TO RELATED APPLI ATIO S

[0003] [0001 f This application claims the benefit of priority to US Provisional Application No. 62 / 550,983, filed August 28, 201.7, ami US Provisional Application No,

[0004] 62 / 621,730, filed January 25, 2018, each of which is incorporated by reference in sis entirety.

[0005] BACKGROUND

[0006] Technical Field

[0007] {00021 T e present invention relates to a drug delivery system for delivery of a sustatned-release anesthetic composition. The present invention relates to a meihod of preparing the drag delivery system. The present invention also relates to a sustained- release pharmaceutical composition adapted to a drug delivery system, which has a prolonged duration of efficacy.

[0008] Description of Related Art

[0009] 1 0031 Several approaches for developing sustained-released local anesthetics have been repotted, including 1 ) preparing multilamellar liposomal local anesthetics using a dehydraUon-rehydration method (US Patent No, 6,926,905), 2) preparing giant multivesicular (GMV) liposomal local anestheti.es using an ammonium sulfate gradient, loading procedure (US Patent No. 7,357,944), and 3) preparing

[0010] multivesicular liposomal (MVL) local anesthetics using a water-in-oil procedure (US Patent No. 8,182,835).

[0011] {O0O4J To prepare multilamellar liposomal local anesthetics by the dehydration- rehydration method, phospholipid and cholesterol dissolved in tot-butanol are lyophili ed and then hydrated to form multilamellar vesicles (MLVs) and the MLVs are homogenized to obtain small unilamellar vesicles (SUVs). A local anesthetic, for

[0012] I example, bupivacaine, is then dissolved in the SUV solution, followed by

[0013] lyophiiization, hydration, and washing with hyperosmotic saline to remove free bupivacaine.

[0014] [0TO5J To prepare GMV liposomal local anesthetics, a thin lipid film is obtained by dissolving lipid in a solvent, removing the solvent, and hydrating with an ammonium, sulfate solution to form the MLVs, MLVs are then homogenized to obtain. SUV's, which are freeze-tbawed to produce GMVs. The external liposomal medium is replaced to create a gradient, an anesthetic— for example, bupivacaine— is actively loaded into the GMVs, and unencapsu!ated bupivacaine is removed.

[0015] j0fl06| To prepare MVL local anesthetics, bupivacaine, for example, is converted to a suitable salt form s thai it may be readily dissolved in an aqueous solution, and. then the aqoeoits biipivacaine solution is mixed with a lipid component in an organic solvent with mechanical turbulence to form a water-in-oil emulsion. The water-in-oil emulsion is then dispersed into a second aqueous phase to form solvent spherules. Finally, a MVL local anesthetic is obtained after removing the organic solvent, f flfl?! In 1 1, Legros et al. (US Patent No. 5,244,678) disclosed preparing MLV liposomal bupivacaine comprising L-a-phosphatidykholine (EPC) and cholesterol in a molar ratio of 4:3. Afterward, this group disclosed the preparation of liposomal anesthetics by making a lipid film comprised of EPC and an apolar anesthetic, followed by hydrating the lipid film with a pH-contro!led buffer in which the apolar anesthetic remains in an uncharged form (US Patent No. 6,1 9,937). For example, in preparing liposomal bupivacaine, the lipid film is preferably hydrated with a p.H 8.1, buffer (the pKa of bupivacaine is 8.1), which maintains 50% of the bupi vacaine in an uncharged form. Legros et al. also disclosed a process for preparing freeze-dried liposome-eneapsulated amphophilic drug compositions (WO 1997042936), which are obtained by producing a thin film comprising lipid components and an amphophilic drug composition, particularly bupivacaine, hydrating the thin film with H 8.1 buffer solution to form liposome-eneapsuiated bupivacaine, freeze drying the liposome-eneapsuiated bupivacaine together with sorbitol as a membrane stabilizer, and then rehydrating before use to obtain MLV liposomal bupivacaine.

[0016] j 00081 Some of the above-mentioned examples of prior art fail to achieve high entrapment of a drug, i.e., a high drug-to-lipid ratio. Even thoug some of the examples of prior art iliusirate a formulation with a putative high drug-to-lipid ratio, manufacturing these formulations involves tedious procedures and high production costs. There is therefore an unmet need for improved and simplified manufacturing processes for making sustained-release .liposomal local anesthetics.

[0017] SUMMARY

[0018] {0009} The present invention provides a method of preparing a sustained-release anesthetic composition using one-step lyophiiization to obtain a highly entrapped lipid structure (HELS) comprising a local anesthetic and a. lipid mixture including one or more phospholipids and / or cholesterol, and then hydrating the HELS with a pH- con trolled buffer solution to form the multilamellar vesicles (MLV) with entrapped local anesthetic and optionally untrapped local anesthetic. This sustained-release anesthetic composition provides a rapid onset of anesthesia and a prolonged duration of local anesthesia with minimal toxic ity, in some embodiments, the local anesthetic is an amide-type anesthetic.

[0019] f OOi J An exemplary local anesthetic according to the present invention is an amide-type anesthetic, such as ropivacaine. Other local anesthetics that may be used include bdocaine, bupivacaine, and levobupivaeaine. hi some embodiments, the HELS according to the present invention is prepared by dissolving apolar ropi acaine, phospholipid, and cholesterol in a solvent system., e.g., fe / t-butanol alone or a tert- butanol / water cosolveni, followed b removing the solvent system using a lyophilization technique, in some embodiments, a ropivacaine composition is formed by hydrating the HELS with, a pharmaceutically acceptable buffer solution at a pH higher than 5.5. The theoretically uncharged ropivacaine is 0.8% of available ropivacaine at pH 6.0 based on the calculation from / ?Ka (the pK& of ropivacaine is 8. !.), However, when a pH 6.0 buffer is selected as a hydration solution, the association efficiency (AE) of the resulting anesthetic composition is greater than 64%, which demonstrates that the percentage of uncharged amide-type anesthetic does not make a critical contribution to AE.

[0020] [00111 The pH value of a pharmaceutically acceptable buffer solution can nevertheless be selected to adjust the ratio of entrapped anesthetic to uutrapped anesthetic in the MLVs of an anesthetic composi tion. In certain embodiments, the molar ratio of amide-type anesthetic to phospholipid in the M.LV with, entrapped amide-type anesthetic of the anesthetic composition is at least 0.5: 1, and can provide a sufficient amount of the amide-type anesthetic to a subject in need thereof to prolong the duration of anesthesia after in vivo local administration, in addition, limiting the amount of uutrapped amide-type anesthetic can achieve rapid onset anesthesia with minimized maximum plasma, concentration (C ) exposure, f (M)t2| Other objec ti ves, advantages and novel features of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] j 013| Fig. 1 is a graph showing the plasma concentration of ropivacaine in rats after subcutaneous (SC) injection of a ropivacaine composi tion (hydraied with a pH 5.5 histidine solution, closed square; hydraied with a pH 6.0 histidine solution, closed triangle; and hydraied with a pH 6.5 histidine solution, closed circle) or after SC injection of unformulated ropivacaine (open diamond) (all date are shown as mean ± standard deviation (SD)};

[0023] (0014} Figs. 2 A and 2B are a series of graphs depicting the effect after SC administration of a ropivacaiiie composiiion (circle), ropivacaiiie (sqiiare) and vehicle (triangle) on mouse paw withdrawal thresholds to mechanical stimuli (all data are shown as mean standard error of the mean (SEM)); Fig. 2 A is graphical plot of time versus the withdrawal threshold (g); Fig. 2B is a graphical plot of time versus the change in mechanical threshold (%); and

[0024] |001S] Figs. 3 A and 3 B are a series of graphs depicting the anesthetic effect over time after stogie intracutaneous (IC) injection of a ropivacaine composition compared with the same dosage of ropivacaine (all data are shown as mean .-i-. SEM); Fig. 3 A illustrates the anesthetic effect on the guinea pig cohort dosed at 3.0 nig per IC wheal of ropivacaine composition (closed square) or ropivacaine (open square); Fig, 3B illustrates the anesthetic effect on. the guinea pig cohort dosed at 1 .5 mg per IC wheal of ropi vacaine composition (closed triangle) or ropivacaine (open triangle) .

[0025] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] f 00161 As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. (00!7| As used herein, the singular forms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise.

[0027] |WI8j All numbers herein may be understood as modified by "about," which, when, referring to a measurable value such as an amount, temporal duration, and the like, is meant to encompass variations of ±10%, preferably ±5%, more preferably ± 1%, and even more preferably 0.1% from the specified value, as such variations are appropriate to obtain a desired amount of drug, unless other specified.

[0028] |ΘΘ19| "Association efficiency" (AE) represents the amount of drug substance entrapped by multilamellar vesicles (MLVs) in an anesthetic composition and is calculated by the ratio of the drug substance amount entrapped in M'LVs to the drug substance amount in the original anesthetic composition. MLV with entrapped drug can be obtained by known methods in the art according to the physical properties of the MLV and the general knowledge in the field of the art. Preferably; MLV with entrapped drug substance can be obtained by separating the untapped drug from an anesthetic composition using eentrifugation methods, e.g., traditional eentrifugation, density gradient eentrifugation, differential eentrifugation, or by filtration methods, e.g., diafiitration, gel filtration, membrane .filtration.

[0029] Multilamellar vesicle

[0030] {00201 The term "multilamellar vesicle (MLV)" or ''multilamellar vesicles (MLVs)" as used herein refers to a particle characterized by having an aqueous interior space sequestered from an outer .medium by a membrane of one or more bilayers forming a vesicle. Si layer membranes of multilamellar vesicles are typically formed by lipids, i.e., a phipkilic molecules of synthetic or natural origin that comprise spatially separated hydrophobic and hydrophiiic domains. In certain embodiments of the present invention, a multilamellar vesicle forms by more than one layer of lipid bilayer membrane.

[0031] [00 11 i general, bilayer membranes of MLV comprise a lipid mixture typically including dialiphafic chain lipids, such as phospholipids, diglycerides, dialiphatie glycolipids, single lipids such as sphingomyelin and glycospSiingolipid, steroids such as cholesterol and derivates thereof, and combinations thereof. Examples of phospholipids according to the present invention include, but are not limited to, 1,2- dilauroyl-*»-glycero-3-phosphochoIine (DLPC), l,2-dimyristoyl $»-gJycero-3- phosphochotme ( MPC), l,2Ktipaimitoy]-.s7i-giycero~3-phosphocbo.iine (DPPC), 1~ pa'lniitoyl.-2-st.earoyl-5 / i-gi.ycero-3-phosphoeho'iine (PSPC), 1 -palmitoyl-2-oleoy w- glycero~3--phospl)atidylcholine (POPC), l,2-distearoyl-5«~glycero-3-phosphocholine (DSPC), 1 ,2~dioleo l-;s?j-glycefO-3-phosphocholiiie (DOPC), hydrogenated soy phosphatidylchoSise (HS.PC), 1 ,2-dirayristoykw-glycero-3-phospho-(1. '-rac-glycerol) (sodium salt) ( MPG), 1 ^^ijmlraito l-iw-gl cero-S-pbos ho-CJ '-rae-glyceroi) (sodium salt) (DFPG), 1 -palmitoy1-2-stearoyl-i»-glycero-3-phospho-(l '-rac-glycero!) (sodium salt) (PSPG), .1 ,2-distearoyi~^?i-gIycero-3-pho8p!io-( ~rac~glycerol) (sodium salt) (DSPO), 1 ,2Hiioieoyl-v«-glycero-3-p'hospho-(l '-rae-g!ycerol) (DOPG), 1,2- dimyristoyl-A-«-giycero-3-phospho-L~serine (sodium salt) (DMPS), 1 ,2-dipalmitoyl- .¥H-glycero-3 -phospho-L-serine (sodium salt) (DPPS), 1 , 2-distearo I -.v«-glycero~3 - phospho-L-serme (sodium salt) (DSPS), 1 ,2-dioleoyl-,SH-g!ycero~3~phospho-L-serine (DOPS), l,2-dimyristoykw-glycero-3-pbosphate (sodium salt) (DMPA), 1 ,2- dipalmitoyki«-glycero-3~phospbate (sodium salt) {DPPA), 1 ,2~distearoyl~.i«-giyeero~ 3 -phosphate (sodium salt) (DSPA), 1 , 2 ~dioSeoyl-s?i-g.lyceiO-3 -phosphate (sodium salt) (DOPA), i .2-dipaSmitoyS.-,s?i-g1ycero-3 -phosphoethanol amine (DPP E), 1 ~palmitoyl-2- oieoyl-s«-glycero-3-phosphoetbanokmine (POPE), l(2-distearoyI-j?j-giycero--3- phosphoeraanokmine (DSPE), 1 ,2-dioi eoyl-sn-glycero-3-phosphoethanoIamme (DOPE), 1 ,2~dipalmitoyl-vV«~glycero~3-phi0sp o-( 1 '-myoinositol) (ammonium salt) (DPP!), l,2-distearoyl-.vR-glycero-3-phosphoinosito! (ammonium salt) (DSPi), 1,2- dioleoykv»-glycer ^3-phospho^ -Hiyo-mositol) (ammonium salt) (DOPi), cardiolipiii, L-a-phospbatidyicholine (EPC), and L-a-phosphatidyletbauolamine

[0032] (EPE).

[0033] Local anesthetics

[0034] |0022| The term "local anesthetics'*refers to one or more groups of substances causing loss of sensation in a c ircumscribed area of a subject caused by depression of excitation in nerve endings or inhibition of the conduction process in peripheral nerves. In some embodiments, the local anesthetics are amide-type anesthetics. The typical amide-type anesthetic structure contains a lipophilic part and a hydrophilic part that connect by an HCO linkage near the center of the .molecule. Suitable amide-type anesthetics include, but are not limited to, iidocame, bupivacaine, levobupivacainc, ropivacaine, mepivacaine, pyrrocaine, artieaine, and prilocaine. in certain embodiments, the amide-type- anesthetic is ropivacaine base.

[0035] Eighty entrapped lipid structure

[0036] {0023} The term "highly entrapped lipid structure" (HELS) refers to a solid phase lyophilized cake or dried powder containing a lipid mixture and one or more amide- type anesthetics, which can be manufactured, stored long-term so as to extend the shel f life of the composition, and hydrated immediately prior to clinical use. The lipid mixture described above can comprise one or more phospholipids without cholesterol or can comprise one or more phospholipids with a mole percentage of cholesterol of no more than 50% relative to the amount of the total lipid mixture. Alternatively, the mole percentage of cholesterol on the basis of the lipid mixture is from about 0% to about 50%, and optionally from about 33% to about 40%, hi some embodiments of the present invention, the phosphoiipid(s) and cholesterol are at a molar ratio of from 1 :1 to 3: 1.

[0037] |0024J The 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 comprising one or more solvents to form a homogeneous solution, and 2) removing the solvents) to solidify the formulation of the lipid mixture and the amide-type anesthetie(s). Solvent removal can be performed using known techniques such as freeze drying

[0038] (lyophilizatkm) or spray drying. Examples of sol vent systems suitable for freeze drying include, but are not limited to, feri-botanoJ and &? f-butanoi / water cosolvent systems with or without other non-aqueous solvents such as acetone, acetonitrile, ethauol, «-propanol, isopropanol, κ-butanol, .methanol, dichloromethane, dimethyl sulfoxide, and carbon tetrachloride. Examples of solvent systems suitable for spray drying include, but are not limited to, water, ethanol, methanol, chloroform, diehloromemane, diethyl ether, carbon tetrachloride, ethyl acetate, and dioxane. Anesthetic composition

[0039] {00251 The term "anesthetic composition" reiers to a multilamellar vesicle (MLV) product suitable for local administration, in certain embodiments, an anesthetic composition comprises an amide-type an esthetic entrapped by MLVs as well as ontrapped amide-type anesthetic. The term "entrap" or ''entrapment" refers to bilayer membrane of MLVs encapsulating, embedding, or associating with a. target drug substance. The MLVs with entrapped amide-type anesthetic can be obtained by known methods in the art, preferably, by separating the unirapped amide-type anesthetic from anesthetic composition using eentri&gation methods, e.g.. traditional centrifugation. density gradient centrifugation, differential ceniriragation, or by filtration methods, e.g., diafihration, gel filtration, membrane filtration. The size distribution of the MLVs with entrapped amide-type anesthetic according to the present invention can be determined by various known methods in. the art. An exemplary particle size of MLVs with, entrapped amide-type anesthetic is no less than 1. μηι; and optionally, is more than 5 μηι, such as at. a range from 5 μηι to 50 μ*η, or from 10 μιη to 25 μτη. Alternatively, the median diameter (D50) of the MLVs with entrapped amide-type anesthetic of the anesthetic composition is no less than 1 μηι; and, optionally, is more than 5 μιη, such as at a range from 5 pra to 50 pm, or from 10 μπι to 25 μιη,

[0040] (0026} To prepare the anesthetic composition for use. the HBLS is hydrated with an aqueous buffer solution at a pH value higher than 5.5. In some embodiments, the aqueous buffer solution is at a pH range of from 5,5 to 8.0, and optionally of irom 6.0 to 7.5.

[0041] (00271 Suitable aqueous buffer solutions according to the present invention include, but are not limited to, citrate, acetate, raa!aie, piperazine, succinate, 2-(N- morphohiio)ethanesulfonie acid (MES), histidme, bis-tris, phosphate, ethanolamine, / '-(2-acetamido)iminodiacetic acid (ADA), carbonate, N-(2~acetamido)-2- a moethanesulfomc acid (ACES), 1 ,4-piperazjnediethanesult nic acid (PIPES), 3- morpholino>2-hydroxypropanesalfonjc acid (MOPSO), imidazole, A',Af~bis(2- hydroxyethyl)-2-antinoethanesulfonic acid (BES), 4-(2~hydroxyethyi)piperazine-i - ethanesi fonic acid (HEPES), triethanolam ne, lysine, tris, and glycylglycine. The amount of untrapped amide-type anesthetic in the composition can. be adjusted based on the distribution-coefficient of the anesthetic by selecting an appropriate pH value for the aqueous bofier solution based on the clinical indication and the total, injection dosage.

[0042] j0028| hi some embodiments, the aqueous buffer solution comprises histidme at a concentration ranging from .1 mM to 200 mM, from 10 mM to 150 mM, or from 40 niM to 120 mM.

[0043] {0029J The amount of untrapped amide-type anesthetic is a function of the assoc iation efficiency (AE) of the anesthetic composition, which is determined, by a centrifugation method. Mathematically, the amount of unt apped amide-type anesthetic is expressed as follows;

[0044] (0Θ30| Amtra,pped ~ &tatalx(1 ~

[0045] {0031 f wherein Ams is the amount of untrapped amide-type anesthetic; Ακ,κ,ι is the total amount of amide-type anesthetic in the anesthetic composition; and . AE is obiaiired by dividing the amount of amide-type anesthetic entrapped in MLVs by the total amount of amide-rype anesthetic in the anesthetic composition. AE according to the present invention is at least 60%, and, optionally, from 70% to 95%.

[0046] £00321 The molar ratio of amide-type anesthetic to phospholipid

[0047] D:PL) of the MLVs with entrapped amide-type anesthetic is preferably at least 0.5:1, including but not limited to 0,7:1 , 0.9: 1 , 1.2:1, or 1.4: ! , and the median diameter (D50) of the MLVs with entrapped amide-type anesthetic is preferably not less than I pm, for example, not less than 5 um; and, optionally, within a range from 5 μι.π to 20 μηι, or from 5 pm to 1.5 pm.

[0048] jO033{ The amide-type anesthetic concentration of the anesthetic composition should be higher than 2 mg / mL to achieve a clinical therapeutic benefit. Suitable amide-type anesthetic concentrations include but are not limited to from 2 mg / mL to 30 mg mL and from 30 mg mL to 20 mg mL. The restricted amount of tmtrapped anesthetic in the anesthetic compositions of the invention can provide the benefit of achieving a higher maximum tolerance dosage (depending on the plasma anesthetic concentration that causes central nervous system and cardiovascular system toxicity) and can be used to provide rapid-onset efficacy. In some embodiments, the C s after administration of a ropivacaine composition is 16.7% of that after administration of unformulated ropivacaine, which indicates thai a 6-fold higher approved clinical dosage may be used within the safety window of this anesthetic.

[0049] [O034J For clinical use, AE in certain embodiments of the invention ranges from 70% to 95%. The remaking MLVs with entrapped, amide-type anesthetic act as a depot to release the amide-type anesthetic into the local environment gradually in a manner that maintains the therapeutically effective dosage at the local she. In some embodiments, the half-life of ropivacaine derived fern a single SC administration of a ropivacaiiie composition according to the invention is prolonged at least 1 -foid compared to that of unformulated ropivacaiiie. The duration of the anesthetic effect after administration of the ropivacame composition, of the in vention significantl extends beyond that of unformulated ropivacaiiie,

[0050] {0035} The disclosure will he further described with reference to the following specific, non-limiting examples.

[0051] EXAMPLES

[0052] |0036] The following examples illustrate the preparation and properties of certain embodiments of the present invention.

[0053] Example 1

[0054] .Preparation of ropivacatne compositions

[0055] {0037] HSPC and DMPC were purchased from NOP Corporation. Cholesterol was purchased from Sigma- Aklrich and ropivacame was purchased from. Apollo Scientific or Dishman. All other chemicals were purchased from Sigma- Aklrich.

[0056] {0038f To prepare several HELSs, different lipid mixtures with ropivacaiiie at the following molar ratios; HSPC holestero ropivacain:;;1.5:1:2.2,

[0057] HSPC:cholesteroi:ropi.vaca.ine ~ 2: 1 :2.9, DMPC:cholesterol:ropivacame - 2: 1 :2.9. and DMPC;DPPG:cholesteiOi:ropivacaii¾e:::.1.85:0.15: 1 :2.9 were used. The lipids and ropivacame were mixed and then dissolved in fen~butanoi or a r ri-butaiioi / water cosoSvent system (1 / 1 , vol / vol) to form the liquid structures. Each liquid structure sample was frozen for 30 to 60 minutes and then was lyophilized overnight to obtain HELS in a i ophiii ecl cake form.

[0058] {O039J To prepare the lipid structures for the vehicle control, a lipid mixture with a molar ratio of DMPC cholesterol - 2:1 was weighed and then dissolved in ten- butanol. The resulting sample was frozen for 60 minutes and then was SyopMMzed overnight to obtain a lyophilized cake of vehicle. {00401 The lyophiiized cakes were hydrated with different buffers at different pH values at suitable temperatures (for instance, higher than 25¾C / arabiem temperature (AT) for DMPC and higher than. 60*C for HSPC) for 2 to JO minutes to form ropivacaine compositions and vehicle compositions, respectively.

[0059] Example 2

[0060] Characterization of ropivacaine compositions

[0061] {00411 The association efficiency (AE) of each above-described preparation was determined as follows. Two hundred microliters of each ropivacaine composition were transferred to a. centrifuge and spun for 5 mm at 3000 * g at 4°C. After decanting the supernatant, MLVs with entrapped ropivacaine were obtained and re- suspended to a final volume of 200 ΐ,. A reference absorhasiee standard was established for each drug substance (e.g., ropivacaine) based on solutions of the test drug substance of known concentration. The drug amounts of both the original ropivacaine composition and the MLVs with entrapped ropivacaine were measured using an ultravi let / visible (U'V / Vis) spectrophotometer. The AE represents the ratio of the drug amount in the MLVs with entrapped ropivacaine to the drug amount in the ropivacaine composition. The D:PL of MLVs with entrapped ropivacaine was caiculated by multiplying the D;PL of the HELS by AE, A summary of the results is shown in Table 1.

[0062] 10O42'{ The particle size of each ropivacaine composition was measured using a laser diffraction analyzer (LA-950V2, Horiba). The median diameter (DSO) of the MLVs with entrapped ropivacaine formed by hydrat g the .HELS (DM'PCxhoJesterol - 2: 1 > with 50 raM histidine buffer (pH 6.5) was 11.1 ± 0.3 pm (w - 3). 1 (00431 Table 1 . The AE and calculated molar ratio of amide-type anesthetic to phospholipid (D:PL) from various formulations

[0063] Hydration endi!ioii Roptvaeaine concentration Calculated D:PL ratio «f

[0064] AE

[0065] Lipid cottijpositum (mg / mL) of ropivacaine Vtl A's with entrapped iiffc-fi pH cantptn¾t« r<> vaca»»<e fiSPCxholestera -· ! 00 mM citrate buffer in 0.9% NaO solution 5.0 60 17.6 0 2 HSPCxhoiosierol - 100 mM stidinss buffer in 0.9% NaCl solution. 6.0 60 17 Λ 64 0.93 HSP xholesterol÷50 mM phosphate buffer 6.5 60 1 . '? Si 1 .3

[0066] HSPC'xholc¾ierol■·- 50 mM istidme buffer 6.5 60 17.9 93 1.36 HSPCehoiesfero! = 100 mM histidme buflcr in 0.9% NaCl solution 7,0 60 18.3 86 1.25 HSPCxholesterol -- 100 rnM iris buffer in 0.9% NaCl solution 8.0 60 i O i .46 HSPCxhoiesterol - 50 mM istidtne buffer 6.0 60 1 .2 94 1.37 DMPCxhoksteroi 50 rnM isiidiirc buffer 5.5 AT 18.6 71 Ϊ 03 MPCxiwiestcroi 50 mM iiistidme buffer 6,0 AT 17.8 84

[0067] DMP olesterol (coso!veirt) 50 niM iiistid ne buffer 6,0 AT 17. ! 82

[0068] DMPCxholesterol 50 mM histidine buffer 6.5 AT 17.5 90

[0069] Exa mple 3

[0070] Pharmacokinetic study of ropivaeaine compositions

[0071] 00 4) Jugular vein cannulated (JVC) female Sprague-Dawley rats were used for a pharmacokinetic (PK) study. The rats were housed in a holding room that operates on a J 2-hr S.ight / 12-hr dark circadian cycle and thai provides free access to water and food. The ropivaeaine compositions were prepared according to Example Ϊ , wherein HELSs of DMPCtchoiesterokropivacaine:::2: 1 :2.9 were hydrated with 50 raM histid.be buffers at pH 5.5, 6.0, and 6.5, respectively. Unformulated ropivaeaine was prepared by dissolving ropivaeaine hydrochloride monohydrate in 0.9% NaCI to 24.0 mg / niL, The in vivo PK profiles of the respecti ve liposomal ropivaeaine compositions and of unformulated ropivaeaine administered to groups of rats (n ~ 3 or 4 per group) were compared following subcutaneous (SC) injection at a dosage of 20.0 mg / kg of ropivaeaine. Blood samples were collected at 15 tiiin, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, and 72 hours post-injection. Plasma samples were obtained by cenirifugation and were kept frozen at -80°C until analysis. PK data obtained from the samples were analyzed using a noucompartmeiHal model (Win onlin® software). The PK parameters derived from this model are shown in Table 2.

[0072] )0045) Table 2. PK parameters deri ved from rats after single SC administration of ropivaeaine compositions or unformulated ropivaeaine

[0073] optvacaroe eoaaposrtKW

[0074] Hydrated with Hydraied with Hydraied wiili

[0075] Parameter Ropivaeaine

[0076] " pH 5,5"pH 6.0'pH 6.5 ti tidine sohiikui histidine solution hisddins solution ft 4 4 4

[0077] Tw (b) 3.1 -i-. 1.4 33.9 ± 24,3 31. $ 16, 34.9 -i.- Ϊ 6.0 CiMxing / mL) 801 ,8 ± 208.0 444,7 * 74.0 284,5 * 1.3 133.5 ± 34.8

[0078] I :::>:\ t · 1.8 * 0,5 1.7 * 0.6 1 ,5 - 0.6 1.5 A 0.6

[0079] Λ I ;<.';:.: (fax ng / mL) 4035 A 632 3415 142 351 ! ά 462 3665 ά 930

[0080] [0Θ 6| The Cx x of ropi vaeaine composition was lower when the pH value of the hydration solution was more alkaline. Compared to the unformulated ropivaeaine group, the Ctnax was 55,5% for ropivacaine composition hydrated with the pH 5.5 hixtMine solution, 35.5% for ropivacaine composition hydrated with the pH 6,0 histidioe solution, and 16,7% for ropivacaine composition hydrated with the pH 6.5 histidine solution. The half-life (Tin) of ail three ropivacaine compositions was significantly prolonged compared with that of unfo mul ted ropivacaine. Based on ihe area under the curve (AUCCM). 84.6% to 90.8% of the ropivacaine was released 72 hours after injection of the ropivacaine composition. The results of the P study are shown in Fig. 1. After administration of the same dosage, ropivacaine in the plasma could be detected until 72 hours in all ropivacaine composition groups, however, ropivacaine could not be detected in the plasma after 24 hours in the unformulated ropivacaine group.

[0081] Example 4

[0082] Anesthetic effect in a paw incision mouse model

[0083] [0047 j Wild type male C57 / BL6 mice (8-week-oid, Envigo) were used for evaluating the anesthetic efficacy after paw incision as described in Anesthesiology, 2003 Oct: 99(4); 1023-7 and JNeurosci Methods. 1994 Jul; 53(1): 55-63, The mouse holding room, operates on a ! 2-hour light / 12-hour dark circadian cycle to ensure lights are not used and that researchers and technicians do not enter the mouse room during the dark cycle. The ropivacaine composition and vehicle were prepared according to Example 1, wherein a HELS of DMPC:cholesierol:roptvacaine ~ 2:1 :2,9 and a vehicle's lipid structure of D PC:cholesterot™2; 1 were each hydrated with 50 niM hisi'idine buffer at pH 6.0. Unformulated ropivacaine was prepared by dissolving ropivacaine in a 9.4% sucrose solution containing 0.1 N HCl at 18.3 rng / mL. The in vivo efficacy study of the ropivacaine composition, unformulated ropivacaine, and vehicle (n ~ 8 per group) were compared following SC injection after paw incision at the dosage of 0.18 mg ropivacaine per incision. {0Θ 8| Baseline (T ~ -2 hours) mechanical (von Frey) thresholds of 32 mice were taken prior to surgery; baseline thresholds were measured on the mouse's left hind paw. All 32 mice received a -plantar incision (5 mm long and 5 mm depth) on their left hind paw. Two-hoars post -surgery (T~ 0 hour), the mechanical threshold of each mouse was reassessed and the presence of mechanical allodyiita in each mouse was confirmed. Thirty-two mice were randomized into 4 groups (8 mice per group). While anesthetized with 2.5% isollurane anest esia, each mouse received a SC injection of vehicle (10 μΐ,), ropivacaine composition (10 pL f 18.3 mg / mL) or unformulated ropivacaine (10 uL of 18.3 mg / mL). The 50% paw withdrawal threshold of each mouse was obtained using the up-down method at baseline time point (-2), and the designated t me points (0, 1 , 2» 3, 4, 5, 6, 8, and 24 hours) after SC injection treatment.

[0084] {0049J The anesthetic efficac of the ropivacaine composition f circle), unformulated ropivacaine (square) and vehicle (triangle) after paw incision is shown in Figs. 2A and 2B, The average 50% withdrawal threshold for each treatment group was graphed; data presented as a withdrawal threshold (g) were plotted against time {Fig, 2A). To account for variabilit among the baseline mechanical sensitivities of individual mice, each mouse's 50% paw withdrawal thresholds after surgery and treatment were normalized to its own baseline 50% withdrawal threshold (T~-2 hours). The average normalized 50% withdrawal threshold for each treatment group was graphed; data presented as a % change in mechanical threshold relative to baseline thresholds were plotted against time (Fig. 2B), The onset time of ropivacaine composition and unformulated ropivacaine anesthesia after administration was similar, with the withdrawal threshold increased from 0,04 g to 0,26 g and 0.22 g, respectively, at the first time point {T ~ 1 hour), The ropivacaine composition group produced the largest (--88%) and longest (at least 5 hours) analgesic action, and the i? unformulated ropivacaine group also produced some degree of analgesia compared to vehicle group.

[0085] Exa mple S

[0086] A nesthesia effect on guinea pigs using a modified iC wheal pin-prick ttwdel jOflSOJ Male guinea pigs (8-week.s~o.kl, around 500 g, Charles River Laboratories) were employed to evaluate the anesthetic efficacy as described in J Pharmacol Exp Then .1945; 85: 78-84. All guinea pigs were housed in group cages with 2 animals per cage, with Guinea Pig food {.Healthy Pet®) and water ad libitum in order to ensure proper nurturing and enrichment The housing condition was controlled at 65-75°F (~J 8-23°C) with a 12-hour light i 2-hour dark eircadian cycle. After an initial period of acclimatization to laboratory conditions for 12 days, the guinea, pigs were randomly designated as No. 1 to No, 8. The ropivacaine composition was prepared according to Example 1 , wherein a HBLS of DMPCxholestero ropjvacaine -~ 2:1 :2,9 was hydrated with 50 m'M hisiidine buffer at pli 6.0. Unformulated ropivacaine was prepared by dissolving ropivacaine hydrochloride monohydrate in ultrapure water to 20.5 .rag / rnL.

[0087] jOOS!J This in vivo efficacy study of the guinea pigs (n ~ 4 or 6 per group) compared the ropivacaine composition and unformulated ropi acaine following intracutaneous (IC) injection, at a dosage of 3.0 mg of ropivacaine per 1.C wheal and 1 ,5 mg of ropivacaine per IC wheal, respectively. The backs of the guinea pigs were shaved one day before the experiment. On the experimental day, fou areas were drawn on the back before the drug administration and the sensitivity of these areas was determined by a pin-prick. Each animal received four designated formulations on the back, which created 4 wheals, espectively. The reaction to pin-pricks at the injection site was tested at 0 mm, 15 min, I hour, 2 hours, 4 hours, 5 hours, 6 hours, 8 hours, .10 hours, 12 hours, and 23 hours following injection of the formulation. The pin-pricks were applied first to a control area outside the wheal at each time point. After observing the animal's normal reaction to the pin-prick outside fee wheal, six pricks were applied inside the wheal, and the pricks to which the guinea pig failed to react were recorded as non-responses. Animals that displayed a 100% response for ail pricks were not monitored at further time points,

[0088] j 065 1 The anesthetic effects of the ropivacaine composition group compared with the unformulated ropivacaine group at the same dosage were determined and the results are depicted in Figs. 3A and 3B. Onset of anesthesia for both the ropivacaine composition and unformulated ropivacaine at both the 3,0 mg (Fig. 3 A) and 1,5 mg ropivacaine (Fig. 3B) dosages was observed at the first time point, within 15 .min. The ropivacaine composition group exhibited sustained anesthetic effects compared to what was observed for the unformulated ropivacaine group for both dosages. For a dosage of 3 mg of ropivacaine per IC wheal, a significantly sustained anesthetic effect was observed at 1 hours and 12 hours -post injection (p<0.05} for the ropivacaine composition group compared to the unformulated ropivacaine group. For a dosage of .1.5 nig of ropi acaine per IC wheal, the anesthetic effect was also sustained longer for the ropivacaine composition group compared to the unformulated ropivacaine group, and significant differences (p < 0.05} were observed at 2 hours, 4 hours, and 5 hours post-injection.

Claims

WHAT IS CLAIMED Bi1. A method of preparing a sustained-release anesthetic composition, comprising; creating a highly entrapped lipid structure (HELS) comprising;at least one amide-type anesthetic, anda lipid mixture .including at least one phospholipid, andhydrating the HELS with an aqueous buffer solution, at a pH of 5.5 to 8,0; wherein hydrating the HELS forms multilamellar vesicles (MLVs) with entrapped amide-type anesthetic; and wherein the median diameter of the MLVs with entrapped amide-type anesthetic is at least 1 μηι.

2. The method according to claim 1, wherein the HE LS is in a form of a cake, powder, non-film solid bulk, or a combination (hereof.

3. The method according to claim 1 , wherein the molar ratio of amide-type anesthetic to phospholipid in the MLVs with, entrapped amide-type anesthetic is at least 0.5: 1.

4. The method according to claim 1 , wherein the method of creating the HELS comprises:dissolving the lipid mixture and the at least one amide-type anesthetic in a solvent system to form a liquid structure; andremoving the solvent system from the liquid structure.

5. The method according to claim 4, wherein the step of removing the solvent system includes i>¾philizing or spray drying the liquid structure.

6. The method according to claim 4, wherein the solvent system includes ten- butanol or a teri-butanol water cosolvent.

7. The method according to claim 1 , wherein the lipid mixture comprises cholesterol.

8. The method according to claim 7, wherein the mole percentage of cholesterol inthe lipid mixture is not more than 50%.

9. The method according to claim 7, wherein the at least one phospholipid and cholesterol are at a molar ratio of from 1:0.01 to 1 : 1.I 0. The method according to any of claims I to 9, wherein the at least one amide- type anesthetic is Hdocaine, b pjvacaine, levobupivacaine, ropivacaine, mepivacaine, pyrrocaine, articaine, or priloeaine.1 . The method according to any of claims 1 to 9 wherein the at least one amide- type anesthetic is ropivacaiiie base,52. The method according to any of claims 1 to 9, wherein the aqueous buffer solution comprises hisiidine at a concentration ranging from 1 ra to 200 m .

13. A sustained-release anesthetic composition: for locally administering a local anesthetic to a subject in need thereof wherein the composition is prepared by the method according to any of claims 1 to 9 and wherein, the median diameter of the MLVs with entrapped amide-type anesthetic in the anesthetic composition is not less than 1 μηι..

14. The sustained-release anesthetic composition according to claim 13, wherein the molar ratio of amide-type anesthetic to phospholipid in the MLVs with entrapped amide-type anesthetic is not less than 0,5:1 ,15. The sustained-release anesthetic composition according to claim 1.

3. wherein the at least one amide-type anesthetic is ropivacaine base,.

16. The sustained-release anesthetic composition according to claim. 13, wherein the aqueous buffer solution comprises hisiidine at a concentration ranging from J oiM to 200 mM.

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