Sustained-release liposomal anesthetic compositions

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Sustained-Release Liposomal Anesthetic Compositions

Background of the Invention

This invention relates to liposomal formulations of compounds such as drugs. More particularly this invention relates to methods of encapsulating anesthetics in multivesicular liposomes with high efficiency and sustained in vivo rates of release.

to A local anesthetic's duration of action following administration is usually sufficiently long to cover the pain inflicted during most surgical procedures. However, the duration of action is not long enough to cover most post-operative pain, or pain from many invasive diagnostic procedures, or from injuries. Continuous infusion or repeated infiltration of a local anesthetic into a surgical wound, diagnostic port or injury site is impractical.

is Therefore, a sustained-release formulation of a local anesthetic would be useful for pain management, especially in view of the current trend for out-patient surgeries and emergency care centers. Desirably, such formulations are useful in trauma and diagnostic pain, as well.

Several approaches to develop sustained-release formulations of local anesthetics have been described in the literature. For example, polylactic-co-glycolic acid polymer

2o microspheres containing both bupivacaine and dexamethasone have produced long duration of local anesthesia. Crystalline forms of local anesthetics have also been shown to have long duration of action. Lipophilic bupivacaine free-base incorporated into the membranes of multilamellar liposomes and proton-gradient-loaded large unilamellar liposomes have shown efficacy lasting 6 to 11 hours.

Multivesicular liposomes (MVL) are being developed as a lipid-based sustained-release drug delivery system for local, regional or systemic drug delivery. Sustained release of many water-stable drugs encapsulated into MVL has been shown in animal models via intrathecal, subcutaneous, intraperitoneal and epidural routes of administration, as well as in human patients via intracerebroventricular, intrathecal, subcutaneous and epidural routes. A multicenter, randomized phase III clinical trial of a MVL formulation of cytotoxic agent

-iCA 02304096 2000-06-27 cytarabine has shown that this formulation is more efficacious than free cytarabine in treating leptomengial carcinoma.

MVL are defined as liposomes containing multiple non-concentric chambers within each liposome particle, resembling a ‘‘foam-like matrix. Such particles are to be distinguished from multiiamellar vesicles (MLV), also known as a multilamellar liposome, which contain multiple concentric chambers within each liposome particle. A further distinct particle is the unilamellar vesicle (ULV). also known as a unilamellar liposome, which encloses a single internal aqueous compartment. The present invention relates to MVL. The prior an describes the preparation of MVL (Kim cv cd.. Biochim. Biophys. Acta 72S, 3 39-348. 1983).

Many of the cationic biologically active substances-used in MVL encapsulation techniques are used as salts of mcnoprotic minerai acids (for example, as hydrochloride salts). The prior art has used such commonly available mcnoprotic mineral acid salts of cationic biologically active substances for encapsulation into liposomes without any modification into a salt of diprotic or triprotic mineral acid. The prior an has also used organic acids such as citric or glutamic acids to effect encapsulation.

Summan⁷ of the invention

An object of the present invention is to provide sustained-release liposomal anesthetic compositions. In accordance with an aspect of the present invention, there is provided a pharmaceutical composition comprising:

a) a multivesicular liposome comprising at least one type of amphipathic lipid, and at least one type of neutral lipid; and

b) an aqueous phase comprising polyhydroxy carboxylate salts and di- or tri-protic mineral salts of amide-type anesthetics, wherein the aqueous phase is encapsulated within the multivesicular liposome.

In accordance with another aspect of the invention, there is provided a process for preparing a multivesicular liposome-encapsulated anesthetic composition, the process comprising:

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a) forming a “water-in-oil” type emulsion from a first aqueous phase and a volatile organic phase, wherein the first aqueous phase comprises polyhydroxy carboxylate salts and di-or tri-protic mineral salts of amide-type anesthetices, and the volatile organic phase comprises a volatile organic solvent, at least one type of amphipathic lipid, and at least one type of neutral lipid;

b) dispersing the “water-in-oil” type emulsion into a second aqueous phase to form solvent spherules; and

c) removing the volatile organic solvent from the solvent spherules to form a multivesicular liposome-encapsulated amide-type anesthetic suspended in the second aqueous phase.

In accordance with another aspect of the invention, there is provided a method of increasing drug loading in multivesicular liposomes by converting an amide-type anesthetic into a binary salt mixture wherein the two counterions are derived from a polyhydroxy carboxylic acid and a di- or tri-protic mineral acid.

The invention provides local anesthetics encapsulated in multivesicular liposomes (MVL). that is, lipid vesicles having multiple non-concentric internal aqueous chambers having internal membranes distributed as a network throughout the MVL. The chambers contain acids which are effective to enable the encapsulation of certain anesthetics and to modulate the release rate of the encapsulated anesthetics. The invention also provides «

methods of making such compositions, and of providing local anesthesia to subjects by administering the compositions. _t

The prior art has used commonly available monoprotic (for example, hydrochloride or glutamic) salts of biologically active compounds. Tnis has resulted in either unacceptable formulations for encapsulating the biologically active substances in MVL or very low encapsulation efficiency. The invention results from the surprising finding that inclusion of the free base form of anesthetic compounds solublized with phosphoric acid, or conversion of the commonly available hydrochloride salts of anesthetic compounds into phosphate (salt

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PCT/US98/19583 of triprotic minerai acid) or sulfate salts (salt of diprotic mineral acid) for inclusion into MVL results in markedly improved encapsulation efficiency as well as sustained release in biologically relevant media. Polyalcoholic organic acids such as glucuronic or gluconic acid are also included, wherein such acid is co-encapsulated with anesthetics to assist encapsulation and to effect sustained-release of the anesthetic. Surprisingly, polyalcoholic organic acids are superior to non-polyalcoholic organic acids, giving compositions with high encapsulation efficiency and sustained release of anesthetic. Polyalcoholic organic acids greatly improve the encapsulation of anesthetic and the acceptability of the formulation. Sulfate salts and a number of other salts require the inclusion of such acids to form acceptable formulations.

When the encapsulated anesthetic is administered as a single intracutaneous or subcutaneous dose, the duration or'anesthesia and half-lire of the drug at the local injection site is increased as compared to injection of unencapsulated anesthetic. The maximum tolerated dose of the encapsulated anesthetic is also markedly increased in the liposomal formulation over injection of unencapsulated anesthetic.

The major use for the invention is for making sustained-release formulations of biologically active substances that have high diffusion rates through bilaver lipid membranes. Both the use of diprotic and triprotic mineral acid salts of biologically active substances and con-encapsulation of polyalcoholic organic acids enable these difficult-to-encapsulate drugs to be encapsulated easily and released slowly.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Brief Description of the Drawings

Fig. 1A is a graph showing the anesthetic effect (number of non responses to six pinpricks) as a function of time following a single intracutaneous dose of MVL-encapsulated bupivacaine phosphate containing different concentrations of bupivacaine.

Fig. IB is a graph showing the anesthetic effect (number of non responses to six pinpricks) as a function of time following a single intracutaneous dose of unencapsulated bupivacaine hydrochloride at different concentrations.

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Fig. 2 is a graph showing a comparison of the duration of anesthesia for the formulations of Figs. 1A (MVL-encapsulated bupivacaine phosphate, filled circles) and IB (unencapsulated bupivacaine hydrochloride, open circles) as quantified by “time to half maximal response (R₃)” (ordinate) versus concentration of administered dose (abscissa).

Fig. 3A is a graph showing the total amount of bupivacaine (mg) remaining at an injection site up to 72 hours following a single intracutaneous dose of MVL-encapsulated bupivacaine phosphate (filled circles) or unencapsulated bupivacaine hydrochloride (open circles).

Fig. 3B is a graph showing the serum bupivacaine concentrations (pg/mL) up to 72 hours following a single intracutaneous dose of MVL-encapsulated bupivacaine phosphate at a concentration of 1.0 percent (w/v) of bupivacaine (filled circles) or 0.5 percent (w/v) of unencapsulated bupivacaine hydrochloride (open circles).

Detailed Description

Post-operative or post-trauma pain is thought to be most intense in the immediate postoperative or post-injury and subsequent 24-hour period. It is possible that improved control of post-operative pain can decrease pulmonary and gastrointestinal complications and perhaps shorten hospital stay. Systemic opiates commonly used to control pain during this post-operative period can depress pulmonary function and slow gastro-intestinal recovery. Other antinociceptive agents such as non-steroidal anti-inflammatory agent ketorolac tromethamine can increase bleeding and gastrointestinal irritation in this time of stress. Since nociceptive stimuli arising from surgical interventions or traumatic injury are usually local or regional in origin, prolonged local or regional sensory block for pain control is an intriguing concept. Thus, it is believed that improved treatment with local anesthetics involves maintenance of anesthetic level for a prolonged period. Unfortunately, the half-life of many anesthetics is very short after an intraperitoneal (IP), intravenous (IV), intrathecal (IT), intraartricular (IA), intramuscular (IM), or subcutaneous (SC) dose. Therefore, a slowrelease preparation which provides a prolonged and sustained exposure at a therapeutic concentration of a local anesthetic is needed. The present invention is directed to the production, composition, and use of such a preparation.

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Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the an to which this invention belongs. Although methods and materials similar to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described 5 below.

In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Anesthetics io The present invention provides prolonged release of local anesthetics, particularly of the amide-type anesthetics, from MVL following administration of compositions containing the MVL, The invention utilizes a iocal anesthetic encapsulated in MVL. The local anesthetic generally belongs to the class known as the amide-type anesthetics. The name comes from the presence of the amide (-NHCO-) linkage in the central portion of the is molecule. Tlie group linked to the nitrogen end of the amide is a substituted phenyl ring, especially a phenyl ring containing at least one short chain alkyl group, such as methyl, ethyl, propyl or butyl. Examples of such groups include 2-methylphenyl. 2.6-dimethylphenyl, 2ethylphenvl, 2.6-diethylphenyl. and 2-ethyl-6-methylphenvl. If the substituent group is 2.6dimethylpnenyl. the local anesthetics are also referred to as 2.6-xylidide anesthetics.

The group linked to the CO end of the amide linkage is designated as CHR_tR_:. In the foregoing designation. R. is a secondary or ternary alkyl amine such as N-alkyl amine or Ν,Ν-dialkyl amine. Short chain alkyl groups (from one to four carbon atoms) are preferred.

a

Examples include N-methyiamine. N-ethylamine. N-propyiamine, N-butylamine, N,Ndimethylamine, Ν,Ν-dtethylamine. N-cthyl-N-methylamine, and similarly constructed substituents. The three and four member alkyl chains can be of any configuration, that is, straight chain (n-alkyl), or branched (iso-, sec-, or tert-alkyl). Alternatively, R, can be a secondary or tertiary alkyleneamino group, which further links to R_:. For example, R_t and R_: can be linked by a secondary or tertiary nitrogen-containing alkyl chain, to form an Nalkyl substituted piperidine or pyrrolidine ring. In such examples, the N-alkyl group is preferably a short chain (one to four carbon atoms), such as N-methyl, N-ethyl, N-propyl or

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N-butyl, in which the chain can be straight or branched. The R|-R₂ linking substituent can be

2-piperidyl, 2-pyrrolidyI, 3-piperidyl, 3-pyrrolidyI, 4-piperidyl or 4-pynoIidyl. Preferably, the substituent formed when Rl and R2 are linked by a secondary or tertiary nitrogencontaining alkylene chain is 2-piperidyl or 2-pyrrolidyl. The stereochemistry' of the compounds can be either R or S, depending on the most efficient anesthetic activity. For example, commercially available ropivacaine is found in the (S)(-) configuration. Bupivicaine is also found in the form known as levo-bupivacaine. In the foregoing designation, R₂ is either hydrogen, short chain alkyl (one to four carbon atoms) or a secondary or tertiary alkyleneamino chain which links to Rj, as described above.

The amide-type anesthetics which are useful in the present invention are described by the following structure:

¹¹ '

R₃“NH-C-CH_s

<img file="CA2304096C_D0001.tif" />

wherein R,, and R₂ are as described above, and R₃ is an alkyl-substituted phenyl ring, as described above.

Illustrative of the forgoing description ofthe amide-type anesthetics useful in the present invention are, for example, bupivacaine, levo-bupivacaine, mepivacaine, lidocaine, pyrrocaine, prilocaine, and ropivacaine.

The anesthetics should be present in the compositions ofthe invention in concentrations of from about 0.01% to about 5.0% w/v, or preferably from about 0,5% to about 2.0% w/v. The weight percentages are defined as weight of anesthetic per volume of MVL.

The free base forms of local anesthetics ofthe invention can be solublized. Desirably, the water-soluble salt form is formed for their storage and delivery from MVL. The salt form can be introduced to the first aqueous phase of the MVL as such, or can be formed by adding the free base form, and sufficient acid to solublize the anesthetics to the desired extent. The salt can be any pharmaceutically acceptable di- or tri-protic mineral salt, such as the phosphate or sulfate salt. Also useful are the polyhydroxyl carboxylic acid salts of the

<img file="CA2304096C_D0002.tif" />

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WO 99/13865 PCT/US98/19583 anesthetic, such as the tartarate, gluconate or gluconurate salts. Combinations of such salts are preferable as components of the first aqueous phase of the inverttive compositions. ‘Thus, amide-type anesthetics are present in the pharmaceutical compositions of the invention in the form of polvhydroxy carboxylate salts, and di- and tri-protic mineral salts. Preferred embodiments of the invention are those with a binary mixture of amide-type anesthetic salts, one derived from a polyhydroxy carboxylic acid, and the other derived from a di- or tri-protic mineral acid.

Multivesicular Liposomes

The anesthetic compositions of the invention also include multivesicular liposomes (MVL) which encapsulate and provide modulated and sustained release of the anesthetics described above. The MVL are made by the following process. A “water-in-oil” type emulsion containing a non-hvdrohaiic acid salt of any of the anesthetics described above is formed from two immiscible phases, a lipid phase and a first aqueous phase.

The lipid phase is made up of at least one amphipathic lipid and at least one neutral lipid in a volatile organic solvent. The term “amphipathic lipid” refers to molecules having a hydrophilic “head” group and a hydrophobic “tail” group and may have membrane-forming capability. As used herein, amphipathic lipids include those having a net negative charge, a net positive charge, and zwitterionic lipids (having no net charge at their isoelectric point). The term “neutral lipid” refers to oils or fats that have no vesicle-forming capability by themselves, and lack a charged or hydrophilic “head” group. Examples of neutral lipids include, but are not limited to, glycerol esters, glycol esters, tocopherol esters, sterol esters which lack a charged or hydrophilic “head” group, and alkanes and squalenes.

The amphipathic lipid is chosen from a wide range of lipids having a hydrophobic region and a hydrophilic region in the same molecule. Suitable amphipathic lipids are zwitterionic phospholipids, including phosphatidylcholine, phosphatidylethanolamines, sphingomyelins, lysophosphatidylcholines, and lysophosphatidylethanolamines. Also suitable are the anionic amphipathic phospholipids such as phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, phosphatidic acids, and cardiolipins. Also suitable are the cationic

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WO 99/13865 PCT/US98/19583 amphipathic lipids such as acyl tri methylammonium propanes, diacyl dimethylammonium propanes, and stearylamine.

Suitable neutral lipids are triglycerides, propylene glycol esters, ethylene glycol esters, and squalene. Examples of triglycerides useful in the present invention are triolein, tripalmitolein, trimyristolein, trilinolein, tributyrin, tricaproin, tricaprylin, and tricaprin. The fatty chains in the triglycerides useful in the present invention can be all the same, or not all the same (mixed chain triglycerides), including all different. Both saturated and unsaturated fatty chains are useful in the present invention. The propylene glycol esters can be mixed diesters of caprylic and capric acids.

Many types of volatile organic solvents can be used in the present invention, including ethers, esters, halogenated ethers, hydrocarbons, halohydrocarbons. or Freons. For example, diethyl ether, chloroform, tetrahydrofuran. ethyl acetate. Forane. and any combinations thereof are suitable for use in making the anesthetic compositions of the present invention.

Optionally, but highly desirably, other components are included in the lipid phase. Among these are cholesterol or plant sterols.

The first aqueous phase includes an anesthetic, at least one polyhydroxy carboxylic acid, and at least one di- or tri-protic mineral acid. In some embodiments of the invention, also included is hydrochloric acid. Hydrochloric acid is not an essential constituent, but rather is optional and desirable in some embodiments. The di- or tri-protic mineral acids include sulfuric acid, and phosphoric acid. Also included in the first aqueous phase are such poiyhydroxy carboxylic acids as glucuronic acid, gluconic acid, and tartaric acid. The diand tri-protic mineral acids and the polyhydroxy organic acids are present in the first aqueous phase in concentrations of from 0.01 mM to about 0.5 M, or preferably from about 5 mM to about 300 mM. When hydrochloric acid is used, it is present in lower amounts, from about 0.1 mM to about 50 mM, or preferably from about 0.5 mM to about 25 mM.

The lipid phase and first aqueous phase are mixed by mechanical turbulence, such as through use of rotating or vibrating blades, shaking, extrusion through baffled structures or porous pipes, by ultrasound, or by nozzle atomization, to produce a water-in-oil emulsion. Thus, the anesthetics of the invention are encapsulated directly in the first step of MVL manufacture.

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The whole water-in-oil emulsion is then dispersed into a second aqueous phase by means described above, to form solvent spherules suspended in the second aqueous phase. The term “solvent spherules” refers to a microscopic spheroid droplet of organic solvent, within which are suspended multiple smaller droplets of aqueous solution. The resulting solvent spherules therefore contain multiple aqueous droplets with the anesthetic dissolved therein. The second aqueous phase can contain additional components such as glucose, and/or lysine.

The volatile organic solvent is then removed from the spherules, for instance by surface evaporation from the suspension. When the solvent is substantially or completely evaporated. MVL are formed. Gases which can be used for the evaporation include nitrogen, argon, helium, oxygen, hydrogen, and carbon dioxide. Alternatively, the volatile solvent can be removed by sparging, rotary evaporation, or with the use of solvent selective membranes.

Method of Providing Anesthesia

The invention also provides a method of providing regional aesthesia to a subject by administering the claimed anesthetic compositions either intracutaneously, subcutaneously or via a local or regional nerve block. The dosages can be administered either as a nerve block (including to the limit of acting as a motor block), or as a sensory block.

The term “therapeutically effective” as it pertains to the compositions of this invention means that an anesthetic present in the first aqueous phase within the MVL is released in a manner sufficient to achieve a particular level of anesthesia. Exact dosages will vary depending on such factors as the particular anesthetic, as well as patient factors such as age, sex, general condition, and the like. Those of skill in the art can readily take these factors into account and use them to establish effective therapeutic concentrations without resort to undue experimentation.

Generally however, the dosage range appropriate for human use includes the range of from about 20 mg to about 300 mg of total anesthetic. The upper limit is limited by the toxicity of the particular anesthetic, and the lower limit is approximately 10% of the upper limit.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

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Examples

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

Example 1: Manufacture of Bupivacaine-Dhosphate-containing MVL

Bupivacaine hydrochloride (Sigma Chemical Co., St. Louis, MO) was converted into bupivacaine phosphate by initial precipitation of aqueous bupivacaine hydrochloride with IN sodium hydroxide to prepare the free base. The precipitate was extensively washed with water, and then converted into phosphate salt with an equimolar amount of phosphoric acid.

For each batch of the formulation, 5 mL of a discontinuous first aqueous component containing 60 mg/mL of bupivacaine phosphate, 150 mM glucuronic acid. 15 mN hydrochloric acid, and 20 mM phosphoric acid was added ΐο a mixer vessel containing a lipid component containing 5 mL of USP chloroform (Spectrum Chemical Co., Gardena. CA) as solvent, and 18.6 mM l.2-dierucoyl-sn-glycero-3-phosphocholine (DEPC). 4.2 mM dipalmitoyl phosphatidylglycerol (Avanti Polar-Lipids, Inc.. Alabaster, AL) (an anionic amphipathic lipid), 30 mM cholesterol (Avanti Lipids), and 10.8 mM tricapiylin. The immiscible first aqueous component and lipid component were mixed at 16,000 rpm in an Omni-mixer (OMNI International. Gainesville, VA) for 9 minutes. The resulting water-inoil emulsion was transferred to a 50 mL mixing vessel containing 25 mL of a continuous second aqueous component containing 32 mg/mL of glucose and lOmM free-base lysine (Sigma Chemical Co.. St. Louis. MO). The mixture was then mixed for 20 seconds at 4000 rpm in an Omni mixer.

The resulting water-in-oil-in-water double emulsion was transferred to a 1 L Erlenmeyer flask containing 275 mL of the continuous second aqueous phase (glucose, 32 mg/mL; freebase lysine, 40 mM). The chloroform was evaporated for 15 minutes under a constant flow (90 L/min) of nitrogen gas at 37 °C to form MVL particles in suspension. The MVL particles were isolated by centrifugation at 200 x g for 10 minutes, then washed three times with a 0.9 percent (w/v) solution of NaCl. Each batch was stored at 2-8°C and used for subsequent studies within 48 hours.

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Example 2: Recovery of Bupivicaine from Different MVL Formulations

The bupivicaine samples were solublized by adding an equimolar volume of a 1M * solution of the indicated acid and then slowly adding, with stirring, additional water until 60 mg/mL or a clear solution was achieved. The pH was then adjusted to approximately 5. The 5 final bupivicaine concentration was determined by HPLC against an internal standard.

For each formulation attempt, the first aqueous phase solution contained the bupivicaine counterion at 60 mg bupivicaine per mL, or the limit of solubility of the bupivicaine counterion, at pH 5. Other parameters for MVL manufacture was as described above. Recover}’ refers to percent of bupivicaine in counterion solution encapsulated and recovered to in final MVL product. For study 2, the first aqueous phase also contained 150 mM glucuronic acid. The results are shown in Table 1.

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Table 1- Recovery of MVL-encapsulated bupivicaine from Formulations Containing Various Acids

<td> Bupivicaine (mg/mL)</td><td> acid included</td><td> additional acid</td><td> recovery (%)</td>

<td> Study 1</td><td></td><td></td><td></td>

<td> 60</td><td> phosphoric</td><td></td><td> 5.7</td>

<td> 60</td><td> sulfuric</td><td></td><td> (clumped)</td>

<td> 23</td><td> nitric</td><td></td><td> no MVL formed</td>

<td> 40</td><td> hydrochloric</td><td></td><td> no MVL formed</td>

<td> 26</td><td> glucuronic</td><td></td><td> 34</td>

<td> 60</td><td> tartaric</td><td></td><td> clumped</td>

<td> 41</td><td> acetic</td><td></td><td> no MVL formed</td>

<td> 2.2</td><td> perchloric</td><td></td><td> no MVL formed</td>

<td> Study 2</td><td></td><td></td><td></td>

<td> 60</td><td> phosphoric</td><td> 150 mM glucuronic</td><td> 35</td>

<td> 60</td><td> sulfuric</td><td> 150 mM glucuronic</td><td> 16</td>

<td> 23</td><td> nitric</td><td> 150 mM glucuronic</td><td> 45</td>

<td> 40</td><td> hydrochloric</td><td> 150 mM glucuronic</td><td> 16</td>

<td> 26</td><td> glucuronic</td><td> 150 mM glucuronic</td><td> 48</td>

<td> 60</td><td> tartaric</td><td> 150 mM glucuronic</td><td> 20</td>

<td> 41</td><td> acetic</td><td> 150 mM glucuronic</td><td> 18</td>

<td> 2.2</td><td> perchloric</td><td> 150 mM glucuronic</td><td> no MVL formed</td>

<td> 60</td><td> citric</td><td> 150 mM glucuronic</td><td> 13</td>

<td> 60</td><td> malic</td><td> 150 mM glucuronic</td><td> 19</td>

<td> 60</td><td> succinic</td><td> 150 mM glucuronic</td><td> 20</td>

The results in Table I demonstrate clearly that the addition of a polyhydroxy organic acid (in this case, glucuronic acid) in addition to one of a number of other acids, including

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WO 99/13865 PCT/US98/19583 triprotic mineral acids such as phosphoric acid, or polyhydroxy organic acids such as glucuronic acid provides a remarkable synergistic effect. This surprising discovery leads to higher loading and recovery of the MVL of the invention than previously found.

Example 3: In vivo Animal Studies using Intracutaneous Injections

Male guinea pigs weighing 800-1000 grams (Harlan Sprague-Dawley, San Diego, CA) were used for efficacy studies. Male guinea pigs (Harlan-Sprague-Dawlev) weighing 400600 grams were used for pharmacokinetic studies. The animals were housed, 1 per cage, in a temperature-controlled environment with alternating 12-hour periods of light and darkness io and given unrestricted access to food and water. Prior to each study, animals were habituated to the environment for at least 7 days. Female CD1 mice (Sprague-Dawley) weighing 22-28 grams were used for determination of maximum tolerated dose (MTD) All animals were maintained in accordance with guidelines of the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research

Council.

The formulations of MVL-encapsulated bupivacaine and bupivacaine hydrochloride prepared as described above were diluted in normal saline so that a constant volume of 1 mL contained a dose at concentrations of 2.1%. 1.0%, or 0.5% (w/v) bupivacaine. Concentrations were confirmed by solubilizing a 50μ1 aliquot of the MVL formulation in 1 20 mL of isopropyl alcohol followed by dilution in water and assay by a previously published HPLC method as described (P. Le Guevello et ai. J. Chromatography 622:2M-290. 1993). The HPLC analysis of the MVL formulations revealed that less than 5% of total bupivacaine was present in the formulation as unencapsulated bupivacaine.

Infiltration anesthesia studies were performed in the test guinea pigs using a modified intracutaneous wheal pin-prick model as described (R. H. de Jong et al., Anesth. Analog 59:401-5,1980). On the day preceding the experiment, hairs on the backs of the animals were clipped. Each animal received either a dose of MVL-encapsulated bupivacaine (concentrations of 0.5,1.0 or 2.1 percent (w/v) bupivacaine) or unencapsulated bupivacaine (concentrations of 0.25, 0.5, 0.75 or 1.0 (w/v) percent bupivacaine), which created a wheal.

The margin of the wheal was marked with indelible ink. The reaction to pin pricks at the site

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WO 99/13865 PCT/US98/19583 of injection was tested just prior to injection (time zero) and 15 minutes, 3, 6, 12, 18,24, 30 and 36 hours following injection of MVL-encapsulated bupivacaine, and zero, 5, 15, . minutes, 1, 1.5,2, 3,4, 5, 6, 7 and 8 hours following injection of bupivacaine hydrochloride. 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 (vocalization response), six pricks were applied inside the wheal and the number of pricks to which the guinea-pig failed to react were recorded as non-responses. Each prick was applied at an interval of 3-5 sec. All animals responded with vocalization to all six pin pricks at baseline.

The animal data obtained indicate a rapid onset of anesthesia following a single intracutaneous dose of bupivacaine encapsulated in MVL, followed by a prolonged duration of sensory anesthesia lasting up to 28 hours, depending on the concentration of bupivacaine in the MVL administered. The rapid onset of anesthesia is attributable, in part, to a low, but a significant fraction of unencapsuiated bupivacaine (approximately 5 % of the total) in the batches of MVL encapsulating bupivacaine used in these experiments. The duration of anesthesia obtained by use of these formulations may cover the worst post-operative period, the first 24 hours. A longer anesthesia duration, perhaps 7 days or longer, would be more suitable for chronic pain, such as cancer or neuropathic pain.

Example 4: Data Analysis of Efficacy Studies

Anesthetic efficacy curves were plotted as the number of non-responses as a function of time. Areas under the curve (AUC) were calculated by the trapezoidal rule to the last data point. With regard to Fig. 1 A. the MVL-encapsulated bupivacaine concentrations by weight per volume percent (w/v%) were 2.1% (·). 1.0% (), and 0.5% (A). With regard to Fig. IB. unencapsulated bupivacaine concentrations by weight per volume percent were 0.25%(V), 0.5%(Δ), 0.75%( 0 ), and 1 .()%(□). Each data point represents the average for 5 to 6 animals. The error bars represent the standard error of mean (SEM).

Evaluation of response to pin pricks showed that complete local anesthesia (no response condition) was achieved within 15 minutes following intracutaneous administration of either the MVL formulation of bupivacaine (Fig. 1 A) or of unencapsulated bupivacaine hydrochloride (Fig. IB).

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Fig. 2 shows the duration of anesthetic effect as measured by the time to half maximal response (R₃) for the various doses of the MVL formulation (filled circles) and for unencapsulated drug (open circles). Each data point represents the average and standard error of mean (SEM) from 5 to 6 animals. These results show that the duration of anesthetic effect was concentration dependent in both cases. However, the MVL formulations containing concentrations 0.5 and L0 percent by weight of bupivacaine phosphate were prolonged 3.2 and 2.9-fold, respectively, as compared to comparable doses of bupivacaine hydrochloride.

Example 5: Determination of Maximum Tolerated Dose (MTD)

Determination of maximum tolerated dose (MTD) was done in mice using a subcutaneous test known in the art (R. H. de Jong et al.. Anesthesiology 54:177-81, 1981). Groups of three mice each were given injections of either 780 or 980 mg per kg body weight of the above-described MVL formulation containing bupivacaine sulfate as two divided doses of 500 μΐ each (total 1.0 mL volume). The doses were administered in rapid sequence into each flank. Control groups of 3 mice each received one of the test doses as a single dose of 10, 20,30 or 50 mg/kg body weight of unencapsulated bupivacaine hydrochloride. MTD was defined as the highest dose at which none of the animals experienced systemic toxicity.

These studies showed that none of the mice that received free bupivacaine hydrochloride subcutaneously showed any signs of systemic toxicity at doses of 10 and 20 mg/kg. However, at 30 and 50 mg/kg doses, two out of three, and three out of three animals, respectively, developed toxicity. On the other hand, the MVL formulation of bupivacaine sulfate administered subcutaneously at a dose of 780 mg/kg showed no sign of systemic toxicity in any of the animals; whereas three out of three animals had toxicity at a dose of 980 mg/kg. Therefore, the MTD for unencapsulated bupivacaine hydrochloride was estimated to be about 20 mg/kg of body weight, and that for MVL-encapsulated bupivacaine sulfate was estimated at about 780 mg/kg of body weight.

The most serious toxicity arising from the use of local anesthetics is seizure or cardiovascular collapse. Consistent with the lower peak serum concentration found following administration of the MVL formulations of bupivacaine, the maximum tolerated

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WO 99/13865 PCT/US98/19583 dose for the MVL-encapsulated bupivacaine was many times higher than that for bupivacaine hydrochloride. These data would predict an increased systemic safety profile for the compositions produced by the method of this invention. The toxicity profiles of the active and inactive ingredients are well-defined, reducing the likelihood of finding unexpected toxicity.

Example 6: Pharmacokinetic Studies

The in vivo pharmacokinetics of the MVL formulations of bupivacaine and free bupivacaine hydrochloride were compared following a single 1 mL intracutaneous dose of the MVL formulation containing 1.0 percent (w/v) of bupivacaine, or a dose of 0.5 percent (w/v) of unencapsulated bupivacaine hydrochloride to a group of guinea pigs. The lower concentration was selected for bupivacaine hydrochloride because the 400-600 gram animals were unable to tolerate a 1.0% concentration dose of the unencapsulated drug. For the animals that received free bupivacaine hydrochloride, samples were collected at 0 and 30 minutes, and 1,3, 6, and 9 hours following injection, while the animals that received the MVL formulations of bupivacaine were sampled 0,6, 12,18, 24,48, and 72 hours following injection. At each time point, 3 or more animals were first anesthetized with halothane and then exanguinated by cardiac puncture. Serum samples were obtained by centrifugation of clotted whole blood. Skin was collected around the injection site with 3 cm margins, together with a 2-3 mm layer of underlying subcutaneous tissue. The skin and serum samples were kept frozen at -20°C until analysis.

The amount of total bupivacaine remaining in the injection site was obtained by mincing the tissue followed by in toto homogenization in water using a Polytron homogenizer, (Brinkman. Littau, Switzerland). Bupivacaine was extracted from the homogenate and analyzed by HPLC using a previously published method (Le Guevello et al., J. Chromatography 622:284-290, 1993). Bupivacaine concentration in serum was determined by extraction followed by HPLC (Le Guevello et aL, supra). Tetracaine spiked into each sample before extraction was used as an internal standard. The limit of detection of the assay was 20 ng/mL.

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Pharmacokinetic data obtained from the samples were analyzed using a noncompartmental model (WinNonlin software, Scientific Consulting, Inc., Apex, NC). Parameters calculated were the drug amount remaining at the injection site, the area under the “amount vs. time” curve (AUC), and drug half-life (t.J. In addition to the AUC and half5 life, peak concentration (C_max) was also reported for serum bupivacaine pharmacokinetics.

One way analysis of variance (ANOVA) was used to separately determine dose dependency for the different drug formulations and route of administration (via MVL or free drug) as well as for comparison between formulations. Student-Newman-Keuls tests were used on all ANOVA analyses. The pharmacokinetic parameters obtained by these methods io are summarized in Table 2 below.

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Table 2

Pharmacokinetic Parameters Following Either a Single 1.0% DepoBupivacaine or 0.5%

Bupivacaine Hydrochloride Intracutaneous Injection

<td></td><td> MVL-encapsulated Bupivacaine</td><td> Bupivacaine hydrochloride</td>

<td> Drug Concentration Administered</td><td> 1.0%</td><td> 0.5%</td>

<td> Peak Amount (mg), Skin</td><td> 11.6</td><td> 3.8</td>

<td> t_w (hr), Skin</td><td> 12.0</td><td> 1.3</td>

<td> AUC (mg*hr), Skin</td><td> 236</td><td> 2.9</td>

<td> C™, Og/mL), Serum</td><td> 2.9</td><td> 6.5</td>

<td> t.₄(hr), Serum</td><td> 20.5</td><td> 2.1</td>

<td> AUC (/zg*hr*mL*‘), Serum</td><td> 56.1</td><td> 21.2</td>

<td> r. Skin</td><td> 0.97</td><td> 0.85</td>

<td> r. Serum</td><td> 0.89</td><td> 0.93</td>

The “drug concentration administered” is in units of weight of anesthetic per volume of MVL. The “peak amount” shows the maximum amount of the indicated substance in the skin sample. The “Cmax” is the maximum concentration of the indicated substance in serum. The “t'/i” is the drug half-life. The “AUC area” is the area under the “amount vs. time” curve. The “r is the square of the sample correlation coefficient.

As these results show, following intracutaneous administration of the MVL formulation, the total amount of drug in the injection-site tissue decreased with a half-life of 12 hours compared to 1.3 hours for unencapsulated bupivacaine hydrochloride. Peak serum concentration of bupivacaine following a single intracutaneous dose of the 1.0% MVL formulation was decreased 2.2 fold (4.4 fold when corrected for the dose) compared to that following 0.5% bupivacaine hydrochloride. Similarly, the terminal serum half-life for 1.0 percent (w/v) MVL formulations was 20.5 hours compared to 2.1 hours for unencapsulated bupivacaine hydrochloride at a concentration of 0.5 percent (w/v).

The local injection site AUC for the MVL formulation was 81 times (41 times when corrected for the dose) that for unencapsulated bupivacaine hydrochloride, and the serum

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AUC was 2.6 times (1.3 times when corrected for the dose) that for bupivacaine hydrochloride.

Figs. 3A and 3B show the result of the pharmacokinetic studies. Fig. 3 A shows the amount of either MVL-encapsulated bupivacaine at a concentration of 1.0 percent (w/v) of 5 bupivacaine (filled circles) or unencapsulated bupivacaine hydrochloride at a concentration of 0.5 percent (w/v) of bupivacaine (open circles) remaining at an injection site at time points tested over a period of 72 hours. Fig. 3B shows the serum concentration (/xg/mL) of bupivacaine following a single intracutaneous dose of the MVL-encapsulated formulation at 1.0 percent (w/v) of bupivacaine (filled circles) or unencapsulated bupivacaine io hydrochloride at a concentration of 0.5 percent (w/v) of bupivacaine (open circles). Each data point represents the average and standard error of mean (SEM) from 3 to 6 animals. A statistical significance level of 0.05 was used for all tests.

The pharmacokinetics data obtained in the Examples herein were consistent with a prolonged duration of anesthetic effect. The anesthesia duration was 2.9- to 3.2-fold longer 15 for bupivacaine encapsulated in MVL, and the half-life at the injection site was 9.2-fold longer compared to bupivacaine hydrochloride. The peak serum concentration was decreased by 4.5 fold (normalized to equivalent doses), and the terminal serum half-life was increased by 9.8-fold following administration of bupivacaine encapsulated in MVL compared to bupivacaine hydrochloride.

In conclusion, a single intracutaneous dose of bupivacaine encapsulated in MVL resulted in a prolonged duration of anesthesia (up to 28 hours) and a 9.2-fold (uncorrected for dose) increase in local injection-site half-life compared to bupivacaine hydrochloride. The maximum tolerated dose was increased by 39 fold compared to bupivacaine hydrochloride. Therefore the formulations of the invention have utility for sustained infiltration anesthesia without the need for continuous infusion and may increase patient satisfaction.

A number of embodiments of the present invention have been described. Nevertheless, it will be understood that various aspects, advantages and modifications of the invention may be made without departing from the spirit and scope of the invention. The foregoing description is intended to illustrate and not limit the scope of the invention, which is defined 30 by the scope of the appended claims.