United States Patent [19]
`Desai et al.
`
`US005439686A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,439,686
`Aug. 8, 1995
`
`[54] METHODS FOR IN VIVO DELIVERY OF
`SUBSTANTIALLY WATER INSOLUBLE
`PHARMACOLOGICALLY ACTIVE AGENTS
`AND COMPOSITIONS USEFUL THEREFOR
`[75] Inventors: Neil P. Desai; Patrick Soon-Shiong;
`Paul A. Sandford, all of Los Angeles;
`Mark W. Grinstaff, Pasadena, all of
`Calif; Kenneth S. Suslick,
`Champaign, Ill.
`[73] Assignee: VivoRx Pharmaceuticals, Inc., Santa
`Monica, Calif.
`[21] Appl. No.: 23,698
`[22] Filed:
`Feb. 22, 1993
`
`[51] Int. Cl.6 . . . . . . . . .
`
`. . . . . . . . . . . .. A61K 9/48
`
`[52] US. Cl. ............................ .... .. 424/451; 424/465;
`424/489
`[58] Field of Search ............. .. 424/451, 465, 450, 439;
`260/403
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`‘
`
`3,526,074 10/1970 Aufhauser ......................... .. 424/451
`3,959,457 5/1976 Speaker et al.
`.... .. 424/469
`4,073,943 2/1978 Wretlind et al. ..
`.... .. 514/772
`4,247,406 1/1981 Widder et a1
`.... .. 424/489
`
`4,534,899 8/1985 Sears . . . . . . . . . .
`
`. . . . .. 260/403
`
`4,572,203 2/1986 Feinstein . . . . . . . .
`
`. . . . .. 424/9
`
`4,671,954 6/1987 Goldberg et a1. .
`
`.... .. 424/450
`
`4,718,433 1/ 1988 Feinstein ....... ..' . . .
`4,789,550 12/1988 Hommel et a1.
`4,844,882 7/1989 Widder et a1. .
`
`. . . . .. 530/427
`.... .. 424/493
`........ .. 424/9
`
`4,929,446 5/1990 Bartolucci . . . . . . . . . .
`
`. . . . .. 424/439
`
`. 549/511
`5,059,699 10/1991 Kingston et a1. ..
`5,110,606 5/ 1992 Geyer et al. ...................... .. 424/489
`
`FOREIGN PATENT DOCUMENTS
`0129619Al 1/l985 European Pat. Off. .
`0295941A2 12/1988 European Pat. Off. .
`O391518A2 2/1990 European Pat. Off. .
`0361677A1 4/ 1990 European Pat. Off. .
`0418153A1 3/1991 European Pat. Off. .
`0190050B1 5/ 1991 European Pat. Off. .
`0213303B1 9/1991 European Pat. Off. .
`85/00011 l/l985 WIPO .
`87/01035 2/ 1987 WIPO .
`
`WIPO .
`
`88/01506 3/1988
`88/07365 10/1988
`89/03674 5/ 1989
`90/13285 ll/1990
`WIPO .
`90/13780 11/1990 WIPO .
`91/15947 lO/l99l WIPO .
`
`WIPO .
`
`WIPO .
`
`OTHER PUBLICATIONS
`Burgess et al., “Potential use of albumin microspheres as
`a drug delivery system. 1. Preparation and in vitro re
`lease of steroids,” International Journal of Pharmaceuti
`cals, 39:129-136 (1987).
`Chen et al., “Comparison of albumin and casein micro
`spheres as a carrier for doxorubicin,” J. Pharm. Phar
`macoL, 39:978-985 (1987).
`Feinstein et al., “Two-Dimensional Contrast Echocar
`diography. I. In Vitro Development and Quantitative
`Analysis of Echo Contrast Agents,” JACC 3(1):14—20
`(1984).
`Grinstaff & Syslick, “Nonaqueous Liquid Filled Micro
`
`(List continued on next page.)
`Primary Examiner-éThurman K. Page
`Assistant Examiner-Benston, Jr. William E.
`Attorney, Agent, or Firm-Stephen E. Reiter; Pretty,
`Schroeder, Brueggemann & Clark
`[57]
`ABSTRACT
`In accordance with the present invention, there are
`provided compositions for the in vivo delivery of sub
`stantially water insoluble pharmacologically active
`agents (such as the anticancer drug taxol) in which the
`pharmacologically active agent is delivered in a soluble
`form or in the form of suspended particles. In particular,
`the soluble form may comprise a solution of pharmaco
`logically active agent in a biocompatible dispersing
`agent contained within a protein walled shell. Alterna
`tively, the protein walled shell may contain particles of
`taxol. In another aspect, the suspended form comprises
`particles of pharmacologically active agent in a biocom
`patible aqueous liquid.
`
`17 Claims, N0 Drawings
`
`CIPLA EXHIBIT 1003
`Page 1 of 12
`
`

`

`5,439,686
`Page 2
`
`OTHER PUBLICATIONS
`capsules,” Polym. Prepn, 32:255-256 (1991).
`Gupta et al., “Albumin microspheres. III. Synthesis and
`characterization of microspheres containing adriamycin
`and magnetite,” International Journal of Pharmaceutics,
`43:167-177 (1988).
`Ishizaka et al., “Preparation of Egg Albumin Microcap~
`sules and Microspheres,” Journal of Pharmaceutical
`Sciences, 70(4):358-363 (1981).
`Klibanov et al., “Amphipathic polyethyleneglcols ef
`fectively prolong the circulation time of liposomes,”
`FEBS, 268(l):235—237 (1990).
`Koenig & Meltzer, “Effect of Viscosity on the Size of
`Microbubbles Generated for Use as Echocardiographic
`Contrast Agents,” Journal of Cardiovascular ultrasonog
`raphy, 5(l):3—4 (1986).
`Leucuta et al., “Albumin microspheres as a drug deliv
`
`ery system for epirubicin: pharmaceutical, pharmaco
`kintetic and biological aspects,” International Journal of
`Pharmaceutics, 41:213-217 (1988).
`Molecular Biosystems, Inc., “ALBUNEX”—Preclini
`cal Investigator’s Package.
`Moseley et al., “Microbubbles: A Novel MR Suscepti
`bility Contrast Agent,” 10th Annual Meeting of Society
`of Magnetic Resonance in Medicine (1991).
`Suslick & Grinstaff, “Protein Microencapsulation of
`Nonaqueous Liquids,”
`J.
`Am. Chem.
`Soc,
`112(2):7807-7809 (1990).
`Willmott & Harrison, “Characterization of freeze-dried
`albumin microsperes containing the anti-cancer drug
`adriamycin,” International Journal of Pharmaceutics,
`43:161-166 (1988).
`. . . , “Serum Albumin Beads: An Injectable, Biodegrad
`able System for the Sustained Release of Drugs,” Sci
`ence, 213(10):233-235 (1981).
`
`CIPLA EXHIBIT 1003
`Page 2 of 12
`
`

`

`1
`
`5,439,686
`
`METHODS FOR IN VIVO DELIVERY OF
`SUBSTANTIALLY WATER INSOLUBLE
`PHARMACOLOGICALLY ACTIVE AGENTS AND
`COMPOSITIONS USEFUL THEREFOR
`
`5
`
`The present invention relates to in vivo delivery of
`substantially water insoluble pharmacologically active
`agents (e.g., the anticancer drug taxol). In one aspect,
`the agent is dispersed as a suspension suitable for admin
`istration to a subject, or is dissolved in a suitable bi
`ocompatible liquid. In another aspect, water insoluble
`pharmacologically active agents (e.g., taxol) are en
`cased in a polymeric shell formulated from a biocom
`patible polymer. The polymeric shell contains particles
`of pharmacologically active agent, and optionally a
`' biocompatible dispersing agent in which pharmacologi
`cally active agent can be either dissolved or suspended.
`
`2
`dissolved in an innocuous carrier such as normal saline.
`Such modi?cations, however, add to the cost of drug
`preparation, may induce undesired side-reactions and
`/or allergic reactions, and/or may decrease the ef?
`ciency of the drug.
`Microparticles and foreign bodies present in the
`blood are generally cleared from the circulation by the
`‘blood ?ltering organs’, namely the spleen, lungs and
`liver. The particulate matter contained in normal whole
`blood comprises red blood cells (typically 8 microns in
`diameter), white blood cells (typically 6-8 microns in
`diameter), and platelets (typically l-3 microns in diame
`ter). The microcirculation in most organs and tissues
`allows the free passage of these blood cells. When mi
`crothrombii (blood clots) of size greater than 10-15
`microns are present in circulation, a risk of infarction or
`blockage of the capillaries results, leading to ischemia
`or oxygen deprivation and possible tissue death. Injec
`tion into the circulation of particles greater than 10-15
`microns in diameter, therefore, must be avoided. A
`suspension of particles less than 7-8 microns, is how
`ever, relatively safe and has been used for the delivery
`of pharmacologically active agents in the form of lipo
`somes and emulsions, nutritional agents, and contrast
`media for imaging applications.
`The size of particles and their mode of delivery deter
`mines their biological behavior. Strand et al. [in Micros
`pheres-Biomedical Applications, ed. A. Rembaum, pp
`193-227, CRC Press (1988)] have described the fate of
`particles to be dependent on their size. Particles in the
`size range of a few nanometers (nm) to 100 nm enter the
`lymphatic capillaries following interstitial injection, and
`phagocytosis may occur within the lymph nodes. After
`intravenous/intraarterial injection, particles less than
`about 2 microns will be rapidly cleared from the blood
`stream by the reticuloendothelial system (RES), also
`known as the mononuclear phagocyte system (MPS).
`Particles larger than about 7 microns will, after intrave
`nous injection, be trapped in the lung capillaries. After
`intraarterial injection, particles are trapped in the ?rst
`capillary bed reached. Inhaled particles are trapped by
`the alveolar macrophages.
`,
`Pharmaceuticals that are water-insoluble or poorly
`water-soluble and sensitive to acid environments in the
`stomach cannot be conventionally administered (e.g.,
`by intravenous injection or oral administration). The
`parenteral administration of such pharmaceuticals has
`been achieved by emulsi?cation of the oil solubilized
`drug with an aqueous liquid (such as normal saline) in
`the presence of surfactants or emulsion stabilizers to
`produce stable microemulsions. These emulsions may
`be injected intravenously, provided the components of
`the emulsion are pharmacologically inert. US. Pat. No.
`4,073,943 describes the administration of water-insolu
`ble pharmacologically active agents dissolved in oils
`and emulsi?ed with water in the presence of surfactants
`such as egg phosphatides, pluronics (copolymers of
`polypropylene glycol and polyethylene glycol), poly
`glycerol oleate, etc. PCT International Publication No.
`WO85/000l1 describes pharmaceutical microdroplets
`of an anaesthetic coated with a phospholipid such as
`dimyristoyl phosphatidylcholine having suitable dimen
`sions for intradermal or intravenous injection.
`Protein microspheres have been reported in the liter
`ature as carriers of pharmacological or diagnostic
`agents. Microspheres of albumin have been prepared by
`either heat denaturation or chemical crosslinking. Heat
`denatured microspheres are produced from an emulsi
`
`20
`
`25
`
`30
`
`BACKGROUND OF THE INVENTION
`Taxol is a natural product ?rst isolated from the Pa
`cific Yew tree, Taxus brevifolia, by Wani et al. [J . Am.
`Chem. Soc. Vol. 93:2325 (1971)]. Among the antimi
`totic agents, taxol, which contains a diterpene carbon
`skeleton, exhibits a unique mode of action on microtu
`bule proteins responsible for the formation of the mi
`totic spindle. In contrast with other antimitotic agents
`such as vinblastine or colchicine, which prevent the
`assembly of tubulin, taxol is the only plant product
`known to inhibit the depolymerization process of tubu
`lin, thus preventing the cell replication process.
`Taxol, a naturally occurring diterpenoid, has been
`shown to have signi?cant antineoplastic and anticancer
`effects in drug~refractory ovarian cancer. Taxol has
`shown excellent antitumor activity in a wide variety of
`35
`tumor models such as the B16 melanoma, L12l0 leuke
`mias, MX-l mammary tumors, and CS-l colon tumor
`xenografts. Several recent press releases have termed
`taxol as the new anticancer wonder-drug. Indeed, taxol
`has recently been approved by the Federal Drug Ad
`40
`ministration for treatment of ovarian cancer. The poor
`aqueous solubility of taxol, however, presents a prob
`lem for human administration. Indeed, the delivery of
`drugs that are inherently insoluble or poorly soluble in
`an aqueous medium can be seriously impaired if oral
`delivery is not effective. Accordingly, currently used
`taxol formulations require a cremaphore to solubilize
`the drug. The human clinical dose range is 200-500 mg.
`This dose is dissolved in a 1:1 solution of ethanolzcrema~
`phore and diluted to one liter of ?uid given intrave
`nously. The cremaphore currently used is polyethox
`ylated castor oil.
`In phase I clinical trials, taxol itself did not show
`excessive toxic effects, but severe allergic reactions
`were caused by the emulsi?ers employed to solubilize
`the drug. The current regimen of administration in
`volves treatment of the patient with antihistamines and
`steroids prior to injection of the drug to reduce the
`allergic side effects of the cremaphore.
`In an effort to improve the water solubility of taxol,
`several investigators have modi?ed its chemical struc
`ture with functional groups that impart enhanced wa
`ter-solubility. Among them are the sulfonated deriva
`tives [Kingston et al., US. Pat. No. 5,059,699 (1991)],
`and amino acid esters [Mathew et al., J. Med. Chem.
`Vol. 35:145-151 (1992)] which show signi?cant biologi
`cal activity. Modi?cations to produce a water-soluble
`derivative facilitate the intravenous delivery of taxol
`
`45
`
`50
`
`55
`
`60
`
`65
`
`CIPLA EXHIBIT 1003
`Page 3 of 12
`
`

`

`4
`tration time relative to administration volumes and
`times required by prior art delivery systems (e. g., intra
`venous infusion of approximately one to two liters of
`?uid over a 24 hour period are required to deliver a
`typical human dose of 200-400 mg of taxol).
`In accordance with another embodiment of the pres
`ent invention, we have developed compositions useful
`for in vivo delivery of substantially water insoluble
`pharmacologically active agents. Invention composi
`tions comprise substantially water insoluble pharmaco
`logically active agents (as a solid or liquid) contained
`within a polymeric shell. The polymeric shell is a bi
`ocompatible polymer, crosslinked by the presence of
`disul?de bonds. The polymeric shell, containing sub
`stantially water insoluble pharmacologically active
`agents therein, is then suspended in a biocompatible
`aqueous liquid for administration.
`
`10
`
`15
`
`5,439,686
`3
`?ed mixture (e.g., albumin, the agent to be incorpo
`rated, and a suitable oil) at temperatures between 100°
`C. and 150° C. The microspheres are then washed with
`a suitable solvent and stored. Leucuta et al. [Interna
`tional Journal of Pharmaceutics Vol. 41:213-217 (1988)]
`describe the method of preparation of heat denatured
`microspheres.
`The procedure for preparing chemically crosslinked
`microspheres involves treating the emulsion with glu
`taraldehyde to crosslink the protein, followed by wash
`ing and storage. Lee et al. [Science Vol. 2l3:233-235
`(1981)] and US. Pat. No‘. 4,671,954 teach this method of
`preparation.
`The above techniques for the preparation of protein
`microspheres as carriers of pharmacologically active
`agents, although suitable for the delivery of water-solu
`ble agents, are incapable of entrapping water-insoluble
`ones. This limitation is inherent in the technique of
`preparation which relies on crosslinking or heat dena
`turation of the protein component in the aqueous phase
`of a water-in-oil emulsion. Any aqueous-soluble agent
`dissolved in the protein-containing aqueous phase may
`be entrapped within the resultant crosslinked or heat
`denatured protein matrix, but a poorly aqueous-soluble
`or oil-soluble agent cannot be incorporated into a pro
`tein matrix formed by these techniques.
`
`20
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`In accordance with the present invention, there are
`provided compositions for in vivo delivery of a substan
`tially water insoluble pharmacologically active agent,
`wherein said agent is a solid or liquid substantially
`completely contained within a polymeric shell,
`wherein the largest cross-sectional dimension of said
`shell is no greater than about 10 microns,
`wherein said polymeric shell comprises a biocompati
`ble polymer which is substantially crosslinked by
`way of disul?de bonds, and
`wherein said polymeric shell containing pharmaco
`logically active agent therein is suspended in a
`biocompatible aqueous liquid.
`As used herein, the term “in vivo delivery” refers to
`delivery of a pharmacologically active agent by such
`routes of administration as oral, intravenous, subcutane
`ous, intraperitoneal, intrathecal, intramuscular, inhala
`tional, topical, transdermal, suppository (rectal), pes
`sary (vaginal), and the like.
`As used herein, the term “micron” refers to a unit of
`measure of one one-thousandth of a millimeter.
`As used herein, the term “biocompatible” describes a
`substance that does not appreciably alter or affect in any
`adverse way, the biological system into which it is in
`troduced.
`Key differences between the pharmacologically ac
`tive agents contained in a polymeric shell according to
`the invention and protein microspheres of the prior art
`are in the nature of formation and the ?nal state of the
`protein after formation of the particle, and its ability to
`carry poorly aqueous-soluble or substantially aqueous
`insoluble agents. In accordance with the present inven
`tion, the polymer (e.g., a protein) is selectively chemi
`cally crosslinked through the formation of disul?de
`bonds through, for example, the amino acid cysteine
`that occurs in the natural structure of a number of pro
`teins. A sonication process is used to disperse a dispers
`ing agent containing dissolved or suspended pharmaco
`logically active agent into an aqueous solution of a
`biocompatible polymer bearing sulfhydryl or disul?de
`groups (e.g., albumin) whereby a shell of crosslinked
`polymer is formed around ?ne droplets of non-aqueous
`medium. The sonication process produces cavitation in
`the liquid that causes tremendous local heating and
`results in the formation of superoxide ions that crosslink
`the polymer by oxidizing the sulfhydryl residues (and
`/or disrupting existing disul?de bonds) to form new,
`crosslinking disul?de bonds.
`
`25
`
`30
`
`35
`
`BRIEF DESCRIPTION OF THE INVENTION
`Thus it is an object of this invention to deliver phar
`macologically active agents (e. g., taxol, taxane, Tax
`otere, and the like) in unmodi?ed form in a composition
`that does not cause allergic reactions due to the pres
`ence of added emulsi?ers and solubilizing agents, as are
`currently employed in drug delivery.
`It is a further object of the present invention to de
`liver pharmacologically active agents in a composition
`of microparticles suspended in a suitable biocompatible
`liquid.
`It is yet another object of the invention to deliver
`pharmacologically active agents enclosed within a pol
`ymer shell which is further suspended in a biocompati
`ble liquid.
`These and other objects of the invention will become
`apparent upon review of the speci?cation and claims.
`In accordance with the present invention, we have
`45
`discovered that substantially water insoluble pharmaco
`logically active agents can be delivered in the form of
`microparticles that are suitable for parenteral adminis
`tration in aqueous suspension. This mode of delivery
`obviates the necessity for administration of substantially
`water insoluble pharmacologically active agents (e.g.,
`taxol) in an emulsion containing, for example, ethanol
`and polyethoxylated castor oil, diluted in normal saline
`(see, for example, Norton et al., in Abstracts of the 2nd
`National Cancer Institute Workshop on Taxol & Taxus,
`Sep. 23-24, 1992). A disadvantage of such known com
`positions is their propensity to produce allergic side
`effects.
`The delivery of substantially water insoluble pharma
`cologically active agents in the form of a microparticu
`late suspension allows some degree of targeting to or
`gans such as the liver, lungs, spleen, lymphatic circula
`tion, and the like, through the use of particles of varying
`size, and through administration by different routes.
`The invention method of delivery further allows the
`administration of substantially water insoluble pharma
`cologically active agents employing a much smaller
`volume of liquid and requiring greatly reduced adminis
`
`55
`
`60
`
`65
`
`CIPLA EXHIBIT 1003
`Page 4 of 12
`
`

`

`5,439,686
`5
`6
`In contrast to the invention process, the prior art
`octanes, halocarbons, renografm, and the like), mag
`method of glutaraldehyde crosslinking is nonspeci?c
`netic contrast agents (e.g., ?uorocarbons, lipid soluble
`and essentially reactive with any nucleophilic group
`paramagnetic compounds, and the like), as well as other
`present in the protein structure (e.g., amines and hy
`diagnostic agents which cannot readily be delivered
`droxyls). Heat denaturation as taught by the prior art
`without some physical and/ or chemical modi?cation to
`signi?cantly and irreversibly alters protein structure. In
`accomodate the substantially water insoluble nature
`contrast, disul?de formation contemplated by the pres
`thereof.
`~Examples of agents of nutritional value contemplated
`ent invention does not substantially denature the pro
`tein. In addition, particles of substantially water insolu
`for use in the practice of the present invention include
`amino acids, sugars, proteins, carbohydrates, fat-soluble
`ble pharmacologically active agents contained within a
`shell differ from crosslinked or heat denatured protein
`vitamins (e.g., vitamins A, D, E, K, and the like) or fat,
`microspheres of the prior art because the polymeric
`or combinations of any two or more thereof.
`shell produced by the invention process is relatively
`A number of biocompatible polymers may be em
`thin compared to the diameter of the coated particle. It
`ployed in the practice of the present invention for the
`has been determined (by transmission electron micros
`formation of the polymeric shell which surrounds the
`copy) that the “shell thickness” of the polymeric coat is
`substantially water insoluble pharmacologically active
`approximately 25 nanometers for a coated particle hav
`agents. Essentially any polymer, natural or synthetic,
`bearing sulfhydryl groups or disul?de bonds within its
`ing a diameter of 1 micron (1000 nanometers). In con
`trast, microspheres of the prior art do not have protein
`structure may be utilized for the preparation of a disul
`shells, but rather, have protein dispersed throughout the
`?de crosslinked shell about particles of substantially
`volume of the microsphere.
`water insoluble pharmacologically active agents. The
`The polymeric shell containing solid or liquid cores
`sulfhydryl groups or disul?de linkages may be preexist
`of pharmacologically active agent allows for the deliv
`ing within the polymer structure or they may be intro
`ery of high doses of the pharmacologically active agent
`duced by a suitable chemical modi?cation. For exam
`in relatively small volumes. This minimizes patient dis
`ple, natural polymers such as proteins, oligopeptides,
`polynucleic acids, polysaccharides (e.g., starch, cellu
`comfort at receiving large volumes of ?uid and mini
`lose, dextrans, alginates, chitosan, pectin, hyaluronic
`mizes hospital stay. In addition, the walls of the poly
`meric shell are generally completely degradable in vivo
`acid, and the like), and so on, are candidates for such
`by proteolytic enzymes (e.g., when the polymer is a
`modi?cation.
`protein), resulting in no side effects from the delivery
`As examples of suitable biocompatible polymers,
`naturally occurring or synthetic proteins may be em
`system as is the case with current formulations.
`According to this embodiment of the present inven
`ployed, so long as such proteins have sufficient cysteine
`tion, particles of substantially water insoluble pharma
`residues within their amino acid sequences so that cross
`linking (through disul?de bond formation, for example,
`cologically active agents are contained within a shell
`having a cross-sectional diameter of no greater than
`as a result of oxidation during sonication) can occur.
`Examples of suitable proteins include albumin (which
`about 10 microns. A cross-sectional diameter of less
`contains 35 cysteine residues), insulin (which contains 6
`than 5 microns is more preferred, while a cross-sec
`cysteines), hemoglobin (which contains 6 cysteine resi
`tional diameter of less than 1 micron is presently the
`most preferred for the intravenous route of administra
`dues per (1232 unit), lysozyme (which contains 8 cyste
`ine residues), immunoglobulins, a-Z-macroglobulin,
`tion.
`Substantially water insoluble pharmacologically ac
`?bronectin, vitronectin, ?brinogen, and the like.
`tive agents contemplated for use in the practice of the
`A presently preferred protein for use in the formation
`present invention include pharmaceutically active
`of a polymeric shell is albumin. Optionally, proteins
`agents, diagnostic agents, agents of nutritional value,
`such as a-2-macroglobulin, a known opsonin, could be
`and the like. Examples of pharmaceutically active
`used to enhance uptake of the shell encased particles of
`substantially water insoluble pharmacologically active
`agents include taxol (as used herein, the term “taxol” is
`intended to include taxol analogs and prodrugs, taxanes,
`agents by macrophage-like cells, or to enhance the up
`and other taxol-like drugs, e.g., Taxotere, and the like),
`take of the shell encased particles into the liver and
`spleen.
`camptothecin and derivatives thereof (which com
`Similarly, synthetic polypeptides containing cysteine
`pounds have great promise for the treatment of colon
`cancer), aspirin, ibuprofen, piroxicam, cimetidine, sub
`residues are also good candidates for formation of a
`stantially water insoluble steroids (e.g., estrogen, pred
`shell about the substantially water insoluble pharmaco
`nisolone, cortisone, hy'drocortisone, di?orasone, and
`logically active agents. In addition, polyvinyl alcohol,
`polyhydroxyethyl methacrylate, polyacrylic acid,
`the like), drugs such as phenesterine, duanorubicin,
`polyethyloxazoline, polyacrylamide, polyvinyl pyrroli
`doxorubicin, mitotane, visadine, halonitrosoureas, an
`throcylines, ellipticine, diazepam, and the like, anaes
`dinone, and the like, are good candidates for chemical
`modi?cation (to introduce sulfhydryl and/or disul?de
`thetics such as methoxy?uorane, iso?uorane, en?uo
`linkages) and shell formation (by causing the crosslink
`rane, halothane, benzocaine, dantrolene, barbiturates,
`ing thereof).
`and the like. In addition, also contemplated are substan
`tially water insoluble immunosuppressive agents, such
`In the preparation of invention compositions, one can
`as, for example, cyclosporines, azathioprine, FK506,
`optionally employ a dispersing agent to suspend or
`prednisone, and the like. A presently preferred pharma
`dissolve the substantially water insoluble pharmacologi
`cally active agent. Dispersing agents contemplated for
`ceutically active agent for use in the practice of the
`present invention is taxol, which is commercially avail
`use in the practice of the present invention include any
`nonaqueous liquid that is capable of suspending or dis
`able from the manufacturer as needle-like crystals.
`Examples of diagnostic agents contemplated for use
`solving the pharmacologically active agent, but does
`in the practice of the present invention include ultra
`not chemically react with either the polymer employed
`sound contrast agents, radiocontrast agents (e.g., iodo
`to produce the shell, or the pharmacologically active
`
`65
`
`50
`
`55
`
`20
`
`25
`
`35
`
`45
`
`CIPLA EXHIBIT 1003
`Page 5 of 12
`
`

`

`5,439,686
`7
`8
`ergy pool required for unfolding and/or change of pol
`agent itself. Examples include vegetable oils (e.g., soy
`bean oil, coconut oil, olive oil, safflower oil, cotton seed
`ymer conformation.
`oil, and the like), aliphatic, cycloaliphatic, or aromatic
`Thermal energy input is a function of such variables
`hydrocarbons having 4-30 carbon atoms (e. g., n~dodec
`as the acoustic power employed in the sonication pro
`ane, n-decane, n-hexane, cyclohexane, toluene, benzene,
`cess, the sonication time, the nature of the material
`being subjected to sonication, the volume of the mate
`and the like), aliphatic or aromatic alcohols having 2-30
`rial being subjected to sonication, and the like. The
`carbon atoms (e.g., octanol, and the like), aliphatic or
`acoustic power of sonication processes can vary widely,
`aromatic esters having 2-30 carbon atoms (e. g., ethyl
`typically falling in the range of about 1 up to 1000
`caprylate (octanoate), and the like), alkyl, aryl, or cyclic
`watts/cm2; with an acoustic power in the range of about
`v ethers having 2-30 carbon atoms (e.g., diethyl ether,
`50 up to 200 watts/cm2 being a presently preferred
`tetrahydrofuran, and the like), alkyl or aryl halides
`range. Similarly, sonication time can vary widely, typi~
`having 1-30 carbon atoms (and optionally more than
`cally falling in the range of a few seconds up to about 5
`one halogen substituent, e.g., CH3Cl, CHgClg,
`minutes. Preferably, sonication time will fall in the
`CH2Cl-—CHZCl, and the like), ketones having 3-30
`range of about 15 up to 60 seconds. Those of skill in the
`carbon atoms (e.g., acetone, methyl ethyl ketone, and
`art recognize that the higher the acoustic power ap
`the like), polyalkylene glycols (e.g., polyethylene gly
`plied, the less sonication time is required, and vice
`col, and the like), or combinations of any two or more
`versa.
`thereof.
`The interfacial free energy is directly proportional to
`Especially preferred combinations of dispersing
`the polarity difference between the two liquids. Thus at
`agents include volatile liquids such as dichloromethane,
`a given operating temperature a minimum free energy
`ethyl acetate, benzene, and the like (i.e., solvents that
`at the interface between the two liquids is essential to
`have a high degree of solubility for the pharmacologi
`form the desired polymer shell. Thus, if a homologous
`cally active agent, and are soluble in the other dispers
`series of dispersing agents is taken with a gradual
`ing agent employed), along with a higher molecular
`change in polarity, e.g., ethyl esters of alkanoic acids,
`weight (less volatile) dispersing agent. When added to
`then higher homologues are increasingly nonpolar, i.e.,
`the other dispersing agent, these volatile additives help
`the interfacial tension between these dispersing agents
`to drive the solubility of the pharmacologically active
`and water increases as the number of carbon atoms in
`agent into the dispersing agent. This is desirable since
`the ester increases. Thus it is found that, although ethyl
`this step is usually time consuming. Following dissolu
`acetate is water-immiscible (i.e., an ester of a 2 carbon
`tion, the volatile component may be removed by evapo
`acid), at room temperature (~20° C.), this dispersing
`ration (optionally under vacuum).
`agent alone will not give a signi?cant yield of polymer
`Particles of pharmacologically active agent substan
`shell-coated particles. In-contrast, a higher ester such as
`tially completely contained within a polymeric shell,
`ethyl octanoate (ester of an 8 carbon acid) gives poly
`prepared as described above, are delivered as a suspen
`mer shell-coated particles in high yield. In fact, ethyl
`35
`sion in a biocompatible aqueous liquid. This liquid may
`heptanoate (ester of a 7 carbon acid) gives a moderate
`be selected from water, saline, a solution containing
`yield while the lower esters (esters of 3, 4, 5, or 6 carbon
`appropriate buffers, a solution containing nutritional
`acids) give poor yield. Thus, at a given temperature,
`agents such as amino acids, sugars, proteins, carbohy
`one could set a condition of minimum aqueous-dispers
`drates, vitamins or fat, and the like.
`ing agent interfacial tension required for formation of
`In accordance with another embodiment of the pres~
`high yields of polymer shell-coated particles.
`ent invention, there is provided a method for the prepa
`Temperature is another variable that may be manipu
`ration of a substantially water insoluble pharmacologi
`lated to affect the yield of polymer shell-coated parti
`cally active agent for in vivo delivery, said method
`cles. In general the surface tension of a liquid decreases
`comprising subjecting a mixture comprising:
`with increasing temperature. The rate of change of
`45
`dispersing agent containing said pharmacologically
`surface tension with temperature is often different for
`active agent dispersed therein, and
`different liquids. Thus, for example, the interfacial ten
`aqueous medium containing biocompatible polymer
`sion (by) between two liquids may be A71 at tempera
`capable of being crosslinked by disul?de bonds
`ture T1 and A312 at temperature T2. If A'y1 at T1 is close
`to sonication conditions for a time suf?cient to promote
`to the minimum required to form polymeric shells of the
`crosslinking of said biocompatible polymer by disul?de
`present invention, and if A'yg (at temp. T2) is greater
`bonds.
`than A71, then a change of temperature from T1 to T2
`A nonobvious feature of the above-described process
`will increase the yield of polymeric shells. This, in fact,
`is in the choice of dispersing agent, speci?cally with
`is observed in the case of ethyl heptanoate, which gives
`respect to the polarity of the dispersing agent. The
`a moderate yield at 20°C. but gives a high yield at 10°
`55
`formation of a shell about the part