United States Patent (19(
`Desai et a1.
`
`[54] MEI'HODS FOR IN VIVO DELIVERY OF
`SUBSTANTlALLY WATER INSOLUBLE
`PHAR.l\iACOLOGICALLY ACTIVE AGU-r8
`AND COMPosmONS USEFUL THEREFOR
`
`[75]
`
`Inventors: Neil P. Desai; Patrick Soon-Smoog;
`P:aul A. Sandford, all of Los Angeles;
`Mark W. Grinstaff. Pasadena, all of
`Calif.; Kellll.etb S. Suslick,
`Champaign, TIL
`[73] Assignee: VivoRx Pharmaceuticals, Inc., Santa
`Monica, Calif.
`[21] Appl. No.: 23,698
`[22]
`Filed:
`Feb. 22, 1993
`[51)
`[52]
`
`(58]
`
`[56]
`
`Int. CI.6 ............................................. A61K 9/48
`U.s. a. ............
`. ....... " ...... 424/451; 424/465;
`424/489
`........... 424/45 I, 465, 450, 439;
`260/403
`
`Field of Search
`
`References Cited
`U .S. PATENT DOCUMENTS
`Aufhauser ......................... 424/45]
`Speaker et al. .........•...
`424/ 469
`W,..,Uind et at .................... 5]4/772
`. ....... 424/489
`Widder et al. ..
`. .... 260/403
`Sea.s ...............
`.. 424/9
`Feinstein
`Goldberg et al.
`. ...•••. 424/450
`Feinstein
`530/427
`424/493
`Hom"",} el al
`.. 424/9
`Widder et al.
`.•... 424/439
`Barto]ucci
`King~ton et aI.
`... 549/511
`Geyer et aI.
`. 424/489
`
`3,526,074 1011970
`3,959,4S7 5/1976
`4,073,943 211978
`4,247,406 1/1981
`4,534,899 8/1985
`4,572,203 211986
`4,671,954 6/1987
`4,718,433 111988
`4,789,550 121]98.8
`4,844,882 7/1989
`4,929,446 5/1990
`5,059,699 \0/1991
`S,IIO,606 5/1992
`
`FOREIGN PATENT DOCUMEf'.."'TS
`0129619A] 1/1985 European Pat. Off.
`0295941A2 ]211988 European Pat. Off .•
`0391518A2 211990 European Pat. Off ..
`0361677AI 4/1990 European Pat. orr . .
`0418153AI 3/1991 European Pat. Off •.
`019OO50Bl S/1991 European Pal. Off ..
`02]3303Bl 9/1991 European Pat. Off.
`85fOC()11 1/1985 WlPO.
`81/01035 2/]981 WIPO .
`
`111111111111111111111 111111111111111 11111 111111111111111111111111111111111
`USOOS439686A
`[11] Patent Number:
`[451 Date of Patent:
`
`5,439,686
`A ug, 8, 1995
`
`88/01506 311988 WIPO .
`GG/0736S 10/198& WIPO .
`89/03674 5/1989 WIPO .
`90/13185 11/1990 WIPO
`90/13780 11/1990 WIPO .
`91/15947 10/199 1 WIPO.
`
`OTHER PUBLICATIONS
`Burgess et al., "Potential use of albumin microspheres as
`a drug delivery system. I. Preparation and in vitro re(cid:173)
`lease of steroids," International Joumal of Pharmaceuti(cid:173)
`cals. 39:129- 136 (1987).
`Chen et aI., "Comparison of albumin and casein micro(cid:173)
`spheres as a carrier fOf dOltOfUbicin," 1. Pharm. Phar(cid:173)
`mlUOl. 39:978-985 (1987).
`Feinstein et al., two-Dimensional Contrast Echocar_
`diograpby. I. In Vitro Developmo::nt and Quantitative
`Analysis of Echo Contrast Agentli," JACC, 3(1):14-20
`(1984).
`Grinstalf & Sys.lick, "Nonaqueous Liquid Filled Micro-
`
`(List COntinued on next page.)
`
`Primary Ex.:I'miner'::'ThUJlllan K. Page
`Assistant Examiner-Benston ; Jr. William E.
`Attomey, Agent, or Firm-5tephen E. Rcitcr; Pretty,
`Schroeder, Brueggemann &: Clark
`ABSTRACf
`[57]
`In accordance with the present invention, there are
`provided compositions for the in vivo delivery of sub(cid:173)
`stantially water insoluble phannacologically active
`agen!!; (such ~ th~ IIl1ti<.;atlC<::£ 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 phanuaco(cid:173)
`logically active agent in a biocompatible dispersing
`agent contained within a prolcin walled shell Altema(cid:173)
`tively, the protein walled shell may contain particles of
`taxol. In another aspect, the suspended fonn comprises
`particles ofpbarmacologicaJly active agent in a bioeom(cid:173)
`patible aqueous liquid.
`
`17 Claims, No Drawings
`
`Apotex v. Abraxis - IPR20 18-00 151 , Ex. 1003 , p.O I of 12
`
`

`

`5,439,686
`
`Pa~ 2
`
`O TIiER PUBLIC AT IONS
`c~ul e$," PoIym. Prep'" 32:255-256 (1991).
`G upta et aI., ~Al bumin microspheres. In. Synthesis aDd
`characterization of microspheres containing adriam ycin
`and magnetite," Intematibnal Journal 0/ Pharmouulics,
`43: 167- 177 ( 1988).
`b hiuka et aI., "Preparation of E gg Albumin Microcap.
`sules and Mict ospheres," Journal of PluJnnacl!UtiCIJl
`&iertces. 70(4):358-363 (l 98J).
`Klibanov et ai., " Amphipathic polyethyleneg1cols ef·
`fectively proiong the circulation lime of liposomes,"
`FEBS, 268(1):235-237 (1990).
`Koenii & Meltzer, "Effect of Viscosity on the Size of
`Microbubbles G enerated for Use as Echocardiographic
`Contrast Agents," JOUlna/ a/Cardiovascular ultroscnog·
`raphy, 5(1):3-4 (1 986).
`Leuc uta el aI., " Albumin microsphere$ as a drug deliv.
`
`cry s),stem fo r epi.rubicin: pharmaceutical, phannaco(cid:173)
`kin tetic and biological asp~" International JOUflW/ af
`Pharmaceutics. 4 1:213-217 (1988).
`Mo~ular Biosystems, Inc., ·'ALBUNEX" -Prcclini·
`cal Investigator', Package.
`Moseley et aI., " Microbubbles; A Novel MR Suscepti·
`bility Contrast Agen\," 10th Annual M:eting of Society
`o f Magnetic RC$Onance in Medicine (1991).
`S uslick & G rinstaif, "Protein Microencapsulation of
`Soc.,
`J. A m.
`Nonaqueous Liquids,"
`Ch ern.
`112(2):7807-7809 ( 1990).
`Willmott &. Harrison, " Characterization o f free~ried
`albumin microsperes containing the anti-cancer drug
`adriamycin," intern(Jtional Journal of Phannaceutr"cs,
`43: 161-1 66 (1988).
`. .. , "Serum Albumin Beads: An Injectable, Biodegrad·
`able System for the Sustained Release of Drugs," Sci·
`ence. 213( 10):233-235 ( 1981).
`
`Apotex v. Abrax is -IPR20 18-001 5 1, Ex. 1003, p.02 of 12
`
`

`

`1
`
`5,439,686
`
`MEIHODS FOR IN VIVO DELIVERY OF
`SUBSTANTIALLY WATER INSOLUBLE
`PHARMACOLOGICALLY ACTIVE AGENTS AND
`COMPOSmONS USEFUL TIlEREFOR
`
`The present invention relates to in vivo delivery of
`substantially water insoluble pharmacologically active
`agents (e.g .• the anticancer drug laxO!). In one aspect,
`the agent is dispersed as a suspension suitable for admin(cid:173)
`istration to a subject, or is dissolved in a suitable bi(cid:173)
`ocornpatib\e liquid. II) another aspect, water insoluble
`pharmacologically active agents (e.g .• !axol) are en(cid:173)
`cased in a polymeric shell formulated from a biocom(cid:173)
`patible polymer. The polymeric shell contains particles
`of pharmacologically active agent. and optionally a
`biocompatible dispersing agent in which phannacologi(cid:173)
`cally active agent can be either dissolved or suspended.
`
`2
`di$Wlved in an l.n.nOCl,lOUS carrier such as nonmil saline.
`Such modifications, however, add to the cost of drog
`preparation, may induce undesired side·reactions and(cid:173)
`/or allergic reactions, and/or may decrease the effi(cid:173)
`ciency of the drug.
`Microparticles and foreign bodies present in the
`blood are general!y cleared from the circulation by the
`'blood nltering organs', namely the spleen, lungs and
`liver. The particulate matter contained in normal whole
`10 blood comprises red blood cells (typically 8 microns in
`diameter), white blood cells (typically 6-8 microns in
`diameter), and platelets (typically 1-3 microns in diame(cid:173)
`ter). The microcirculation in most organs and tissues
`allows the free passage of these blood cells. When mi-
`15 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(cid:173)
`tion into the circulation of particles greater than l Q....15
`BACKGROUND OF "IHE INVENTION
`20 microns in diameter, therefore, must be avoided. A
`suspension of particles less than 7-8 microns., is how-
`Taxol is a natural product first isolated from the Pa-
`ever, relatively safe and has been used for the delivery
`dfic Yew tree, Taxus brevi/alia, by Wani et al. [1. Am.
`of pharmacologically active agents in the fonn of lipo-
`Chern. Soc. Vol. 93:2325 (1971)1. Among the antimi-
`somes and emulsions., nutritional agents, and contrast
`totic agents, taxol, which contains a diterpene carbon
`skeleton, exhibits a unique mode of action on microtu- 25 media for imaging applications.
`bule proteins responsible for the formatiou of the mi-
`The size of particles and their mode of delivery deter-
`mines their biological behavior. Strand et aI. [in Micros-
`totic spindle. In contrast with other antimitotic agents
`such as vinblastine or colchicint; which prevent the
`pheres-Biomedicu{ Applications, ed. A. Rembaum, pp
`assembly of tubulin, taxol is the only plant product
`193--227, CRC Press (1988)] have described the fate of
`Mown to inbibit the: depolymeriutioll process of tubu- )0 particles to t.>e dependent on their site. Particles in the
`size range ofa few na.nometers (nm) to 100 nm enter the
`lin, thus preventing the cell replication process.
`T axo!, a naturally occurring diterpenoid, has been
`lymphatic capillaries following interstitial injection, and
`shown to have significant antineoplastic and anticancer
`phagocytosis may occur wiiliin the lymph nodes. After
`effects in drog-refractory ovarian cancer. Taxol has
`intravenOILVintraarterial injection, particles less than
`sho ..... n excel1ent antitumor activity in a wide variety of 35 about 2 microns will be rapidly cleared from the blood
`tumor models such as the BI 6 melanoma, L I210 leuke-
`stream by the reticuloendothelial system (RES), also
`mias, MX-I mammary tumors, and CS-I colon tumor
`known as the mononuclear pbagocyte system (MPS).
`Particles larger than about 7 microns will, after intrave-
`xenografU. Several recent press rcleases have tenned
`taxol as the new anticancer wonder-drug. Indeed, taxol
`nous injection, be trapped in the lung capillaries. After
`h as recentl y been approved by the Federal Drug Ad- 40 intraarterial injection, particles are trapped in the first
`capillary bed reached. Inhaled particles are trapped by
`ministration for treatment of ovarian cancer. The poor
`aqueous solubility of taxo!, however, presents a prob-
`the alveolar macrophages.
`l em for human administration. Indeed, the delivery of
`Pharmaceuticals that are water-insoluble or poorly
`drogs that are inherently insoluble or poorly soluble in
`water-soluble and sensitive to acid environments in the
`an aqueous medium can be serious1y impaired if oral 4~ stomach cannot be conventionally adntinistered (e.g.,
`d elivery is not effective. Accordingly, currently used
`by intravenous injection or oral administration). Thc
`taxol formulations require a eremaphore to solubilize
`parenteral administration of such pharmaceuticals has
`the drug. The human clinical dose range is 200-500 mg.
`been achieved by emulsification of the oil solubilized
`This dose is dissolved in a (:1 solution ofethanol:crema-
`drug with an aqueous liquid (such as normal saline) in
`phore and diluted to one liter of fluid given intrave- SO the presence of swfacta.nts or emulsion stabilizefS to
`nously. The cremaphore currently used is polyethol'
`produce stable microemulsions. These emulsions may
`y lated caStOr oil.
`be injected intravenously, provided the components of
`In phase I clinical trials, taxa! itself did not show
`the emulsion are pharmacologically inert. U.S. Pat. No.
`eJl=sive toxic Jfects, but severe allergic reactions
`4,073,943 describes the administration of water-insolu-
`w ere caused by the emulsifiers employed to solubilize SS ble pharmacologically active agents dissolved in oils
`the drug. The current regimen of administration in-
`and emulsified with water in the presence ofsurfactants
`volves treatment of the patient with antihistamines and
`such as egg phosphatides, pluronics (copolymers of
`steroids prior to injectiou of the drug to reduce the
`polypropylene glycol and polyethylene glycol), poly-
`glycerol oleate, etc. per International Publication No.
`allergic side effects of the cremaphore_
`In an effort to improve the water solubility of taxol, 60 WQ85/OOOli describes pharmaceutical rnicrodroplets
`of an anaesthetic coated w.ith a phospholipid such as
`several investigators have modified its chemical Slroc-
`ture with functional groups that impan enhanced wa-
`dimyristoyl phosphatidylcholine having suitable dimen-
`ter-solubility. Among them are the sulfonated deriva-
`sions for intradermal or intravenous injection.
`tives [Kingston et ai .• U.S. Pat. No. 5,059,699 (1991)1.
`Protein microspheres have been reported in the liter-
`and amino acid esters [Mathew et ai., J. Med. Chern. 6S ature as carriers of pharmacological or diagnostic
`agents. Microspheres of albumin have been prepared by
`Vol. 35:145- 151 (1992)] which show significant biologi-
`cal activity. Modifications to produce a water-soluble
`either beat denaturation or chemical crosslinking. Heat
`derivative facilitate the intravenous delivery of taxol
`denatured microspheres are produced from an ernulsi·
`
`Apotex v. Abraxis - IPR20 18-00151 , Ex. 1003, p.03 of 12
`
`

`

`4
`Iration time relDtive to administration volwnes and
`times required by prior art delivery systems (e.g., intra(cid:173)
`venous infusion of appwximately one to twO liters of
`fluid over a 24 hour period are required to deliver a
`typical human dose of 2QO...4OO mg of taxol).
`In accordance with another embodiment of the pres(cid:173)
`ent invention, we have developed compositions useful
`for in vivo delivery of substantially water insoluble
`pharmacologically active agents. Invention composi(cid:173)
`tions comprise substantially water insoluble pharmaco(cid:173)
`logically active agents (as a solid or liquid) contained
`within a polymeric shell. The polymeric shell is a bi(cid:173)
`ocompatible polymer, crosslinked by the presence of
`disulfide bonds. The polymeric shell, containing sub(cid:173)
`stantially water insoluble pharmacologically active
`agents therein, is then suspended in a biocompatible
`aqueous liquid for administration.
`
`5,439,686
`
`3
`lied mi.l;t\lre (e.g., alblllIlin, the agent to be incorpo(cid:173)
`rated, and a suitable oil) at temperatures between 100'
`C. and l~O' C. The microspheres are then washed with
`a suitable solvent and stored. Leucu.ta et al. [Interna(cid:173)
`tional Journal of Pharmaceutics Vol. 41:213-217 ( 1988)} 5
`describe the method of preparation of heal denatured
`microsphcres.
`The procedure for preparing chemically crosslinked
`microspheres involves treating the emulsion with glu(cid:173)
`taraldehyde to cr~link the protein, followed by wash- 10
`ing and stOTage. Lee et al. {Science VoL 2 13:233-235
`(1981)] and U,S. Pal. No. 4,671,954 teach this method of
`preparation.
`The above techniques for the preparation of protein
`microspheres as carriers of pharmacologically active 1 S
`agents, although suitable for the delivery of water-solu(cid:173)
`ble agents, are incapable of entrapping water-insoluble
`ones. This limitation is inherent in the technique of
`preparation which relies on cIosslinking or heal dena(cid:173)
`Illration of the protein component in the aqueous phase 20
`of a water-in-oil cmulsion. Any aqueous-soluble agent
`dissolved in the protein-containing aqueous phase may
`be entrapped within the resultant crvsslinked or heat-
`denatured pwtein matrix, but a poorly aqueous-soluble
`or oil-s.oluble agent cannot be incorporated into a pro- 25
`ttin matrix fonned by these t~hniques.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`In accordance with the present invention, there are
`provided compositions for in vivo delivery ofa substan-
`tia1ly water insoluble pharmacologically active agent,
`wherein said agent is a solid or liquid substantially
`completely contained within a polymeric shell,
`wherein the largest cros.s-sectional dimension of said
`shell is no greater than about 10 microns.
`wherein said polymeric shell comprises a biocompati-
`BRIEF DESCRIPTION OF THE INVENTION
`ble polymer which is substantially crosslinked by
`Thus it is an object of this invention to deliver phar-
`way of disulfide bonds, and
`mll.C(liogically active agents (e.g., taxol, taxane, Tax- 30
`otere, and the like) in unmodified fonn in a composition
`wherein said polymeric shell containing phannaco-
`logically active agent therein is suspended in a
`that does not cause allergic reactions due to the pres-
`biocompatible aqueous liquid.
`ence of added emulsifiers and solubilizing agents, as are
`As used herein, the teon "in vivo delivery" refers to
`currently employed in drug delivery.
`It is a further object of the present invention to de- 35 delivery of a pharmacologically active agent by such
`routes of administration as oral, intravenous, subcutane-
`liver phannacologica1ly active agents in a composition
`of microparticles suspended in a suitable biocompatible
`OllS, intraperitoneal, intrathecal, intramuscular, inhala_
`tional, topical, transdennal, suppository (rectal), pes-
`liquid.
`It is yet another object of the invention to deliver
`sary (vaginal). and the like.
`pharmacologically active agents enclosed within a pol- 40 As used herein, the term "micron" refers to a unit of
`ymer shell which is further suspended in a biocompati-
`measure of one one-thousandth of a millimeter.
`As used herein, the tenn "biocompatible" describes a
`ble liquid.
`These and other objects of the invention will become
`substance that does not appreciably alter or affect in any
`apparent upon reIJiew of the specification and claims.
`adverse way, the biological system into which it is in-
`In accordance with the present invention, we have 45 treduced.
`discovered that substantially water insoluble pharmaco-
`Key differences betw~n the pharmacologically ac-
`logically active agents can be delivered in the form of
`tive agents contained in a polymeric shell according to
`microparticles that are suitable for parenteral adminis-
`the invention and protein microspheres of the prior an
`tration in aqueous suspension. This mode of delivery
`are in the nature of formation and the final state of the
`obviates the necessity for administration of substantially SO protein after fonnation of the particle, and its ability to
`carry poorly aqueous-soluble or substantially aqueous-
`water insoluble pharmacologically active agents (e.g.,
`taxol) in an emulsion containing, for example, ethanol
`insoluble agents. In accordance with the present inven-
`and polyethoxylated castor oil, diluted in normal saline
`tion, the polymer (e.g., a protein) is selectively chemi-
`(see, for example, Norton et al., in Abstracts of the 2nd
`cally crosslinked through the format ion of disulfide
`National Cancer Institute Workshop on Taxol &. Taxus, 55 bonds through, for example, the amino acid cysteine
`that occurs in the natural structure of a number of pro-
`Scp. 23-24, 1992). A disadvantage of such known com-
`teins. A sonication process is used to disperse a dispers-
`positions is their propensity to produce allergic side
`ing agent containing dissol ved or suspended pharmaco-
`effects.
`The delivery of substantially water insoluble pharma-
`logiCally active agent into an aqueous solution of a
`cologically active agents in the form of a microparticu- 60 bioeompatibJe polymer bearing sulfhydryl or disulfide
`groups (e.g., albumin) whereby a shell of crosslinked
`late suspension allows some degree of targeting to or-
`gans such as the liver, lungs, spleen, lymphatic eircula-
`polymer is formed around fine droplets of non-aqueous
`tion, and the like, through the use of panicles of varying
`medium. The sonication process produces cavitation in
`size, and through administration by different routes.
`the liquid that causes tremendous local heating and
`The invention method of delivery further allows the 6S results in the formation of superoxide ions that crosslink
`the polymer by oxidizing the sulfuydryl residues (and-
`administration of substantially water insoluble pharma·
`cologically active agents employing a much smaller
`lor disrupting existing disulfide bonds) to fonn new,
`volume of liquid and requiring greatly reduced adminis-
`crosslinking disulfide bonds.
`
`Apotex v. Abraxis - [PR20 18-00 lSI , Ex. 1003 , p.04 of 12
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`

`

`5,439,686
`
`5
`6
`OClanes, halocarbons, renograrm, and the like), mag(cid:173)
`In oontra:st to the invention process, the prior an
`netic contrast agents (e.g., fluorocarbons, lipid soluble
`method of glutaraldehyde crosslinking is nonspecific
`and e;ssenlially TC:active with any nucleophilic group
`paramagnetic compounds, and the like), as well as other
`diagnostic agents which cannot readily be delivered
`present in the protein structure (e.g., amines and hy(cid:173)
`without some physical andlor chemical modification to
`droxyls). H eat denaturation as taught by the prior art 5
`accomodate the substantially water insoluble nature
`significantly and irreversibly alters protein structure. In
`thereof.
`contrast, disulfKie formation contemplate<! by the pres(cid:173)
`. Examples of agents of nutritional value contemplated
`cnt invention does not substantially denature the pro(cid:173)
`for usc in the practice of the present invention include
`tein. In addition, particles of substantially water insolu(cid:173)
`ble pharmacologica.1Jy active agents C(lntained within a 10
`amino acids, sugars, proteins, carbohydrates, fat·soluble
`vitamins (e.g., vitamins A, D, E , K, and the lik~) or fat,
`shell differ from crosslinked or heat denatured protein
`microspheres of the prior art because the polymeric
`or combinations of allY twO or more thereof.
`shell produced by the invention process is relatively
`A number of biocompatible polymers may be em·
`ployed in the practice of the present invention for the
`thin compared to the diameter of the coated particle. It
`formatio n of the polymeric shell which surrounds the
`has been determined (by transmission electron micr05- l~
`substantially water insoluble pharmacologically active
`copy) that the "shell thickness" afthe polymeric coat is
`approximately 25 nanometers for a coated panicle hav(cid:173)
`agents. Essentially any polymer, natural or synthetic,
`ing a diameter of 1 micron (1000 nanometers). In con·
`bearing sulfhydryl groups or disulfide bonds within its
`uast, microspheres of the prior an do not have protein
`structure may be utiliud for the preparation of a disul·
`shells, but rather, bave protein dispersed throughout the 20
`fide crosslinked shell about panicles of substantially
`water insoluble pharmacologically active agents. The
`volume of the microsphere.
`The polymeric shell containing solid or liquid cores
`sulfhydryl groups or disulfide linkages may be preexist·
`ing within the polymer structure or they may be intro·
`of pharmacologically active agent allows for the deliv·
`ery of high doses of the pharmacologically active agent
`duced by a suitable chemical modification. For exam·
`pIe, natural polymers such as proteins, oligopeptides,
`in relatively small volumes. TIlls minimizes patient dis· 2S
`polynucleic acids, polysaccharides (e.g., starch, cellu·
`comfort at receiving large volumes of flu id and mini·
`lose, dcxtrans, alginateS, chitosan, pectin, hyaluronic
`mizes hospital stay. In addition, the walls of the poly·
`acid, and the like), and so on, are candidates for such
`meric shell are generally completely degradable in vivo
`by plOteolytic elll:ymes (e.g., when the polymer is a
`modification.
`protein), resulting in no ~ide effect~ from the delivery 30
`As e1i&mplCli of suitable biocompatible polymcr5,
`system as is the case with current formulations.
`naturally occurring or synthetic proteins may be em·
`ployed, so long as such proteins have sufficient cysteine
`According to this embodiment of the present inven(cid:173)
`residues within their amino acid sequences so that cross·
`tion, panicles of substantially water insoluble pbanna(cid:173)
`linking (through disulfide bond formation, for example,
`cologically active agents are contained within a shell
`as a r~nlt of oxidation durin g sonication) can occur.
`hltving II cm!l.~~tional diam~er of no greater than .1S
`Examples of suitable proteins include albumin (which
`about 10 microns. A cross-sectional diameter of less
`than 5 microns is more preferred, while a cross--sec-
`contains 35 cysteine residues), insulin (which contains 6
`cysteines), hemoglobin (which contains 6 cysteine resi·
`tiona! diameter of less than I micron is presently the
`dues per a2fh unit), lysozyme (which contain:s 8 cyste-
`most preferred for the intravenous route of administra-
`40 ine residues),
`immunoglobulins, a-Z-macroglobulin,
`Mn.
`fibronectin, vitronectin, fibrinogen, and the like.
`Substantially water insoluble phartnacologically ae-
`tive agents contemplated for use in the practice of the
`A presently preferred protein for use in the formation
`of a polymeric shell is albumin. Optionally, proteins
`present
`invention
`include pharmaceutically active
`such as a.-Z.macroglobulin, a known opsonin, could be
`agents, diagnostic agent5, agents of nutritional value,
`and the like. Examples of phannaceutically active 45 used to enhance uptake of the shell encased particles of
`agents include taxol (as used herein, the term "taxol" is
`substantially water insolUble pharmacologically active
`intended to include taxol analogs and prodrugs, laAanes,
`agents by macrophage.like cells, or to enhance the up-
`and other taxol·like drug" e.g., Taxotere, and the like),
`take of the shell encased particles into the liver and
`camptothecin and derivatives thereof (which com·
`spleen.
`pounds have great promise for the treatment of colon 50
`Similarly, synthetic polypeptides containing cysteine
`residues are also good candidates for fonnation of a
`cancer), aspirin, ibuprofen, piroxicam, cimetidine. sub-
`stantially water insolllb1e steroids (e.g., estrogen, pred-
`shell about the substantially water insoluble pharmaco-
`nisolone, cortisone, hydrocortisone, diflorasone, and
`logically active agents. In addition, polyvinyl alcohol,
`the like), drugs such as phenesterine, duanorubicin,
`polyhydroxy~hyl methacrylate, polyacrylic acid,
`doxorubicin, mitOtane, visadine, halonitrosoureas, an- 5S po!yethyloxazolinc, polyacrylamide, polyvinyl pyrroli-
`dillone, and the like, are good candidates for chemical
`throcylines, ellipticine, diazepam, and the like, anaes·
`thelies such as methoxyfluorane, isofluorane, enfluo-
`modification (to introduce sulfhydryl and/or disulfide
`rane, halothane, benzocaine, dantrolene, barbiturates,
`linkages) and shell formation (by causing the crosslink-
`and the like. In addition, also contemplated are substan-
`ing thereof).
`tially water insoluble immunosuppressive agents, such 60
`In the preparation of invention compositions, one can
`as, for example, cyclosporines, axathioprine, FK506,
`optionally employ a dispersing agent to suspend or
`prednisone, and the like. A presently preferred pharma-
`dissolve the substantially water insoluble pharmacologi-
`ceutically active agent for use in the practice of the
`cally active agent. Dispersing agents contemplated for
`use in the practice of the present invention include any
`present invention is taxo!, which is commercially avail-
`able from the manufacturer as needle-like crystals.
`65 nonaqueous liquid that is capable of suspending or dis-
`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
`
`Apotex v. Abraxis - IPR20 18-001 S I, Ex. 1003, p.OS of 12
`
`

`

`7
`agent itst':lf. Examples include vegetable oils (e.g., soy(cid:173)
`bean oi1, coconut oil, olive oil, safflower oil, cotton seed
`oil, and the like), aliphatic, cycloaliphatic, or aromatic
`hydrocarbons having 4-30 carbon atoms (e.g., n..dodec(cid:173)
`ane, n·decane, n-haane, cyclohexane, t oluene, bente-ne,
`and the like), a1ipha.tic or Momatic alcohols having 2-30
`carbon atoms (e.g., octanol, and the like). aliphatic or
`aromatic esters having 2-30 carbon atoms (e.g., ethyl
`caprylate (oc:tanoate), and the like), alkyl, aryl, or cyclic
`ethers having 2-30 carbon atoms (e.g., diethyl ether, 10
`tetrahydrofuran, and the like), alkyl or aryl haJides
`having 1-30 carbon atoms (and optionally more than
`one halogen
`substituent, e.g., CH3C1. CH2CI2,
`CHJC!-CH1Q, and the like), ketones having 3-30
`carbon atoms (e.g., acetone, methyl ethyl ketone, and I'
`the like). polyalkylene glycols (e.g., polyethylene gly(cid:173)
`col, and the like), or combinations of any two or more
`thereof.
`Especially preferred combinations of dispersing
`agents include volatile liquids such as dicbloromethane, 20
`ethyl acetate, benzene, and the like (i.e., solvents that
`have a high degree of solubility for the pharmacologi(cid:173)
`cally active agent, and are soluble in the other dispers(cid:173)
`ing agent employed), along with a higher molecular 25
`weight (less volatile) dispening agent. When added to
`the other dispersing agent, these volatile additivC$ help
`to drive the solubility of the pharmacologically active
`agent into the dispersing agent. This is desirable since
`this Step is usually time consuming. Following dissolu- 30
`tion, the volatile component may be removed by evapo(cid:173)
`ration (optionally under vacuum).
`Particles of pharmacologically active agent substan·
`tially completely contained within a polymeric shell,
`prepared as described above, are delivered as a suspen· J5
`sian in a biocompatible aqueous liquid. This liquid may
`be selected from water, saline, a solution containing
`appropriate buffers, a solution containing nutritional
`agents such as amino acids, sugars, proteins, carbohy-
`drates, vitamins or fat, and the like.
`In accordance with another embodiment of the pres·
`ent invention, there is provided a method for the prepa(cid:173)
`ration of a substantially water insoluble pharmacologi·
`cally active agent for in vivo delivery, said method
`comprising subjecting a mixture comprising:
`dispersing agent containing said pharmacologically
`active agent dispened therein, and
`aqueous mediwn containing biorompatible polymer
`capable of being crosslinked by disulfide bonds
`to sonication conditions for a time sufficient to promote SO
`crosslinking of said biocompatible polymer by disulfide
`bonds.
`A nonobvious feature of the above-described process
`is in the choice of dispersing agent, specificaUy with
`respect to the polarity of the dispersing agent. The 55
`formation of a shell about the panicles of phannacologi(cid:173)
`cally active agent involves unfolding and reorientation
`of the polymer at the interface between the aqueous and
`non-aqueous phases such that the hydrophilic regions
`within the polymer are exposed to the aqueous phase 60
`while the hydrophobic regions within the polymer are
`oriented towards the non-aqueous phase. In order to
`effect unfolding of the polymer, or change the confor(cid:173)
`mation thereof, energy must be supplioo to the polymer.
`The interfacial free energy (interfacial tension) between 65
`the two liquid phases (i.e., aqueous and nOD·aqueouS)
`contributes to changes in polymer conformation at that
`interface. Thennal energy also contributes to the en-
`
`40
`
`8
`t:rgy pool rcquin:d for unfolding: and/or changt: of po}.
`ymer conformation.
`Thennal energy input is a function of such variables
`as the acoustic power employed in the sonication pro(cid:173)
`cess, the sonication time, the nature of the material
`being subjected to sonication, the volume of the mate·
`rial being subjected to sonication, and the like. The
`acoustic power of sonication processes can vary widely,
`typically falling in the range of about I up to 1000
`watlS/cml; with an acoustic power in the range of about
`SO up to 200 wattslcm2 being a presently preferred
`range. Similarly, sonication rime can vary widely, typi(cid:173)
`cally falling in the range of a few seconds up to about 5
`minutes. Preferably, sonication time will fall in the
`range of about 15 up to 60 seconds. Those of skill in the
`art recognize that the higher the acoustic power ap(cid:173)
`plied, the less sonication time is required, and vice
`v<=
`The illlerfacial free energy is directly proportional to
`the polarity difference between the two liquids. Thus at
`a given operating temperature a minimum free energy
`at the interface between the twO liquids is essential to
`form the desired polymer shell. Thus, if a homologous
`series o f disperswg agents is taken with a gradual
`change in polarity, e.g., ethyl esters of alkanoic acids,
`then higher homologues are increasingly nonpolar, i.e.,
`the interl'acial tension between these dispersing agents
`and water increases as the number of carbon atoms in
`the ester increases. Thus it is found that, although ethyl
`acetate is water·immiscible (ie., au ester of a 2 carbon
`acid), at room temperature (_20' C.), this dispersing
`agent alone will