United States Patent
`Desai et al.
`
`[19]
`
`US005916596A
`Patent Number:
`Date of Patent:
`
`[11]
`[45]
`
`5,916,596
`Jun. 29, 1999
`
`[54]
`
`PROTEIN STABILIZED
`PHARMACOLOGICALLY ACTIVE AGENTS,
`METHODS FOR THE PREPARATION
`THEREOF AND METHODS FOR THE USE
`THEREOF
`
`0 295 941 A2 12/1988 European Pat. Off. .
`0 391 518 A2 2/1990 European Pat. Off.
`
`(List continued on neXt page.)
`
`OTHER PUBLICATIONS
`
`Inventors: Neil P. Desai, Los Angeles; Chunlin
`Tao, Beverly Hills; Andrew Yang,
`Rosemead; Leslie Louie, Montebello;
`Tianli Zheng; ZhiWen Yao, both of
`Culver City; Patrick S00n-Shi0ng, Los
`Angeles, all of Calif; Shl0m0
`Magdassi, Jerusalem, Israel
`
`Assignee: Vivorx Pharmaceuticals, Inc., Santa
`Monica, Calif.
`
`Appl. No.:
`
`Filed:
`
`08/720,756
`Oct. 1, 1996
`
`Related US. Application Data
`
`Continuation-in-part of application No. 08/412,726, Mar.
`29, 1995, Pat. No. 5,560,933, which is a division of appli
`cation No. 08/023,698, Feb. 22, 1993, Pat. No. 5,439,686.
`
`Int. Cl.6 ..................................................... .. A61K 9/14
`
`US. Cl. ........................ .. 424/489; 424/450; 424/465;
`424/451; 424/439
`Field of Search ................................... .. 424/489, 422,
`424/423, 475, 9.1, 9.3, 9.32, 450, 400
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3,959,457
`4,073,943
`4,247,406
`4,572,203
`
`5/1976 Speaker et al. .
`2/1978 Wretlind et al. .
`1/1981 Widder et al. .
`2/1986 Feinstein .
`
`(List continued on neXt page.)
`
`FOREIGN PATENT DOCUMENTS
`
`Burgess et al., “Potential use of albumin microspheres as a
`drug delivery system. I. Preparation and in vitro release of
`steroids,” International Journal of Pharmaceutics,
`39:129—136 (1987).
`(List continued on neXt page.)
`Primary Examiner—Neil S. Levy
`Assistant Examiner—William E. Benston, Jr.
`Attorney, Agent, or Firm—Gray, Cary, Ware & Freidenrich;
`Stephen E. Reiter
`[57]
`
`ABSTRACT
`
`In accordance With the present invention, there are provided
`compositions and methods useful for the in vivo delivery of
`substantially Water insoluble pharmacologically active
`agents (such as the anticancer drug paclitaXel) in Which the
`pharmacologically active agent is delivered in the form of
`suspended particles coated With protein (Which acts as a
`stabilizing agent). In particular, protein and pharmacologi
`cally active agent in a biocompatible dispersing medium are
`subjected to high shear, in the absence of any conventional
`surfactants, and also in the absence of any polymeric core
`material for the particles. The procedure yields particles With
`a diameter of less than about 1 micron. The use of speci?c
`composition and preparation conditions (e.g., addition of a
`polar solvent to the organic phase), and careful selection of
`the proper organic phase and phase fraction, enables the
`reproducible production of unusually small nanoparticles of
`less than 200 nm diameter, Which can be sterile-?ltered. The
`particulate system produced according to the invention can
`be converted into a redispersible dry poWder comprising
`nanoparticles of Water-insoluble drug coated With a protein,
`and free protein to Which molecules of the pharmacological
`agent are bound. This results in a unique delivery system, in
`Which part of the pharmacologically active agent is readily
`bioavailable (in the form of molecules bound to the protein),
`and part of the agent is present Within particles Without any
`polymeric matrix therein.
`
`0 129 619 A1
`
`1/1985 European Pat. Off. .
`
`31 Claims, 2 Drawing Sheets
`
`4000
`
`3500 -
`
`3000 -
`
`2500 -
`
`2000 -
`
`1500 —
`
`1000 -
`
`500 —
`
`
`
`
`
`Tumor Volume (MW)
`
`0. Treatment Period
`+++++.
`
`n
`
`a
`
`-500
`12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
`Days Postimplant
`
`|
`
`CIPLA EXHIBIT 1026
`Page 1 of 19
`
`

`

`5,916,596
`Page 2
`
`US. PATENT DOCUMENTS
`
`6/1987 Goldberg et al. .
`4,671,954
`1/1988 Feinstein .
`4,718,433
`4,789,550 12/1988 Hommel et al. .
`4,844,882
`7/1989 Widder et al. .
`5,059,699 10/1991 Kingston et al. ..................... .. 549/511
`5,110,606
`5/1992 Geyer et al. .
`5,145,684
`9/1992 Liversidge et al. ................... .. 424/489
`
`. . . . . .. 424/9
`5,362,478 11/1994 Desai et al. . . . . . . .
`424/451
`5,439,686
`8/1995 Desai et al. ..... ..
`424/450
`5,498,421
`3/1996 Grinstaff et al. ..
`424/9.3
`5,505,932
`4/1996 Gristaff et al.
`424/9.322
`5,508,021
`4/1996 Grinstaff et al. ..
`424/9.322
`5,512,268
`4/1996 Grinstaff et al.
`424/489
`5,560,933 10/1996 Soon-Shing et al. .
`424/400
`5,650,156
`7/1997 Grinstaff et al.
`5,665,382
`9/1997 Grinstaff et al. ...................... .. 424/450
`
`FOREIGN PATENT DOCUMENTS
`
`0 361 677 A1 4/1990 European Pat. Off. .
`0 418 153 A1 3/1991 European Pat. Off. .
`0 190 050 B1 5/1991 European Pat. Off. .
`0 213 303 B1 9/1991 European Pat. Off. .
`2660556 10/1991 France .
`WO 85/00011
`1/1985 WIPO
`WO 87/01035
`2/1988 WIPO
`WO 88/01506
`3/1988 WIPO
`WO 88/07365 10/1988 WIPO
`WO 89/03674 5/1989 WIPO
`WO 90/13285 11/1990 WIPO
`WO 90/13780 11/1990 WIPO
`WO 91/15947 10/1991 WIPO
`WO 94/10980 5/1994 WIPO
`
`OTHER PUBLICATIONS
`
`Chen et al., “Comparison of albumin and casein micro
`spheres as a carrier for doXorubicin,” J. Pharm. Pharmacol.,
`39:978—985 (1987).
`Feinstein et al., “TWo—Dimensional Constrast Echocardio
`graphy. I. In Vitro Development and Quantitative Analysis
`of Echo Contrast Agents,” JACC, 3(1):14—20 (1984).
`Grinstaff & Suslick, “Nonaqueous Liquid Filled Microcap
`sules,” Polym. Prepr, 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 Microcapsules
`and Microspheres,” Journal of Pharmaceutical Sciences,
`70(4):358—363 (1981).
`Klibanov et al., “Amphipathic polyethyleneglycols effec
`tively prolong the circulation time of liposomes,” FEBS,
`268(1):235—237 (1990).
`Koenig & MeltZer, “Effect of Viscosity on the SiZe of
`Microbubbles Generated for Use as Echocardiographic Con
`trast Agents,” Journal of Cardiovascular ultrasonography,
`5(1):3—4 (1986).
`Molecular Biosystems, Inc.,
`Investigator’s Package”.
`
`“ALBUNEX—Preclinical
`
`Moseley et al., “Microbubbles: A Novel MR Susceptibility
`Contrast Agent,” 10th Annual Meeting of Society of Mag
`netic Resonance in Medicine (1991).
`Suslick & Grinstaff, “Protein Microencapsulation of Non
`aqueous Liquids,” J. Am. Chem. Soc., 112(21):7807—7809
`(1990).
`Willmott & Harrison, “Characterisation of freeZe—dried
`albumin microspheres 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,” Science,
`213(10):233—235 (1981).
`BaZile et. al., “Body distribution of fully biodegeradable
`[14C]—poly(latic acid) nanoparticles coated With albumin
`after parenteral adminstration to rats” Biomaterials,
`13:1093—1102 (1992).
`Boury et al., “Dilatational Properties of Absorbed Poly(D,
`L—lactide) and Bovine Serum Albumin Monolayers at the
`Dichloromethane/Water Interface” Langmuir; 11: 1636—1644
`(1995).
`Calvo et al., “Comparative in Vitro Evaluation of Several
`Colloidal Systems, Nanoparticles, Nanocapsules, and
`Nanoemulsions, as Ocular Drug Carriers” J. Pharm. Sci.,
`85(5):530—536 (1996).
`Cavalier et al., “The formation and characterization of
`hydrocortisone—loaded poly((+i)—lactide) microspheres” J.
`Pharm. Pharmacol., 38:249—253 (1985).
`Kumar et al., “Binding of TaXol to Human Plasma, Albumin
`and—Acid Glycoprotein” Research Communications in
`Chemical Pathology and Pharmacology, 80(3):337—344
`(1993).
`Lee et al., “Serum Albumin Beads: An Injectable, Biode
`gradable System for the Sustained Release of Drugs” Sci
`ence, 213:233—235 (1981).
`Leucuta et al., “Albumin microspheres as a drug delivery
`system for epirubicin: pharmaceutical, pharmacokinetic and
`biological aspects” International Journal of Pharmaceutics,
`41:213—217 (1988).
`Liversideg—Merisko et al., “Formulation and Antitumor
`Activity Evaluation of Nanocrystalline Suspensions of
`Poorly Soluble Anticacer Drugs” Pharmaceutical Research,
`13(2):272—278 (1996).
`MatheW et al., “Synthesis and Evaluation of Some Water—
`Soluble Prodrugs and Derivatives of TaXol With Antitumor
`Activity” J. Med. Chem., 35:145—151 (1992).
`Norton et al.,Abstracts of the 2nd National Cancer Institute
`Workshop on Taxol & Taxus, Sep. 23—24, 1992).
`Wani et al., “Plant Antitumor Agents. VI. The Isolation and
`Structure of ToXol, a Novel Antileukemic and Antitumor
`Agents from Taxus brevifolial’z” J. Am. Chem. Soc.,
`93:2325—2327 (1971).
`
`CIPLA EXHIBIT 1026
`Page 2 of 19
`
`

`

`U.S. Patent
`
`Jun. 29, 1999
`
`Sheet 1 of2
`
`5,916,596
`
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`CIPLA EXHIBIT 1026
`Page 3 of 19
`
`

`

`US. Patent
`
`Jun. 29, 1999
`
`Sheet 2 0f 2
`
`5,916,596
`
`
`
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`
`CIPLA EXHIBIT 1026
`
`Page 4 of 19
`
`CIPLA EXHIBIT 1026
`Page 4 of 19
`
`

`

`1
`PROTEIN STABILIZED
`PHARMACOLOGICALLY ACTIVE AGENTS,
`METHODS FOR THE PREPARATION
`THEREOF AND METHODS FOR THE USE
`THEREOF
`
`RELATED APPLICATIONS
`
`This application is a continuation-in-part of US. Ser. No.
`08/412,726, ?led Mar. 29, 1995, noW issued as US. Pat. No.
`5,560,933, Which is, in turn, a divisional of US. Ser. No.
`08/023,698, ?led Feb. 22, 1993, noW issued as US. Pat. No.
`5,439,686, the entire contents of both of Which are hereby
`incorporated by reference herein in their entirety.
`
`FIELD OF THE INVENTION
`
`The present invention relates to methods for the produc
`tion of particulate vehicles for the intravenous administra
`tion of pharmacologically active agents, as Well as novel
`compositions produced thereby. In a particular aspect, the
`invention relates to methods for the in vivo delivery of
`substantially Water insoluble pharmacologically active
`agents (e.g., the anticancer drug taXol). In another aspect,
`dispersible colloidal systems containing Water insoluble
`pharmacologically active agents are provided. The sus
`pended particles are encased in a polymeric shell formulated
`from a biocompatible polymer, and have a diameter of less
`than about 1 micron. Invention colloidal systems are pre
`pared Without the use of conventional surfactant or any
`polymeric core matrix. In a presently preferred aspect of the
`invention, there is provided a method for preparation of
`extremely small particles Which can be sterile-?ltered. The
`polymeric shell contains particles of pharmacologically
`active agent, and optionally a biocompatible dispersing
`agent in Which pharmacologically active agent can be either
`dissolved or suspended. Thus, the invention provides a drug
`delivery system in either liquid form or in the from of a
`redispersible poWder. Either form provides both immedi
`ately bioavailable drug molecules (i.e., drug molecules
`Which are molecularly bound to a protein), and pure drug
`particles coated With a protein.
`
`BACKGROUND OF THE INVENTION
`Intravenous drug delivery permits rapid and direct equili
`bration With the blood stream Which carries the medication
`to the rest of the body. To avoid the peak serum levels Which
`are achieved Within a short time after intravascular injection,
`administration of drugs carried Within stable carriers Would
`alloW gradual release of the drugs inside the intravascular
`compartment folloWing a bolus intravenous injection of the
`therapeutic nanoparticles.
`Injectable controlled-release nanoparticles can provide a
`pre-programmed duration of action, ranging from days to
`Weeks to months from a single injection. They also can offer
`several profound advantages over conventionally adminis
`tered medicaments, including automatic assured patient
`compliance With the dose regimen, as Well as drug targeting
`to speci?c tissues or organs (Tice and Gilley, Journal of
`Controlled Release 2:343—352 (1985)).
`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
`1—3 microns in diameter). The microcirculation in most
`
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`organs and tissues alloWs the free passage of these blood
`cells. When microthrombii (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. Injection into
`the circulation of particles greater than 10—15 microns in
`diameter, therefore, must be avoided. A suspension of par
`ticles less than 7—8 microns, is hoWever, relatively safe and
`has been used for the delivery of pharmacologically active
`agents in the form of liposomes 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
`Microspheres-Biomea'ical 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 mono
`nuclear phagocyte system (MPS). Particles larger than about
`7 microns Will, after intravenous 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 administra
`tion 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-insoluble pharmacologically active agents dissolved
`in oils and emulsi?ed With Water in the presence of surfac
`tants such as egg phosphatides, pluronics (copolymers of
`polypropylene glycol and polyethylene glycol), polyglyc
`erol oleate, etc. PCT International Publication No. W085/
`00011 describes pharmaceutical microdroplets of an anaes
`thetic coated With a phospholipid such as dimyristoyl
`phosphatidylcholine having suitable dimensions for intrad
`ermal or intravenous injection.
`An eXample of a Water-insoluble drug is taXol, a natural
`product ?rst isolated from the Paci?c YeW tree, Taxus
`brevifolia, by Wani et al. (J. Am. Chem. Soc. 93:2325
`(1971)). Among the antimitotic agents, taXol, Which contains
`a diterpene carbon skeleton, eXhibits a unique mode of
`action on microtubule proteins responsible for the formation
`of the mitotic 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 tubulin, 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 tumor models such as
`the B16 melanoma, L1210 leukemias, MX-1 mammary
`tumors, and CS-1 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 Administration for treatment of ovarian
`
`CIPLA EXHIBIT 1026
`Page 5 of 19
`
`

`

`3
`cancer. The poor aqueous solubility of taxol, however,
`presents a problem 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 cremaphor to solubiliZe the
`drug. The human clinical dose range is 200—500 mg. This
`dose is dissolved in a 111 solution of ethanol1cremaphor and
`diluted to one liter of ?uid given intravenously. The crema
`phor currently used is polyethoxylated castor oil.
`In phase I clinical trials, taxol itself did not shoW exces
`sive toxic effects, but severe allergic reactions Were caused
`by the emulsi?ers employed to solubiliZe the drug. The
`current regimen of administration involves 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 structure
`With functional groups that impart enhanced Water
`solubility. Among them are the sulfonated derivatives
`(Kingston et al., US. Pat. No. 5,059,699 (1991)), and amino
`acid esters (MatheW et al., J. Med. Chem. 351145—151
`(1992)) Which shoW signi?cant biological activity. Modi?
`cations to produce a Water-soluble derivative facilitate the
`intravenous delivery of taxol 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.
`Protein microspheres have been reported in the literature
`as carriers of pharmacological or diagnostic agents. Micro
`spheres of albumin have been prepared by either heat
`denaturation or chemical crosslinking. Heat denatured
`microspheres are produced from an emulsi?ed mixture (e.g.,
`albumin, the agent to be incorporated, and a suitable oil) at
`temperatures betWeen 100° C. and 150° C. The micro
`spheres are then Washed With a suitable solvent and stored.
`Leucuta et al. (International Journal of Pharmaceutics
`411213—217 (1988)) describe the method of preparation of
`heat denatured microspheres.
`The procedure for preparing chemically crosslinked
`microspheres involves treating the emulsion With glutaral
`dehyde to crosslink the protein, folloWed by Washing and
`storage. Lee et al. (Science 2131233—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-soluble agents,
`are incapable of entrapping Water-insoluble ones. This limi
`tation is inherent in the technique of preparation Which relies
`on crosslinking or heat denaturation of the protein compo
`nent 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 protein matrix formed by these techniques.
`One conventional method for manufacturing drug
`containing nanoparticles comprises dissolving polylactic
`acid (or other biocompatible, Water insoluble polymers) in a
`Water-immiscible solvent (such as methylene chloride or
`other chlorinated, aliphatic, or aromatic solvent), dissolving
`the pharmaceutically active agent in the polymer solution,
`adding a surfactant to the oil phase or the aqueous phase,
`forming an oil-in-Water emulsion by suitable means, and
`evaporating the emulsion sloWly under vacuum. If the oil
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`droplets are suf?ciently small and stable during evaporation,
`a suspension of the polymer in Water is obtained. Since the
`drug is initially present in the polymer solution, it is possible
`to obtain by this method, a composition in Which the drug
`molecules are entrapped Within particles composed of a
`polymeric matrix. The formation of microspheres and nano
`particles by using the solvent evaporation method has been
`reported by several researchers (see, for example, Tice and
`Gilley, in Journal of Controlled Release 21343—352 (1985);
`Bodmeier and McGinity, in Int. J. Pharmaceutics 431179
`(1988); Cavalier et al., in J. Pharm. Pharmacol. 381249
`(1985); and D’SouZa et al., WO 94/10980) While using
`various drugs.
`BaZile et. al., in Biomaterials 1311093 (1992), and Spen
`lehauer et al., in Fr Patent 2 660 556, have reported the
`formation of nanoparticles by using tWo biocompatible
`polymers, one (e.g. Polylactide) is dissolved in the organic
`phase, together With an active component such as a drug, and
`the other polymer, such as albumin is used as the surface
`active agent. After emulsi?cation and removal of the
`solvent, nanoparticles are formed, in Which the drug is
`present inside the polymeric matrix of the polylactide par
`ticles.
`The properties of the polymer solution from Which the
`polymeric matrix is formed are very important to obtain the
`proper emulsion in the ?rst stage. For example, polylactide
`(the polymer commonly used in the preparation of injectable
`nanoparticles), has a surface activity Which causes the rapid
`adsorption thereof at the dichloromethane-Water interface,
`causing reduced interfacial tension (see, for example, Boury
`et al., in Langmuir 1111636 (1995)), Which in turn improves
`the emulsi?cation process. In addition, the same researchers
`found that Bovine Serum Albumin (BSA) interacts With the
`polylactide, and penetrates into the polylactide monolayer
`present at the oil-Water interface. Therefore, it is expected,
`based on the above reference, that emulsi?cation during the
`conventional solvent evaporation method is greatly favored
`by the presence of the surface active polymer (polylactide)
`in the nonaqueous organic phase. In fact, the presence of
`polylactide is not only a suf?cient condition, but it is actually
`necessary for the formation of nanoparticles of suitable siZe.
`Another process Which is based on the solvent evapora
`tion method comprises dissolving the drug in a hydrophobic
`solvent (e.g., toluene or cyclohexane), Without any polymer
`dissolved in the organic solvent, adding a conventional
`surfactant to the mixture as an emulsi?er, forming an oil
`in-Water emulsion, and then evaporating the solvent to
`obtain dry particles of the drug (see, for example, Sjostrom
`et al., in J. Dispersion Science and Technology 15189—117
`(1994)). Upon removal of the nonpolar solvent, precipitation
`of the drug inside the solvent droplets occurs, and submicron
`particles are obtained.
`It has been found that the siZe of the particles is mainly
`controlled by the initial siZe of the emulsion droplets. In
`addition, it is interesting to note that the ?nal particle siZe is
`reported to decrease With a decrease in the drug concentra
`tion in the organic phase. This ?nding is contrary to the
`results reported herein, Wherein no conventional surfactant
`is used for the preparation of nanoparticles. In addition, it is
`noted by the authors of the Sjostrom paper that the drug
`used, cholesteryl acetate, is surface active in toluene, and
`hence may be oriented at the oil-Water interface; therefore
`the concentration of drug at the interface is higher, thus
`increasing the potential for precipitation.
`Formation of submicron particles has also been achieved
`by a precipitation process, as described by Calvo et al. in J.
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`CIPLA EXHIBIT 1026
`Page 6 of 19
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`

`5,916,596
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`5
`Pharm. Sci. 85:530 (1996). The process is based on dissolv
`ing the drug (e.g., indomethacin) and the polymer (poly
`caprolactone) in methylene chloride and acetone, and then
`pouring the solution into an aqueous phase containing a
`surfactant (PoloXamer 188), to yield submicron siZe par
`ticles (216 nm). HoWever, the process is performed at
`solvent concentrations at Which no emulsion is formed.
`
`BRIEF DESCRIPTION OF THE INVENTION
`
`Thus it is an object of this invention to deliver pharma
`cologically active agents (e.g., taXol, taXane, TaXotere, and
`the like) in unmodi?ed form in a composition that does not
`cause allergic reactions due to the presence of added emul
`si?ers and solubiliZing agents, as are currently employed in
`drug delivery.
`It is a further object of the present invention to deliver
`pharmacologically active agents in a composition of micro
`particles or nanoparticles, optionally suspended in a suitable
`biocompatible liquid.
`It is yet another object of the present invention to provide
`a method for the formation of submicron particles
`(nanoparticles) of pharmacologically active agents by a
`solvent evaporation technique from an oil-in-Water emulsion
`using proteins as stabiliZing agents in the absence of any
`conventional surfactants, and in the absence of any poly
`meric core material.
`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 dis
`covered that substantially Water insoluble pharmacologi
`cally active agents can be delivered in the form of micro
`particles or nanoparticles that are suitable for parenteral
`administration 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 poly
`ethoXylated 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 compositions is their
`propensity to produce allergic side effects.
`Thus, in accordance With the present invention, there are
`provided methods for the formation of nanoparticles of
`pharmacologically active agents by a solvent evaporation
`technique from an oil-in-Water emulsion prepared under
`conditions of high shear forces (e.g., sonication, high pres
`sure homogeniZation, or the like) Without the use of any
`conventional surfactants, and Without the use of any poly
`meric core material to form the matriX of the nanoparticle.
`Instead, proteins (e.g., human serum albumin) are employed
`as a stabiliZing agent.
`The invention further provides a method for the repro
`ducible formation of unusually small nanoparticles (less
`than 200 nm diameter), Which can be sterile-?ltered through
`a 0.22 micron ?lter. This is achieved by addition of a Water
`soluble solvent (e.g. ethanol) to the organic phase and by
`carefully selecting the type of organic phase, the phase
`fraction and the drug concentration in the organic phase. The
`ability to form nanoparticles of a siZe that is ?lterable by
`0.22 micron ?lters is of great importance and signi?cance,
`since formulations Which contain a signi?cant amount of
`any protein (e.g., albumin), cannot be steriliZed by conven
`tional methods such as autoclaving, due to the heat coagu
`lation of the protein.
`In accordance With another embodiment of the present
`invention, We have developed compositions useful for in
`
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`6
`vivo delivery of substantially Water insoluble pharmacologi
`cally active agents. Invention compositions comprise sub
`stantially Water insoluble pharmacologically active agents
`(as a solid or liquid) contained Within a polymeric shell. The
`polymeric shell is a crosslinked biocompatible polymer. The
`polymeric shell, containing substantially Water insoluble
`pharmacologically active agents therein, can then be sus
`pended in a biocompatible aqueous liquid for administra
`tion.
`The invention further provides a drug delivery system in
`Which part of the molecules of pharmacologically active
`agent are bound to the protein (e.g., human serum albumin),
`and are therefore immediately bioavailable upon adminis
`tration to a mammal. The other portion of the pharmaco
`logically active agent is contained Within nanoparticles
`coated by protein. The nanoparticles containing the phar
`macologically active agent are present as a pure active
`component, Without dilution by any polymeric matriX.
`A large number of conventional pharmacologically active
`agents circulate in the blood stream bound to carrier proteins
`(through hydrophobic or ionic interactions) of Which the
`most common eXample is serum albumin. Invention meth
`ods and compositions produced thereby provide for a phar
`macologically active agent that is “pre-bound” to a protein
`(through hydrophobic or ionic interactions) prior to admin
`istration.
`The present disclosure demonstrates both of the above
`described modes of bioavailability for TaXol (PaclitaXel), an
`anticancer drug capable of binding to human serum albumin
`(see, for eXample, Kumar et al., in Research Communica
`tions in Chemical Pathology and Pharmacology 801337
`(1993)). The high concentration of albumin in invention
`particles, compared to TaXol, provides a signi?cant amount
`of the drug in the form of molecules bound to albumin,
`Which is also the natural carrier of the drug in the blood
`stream.
`In addition, advantage is taken of the capability of human
`serum albumin to bind TaXol, as Well as other drugs, Which
`enhances the capability of TaXol to absorb on the surface of
`the particles. Since albumin is present on the colloidal drug
`particles (formed upon removal of the organic solvent),
`formation of a colloidal dispersion Which is stable for
`prolonged periods is facilitated, due to a combination of
`electrical repulsion and steric stabiliZation.
`In accordance With the present invention, there are also
`provided submicron particles in poWder form, Which can
`easily be reconstituted in Water or saline. The poWder is
`obtained after removal of Water by lyophiliZation. Human
`serum albumin serves as the structural component of inven
`tion nanoparticles, and also as a cryoprotectant and recon
`stitution aid. The preparation of particles ?lterable through
`a 0.22 micron ?lter according to the invention method as
`described herein, folloWed by drying or lyophiliZation, pro
`duces a sterile solid formulation useful for intravenous
`injection.
`The invention provides, in a particular aspect, a compo
`sition of anti-cancer drugs, e.g., TaXol, in the form of
`nanoparticles in a liquid dispersion or as a solid Which can
`be easily reconstituted for administration. Due to speci?c
`properties of certain drugs, e.g., TaXol, such compositions
`can not be obtained by conventional solvent evaporation
`methods that rely on the use of surfactants. In the presence
`of various surfactants, very large drug crystals (e.g., siZe of
`about 5 microns to several hundred microns) are formed
`Within a feW minutes of storage, after the preparation
`process. The siZe of such crystals is typically much greater
`than the alloWed siZe for intravenous injection.
`
`CIPLA EXHIBIT 1026
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`5,916,596
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`7
`While it is recognized that particles produced according to
`the invention can be either crystalline, amorphous, or a
`mixture thereof, it is generally preferred that the drug be
`present in the formulation in an amorphous form. This
`Would lead to greater ease of dissolution and absorption,
`resulting in better bioavailability.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG. 1 presents the results of intravenous administration
`of paclitaxel nanoparticles to tumor bearing mice (n=5 in
`each group), shoWing a complete regression of tumor in the
`treatment group (I) compared With a control group receiv
`ing saline
`Virtually uncontrolled tumor groWth is seen
`in the control group. Dose for the treatment group is 20
`mg/kg of paclitaxel administered as an intravenous bolus for
`?ve consecutive days.
`FIG. 2 presents the results of intr