United States Patent
`
`19
`
`Desai et at
`
`111 Patent Number
`
`45 Date of Patent
`
`5916596
`Jun 29 1999
`
`54 PROTEIN STABILIZED
`PHARMACOLOGICALLY ACTIVE AGENTS
`METHODS FOR THE PREPARATION
`THEREOF AND METHODS FOR THE USE
`THEREOF
`
`75 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 SoonShiong
`Angeles all of Calif Shlomo
`Magdassi Jerusalem Israel
`
`Los
`
`73 Assignee Vivorx Pharmaceuticals
`Monica Calif
`
`Inc Santa
`
`21 Appl No 08720756
`
`22 Filed
`
`Oct 1 1996
`
`Related US Application Data
`
`60 Continuation in part of application No 08412726 Mar
`29 1995 Pat No 5560933 which is a division of appli
`cation No 08023698 Feb 22 1993 Pat No 5439686
`51 Int C16
`52 US Cl
`
`A61K 914
`424489 424450 424465
`424451 424439
`
`58 Field of Search
`424489 422
`424423 475 91 93 932 450 400
`
`56
`
`References Cited
`
`US PATENT DOCUMENTS
`
`3959457
`4073943
`4247406
`4572203
`
`51976
`Speaker et al
`21978 Wretlind et al
`11981 Widder et al
`21986 Feinstein
`
`List continued
`
`on next page
`
`FOREIGN PATENT DOCUMENTS
`
`0 295 941 A2
`0 391 518 A2
`
`121988
`21990
`
`European Pat Off
`European Pat Off
`
`List continued on next page
`
`OTHER PUBLICATIONS
`
`of Pharmaceutics
`
`Burgess et al Potential use of albumin microspheres
`as a
`and in vitro release of
`drug delivery system I Preparation
`Journal
`steroids
`International
`39129136 1987
`List continued on next page
`Primary ExaminerNeil S Levy
`Assistant ExaminerWilliam E Benston Jr
`Attorney Agent or FirmGray Cary Ware
`Stephen E Reiter
`57
`
`Freidenrich
`
`ABSTRACT
`
`In accordance with the present
`invention there are provided
`compositions and methods useful for the in vivo delivery of
`substantially water
`active
`insoluble pharmacologically
`agents such as the anticancer drug paclitaxel
`in which the
`is delivered in the form of
`pharmacologically active agent
`suspended particles coated with protein which acts
`as a
`In particular protein and pharmacologi
`stabilizing agent
`in a biocompatible dispersing medium are
`cally active agent
`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 specific
`conditions eg addition of a
`composition and preparation
`to the organic phase and careful selection of
`polar solvent
`phase and phase fraction enables the
`the proper organic
`reproducible production of unusually small nanoparticles of
`less than 200 nm diameter which can be sterile filtered The
`system produced according to the invention can
`particulate
`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
`bio available 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 Al
`
`11985
`
`European Pat Off
`
`31 Claims 2 Drawing Sheets
`
`4000
`
`3500
`
`3000
`
`2500
`
`2000
`
`1500
`
`1000
`
`500 I
`
`MM3
`
`TumorVolume
`
`0 I
`
`Treatment Period
`
`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
`
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`1PR201701101 1PR201701103 1PR201701104
`Page 1 of 19
`
`

`

`US PATENT DOCUMENTS
`
`4671954
`4718433
`4789550
`4844882
`5059699
`5110606
`5145684
`5362478
`5439686
`5498421
`5505932
`5508021
`5512268
`5560933
`5650156
`5665382
`
`61987 Goldberg et al
`11988 Feinstein
`121988 Hommel et al
`71989 Widder et al
`101991 Kingston et al
`51992 Geyer et al
`91992
`Liversidge et al
`111994 Desai et al
`81995 Desai et al
`31996 Grinstaff et al
`41996 Gristaff et al
`41996 Grinstaff et al
`41996 Grinstaff et al
`101996 SoonShing et al
`71997 Grinstaff et al
`91997 Grinstaff et al
`
`FOREIGN PATENT DOCUMENTS
`
`549511
`
`424489
`4249
`424451
`424450
`42493
`4249322
`4249322
`424489
`424400
`424450
`
`0 361 677 Al
`0 418 153 Al
`0 190 050 B1
`0 213 303 B1
`2660556
`WO 8500011
`WO 8701035
`WO 8801506
`WO 8807365
`WO 8903674
`WO 9013285
`WO 9013780
`WO 9115947
`WO 9410980
`
`41990
`31991
`51991
`91991
`101991
`11985
`21988
`31988
`101988
`51989
`111990
`111990
`101991
`51994
`
`European Pat Off
`European Pat Off
`European Pat Off
`European Pat Off
`France
`WIPO
`WIPO
`WIPO
`WIPO
`WIPO
`WIPO
`WIPO
`WIPO
`WIPO
`
`OTHER PUBLICATIONS
`
`Grinstaff
`
`Chen et al Comparison of albumin and casein micro
`spheres as a carrier for doxorubicin J Pharm Pharmacol
`39978985 1987
`Feinstein et al TwoDimensional Constrast Echocardio
`graphy I In Vitro Development
`and Quantitative Analysis
`of Echo Contrast Agents JACC 311420 1984
`Suslick Nonaqueous Liquid Filled Microcap
`sules Polym Prepr 32255256 1991
`Gupta et al Albumin microspheres III Synthesis
`and
`containing adriamycin and
`of microspheres
`characterization
`Journal
`of Pharmaceutics
`
`International
`
`magnetite
`43167177 1988
`Ishizaka et al Preparation of Egg Albumin Microcapsules
`and Microspheres
`Journal of Pharmaceutical Sciences
`704358363 1981
`Klibanov et al Amphipathic polyethyleneglycols effec
`liposomes FEBS
`tively prolong the circulation time of
`2681235237 1990
`Meltzer Effect of Viscosity on the Size of
`Koenig
`Microbubbles Generated for Use as Echocardiographic Con
`trast Agents Journal of Cardiovascular ultrasonography
`5434 1986
`Molecular Biosystems
`Investigators Package
`
`Inc ALBUNEXPreclinical
`
`5916596
`Page 2
`
`Suslick
`
`Moseley et al Microbubbles A Novel MR Susceptibility
`Contrast Agent 10th Annual Meeting of Society of Mag
`netic Resonance in Medicine 1991
`of Non
`Grinstaff Protein Microencapsulation
`aqueous Liquids J Am Chem Soc 1122178077809
`1990
`Willmott
`Harrison Characterisation
`the anticancer
`albumin microspheres
`drug
`containing
`Journal of Pharmaceutics
`adriamycin International
`43161166 1988
`Serum Albumin Beads An Injectable Biodegrad
`able System for the Sustained Release of Drugs Science
`21310233235 1981
`Bazile et al Body distribution of
`14 Cpolylatic acid nanoparticles
`after
`adminstration
`parenteral
`
`of
`
`freezedried
`
`fully biodegeradable
`coated with albumin
`rats Biomaterials
`
`to
`
`of Several
`
`of
`
`1310931102 1992
`Boury et al Dilatational Properties of Absorbed PolyD
`Llactide and Bovine Serum Albumin Monolayers at
`the
`1116361644
`DichloromethaneVVater
`InterfaceLangmuir
`1995
`Calvo et al Comparative in Vitro Evaluation
`and
`Colloidal Systems Nanoparticles
`Nanocapsules
`Nanoemulsions as Ocular Drug Carriers J Pharm Sci
`855530536 1996
`Cavalier et al The formation and characterization
`hydrocortisoneloaded poly+ lactide microspheres J
`Pharm Pharmacol 38249253 1985
`to Human Plasma Albumin
`Kumar et al Binding of Taxol
`andAcid Glycoprotein
`Research Communications in
`Chemical Pathology and Pharmacology 803337344
`1993
`Lee et al Serum Albumin Beads An Injectable Biode
`gradable System for the Sustained Release of Drugs Sci
`ence 213233235 1981
`Leucuta et al Albumin microspheres
`as a drug delivery
`and
`system for epirubicin pharmaceutical pharmacokinetic
`biological aspects International Journal ofPharmaceutics
`41213217 1988
`et al Formulation and Antitumor
`LiversidegMerisko
`Activity Evaluation of Nanocrystalline Suspensions
`of
`Poorly Soluble Anticacer Drugs Pharmaceutical Research
`132272278 1996
`of Some
`Mathew et al Synthesis and Evaluation
`Prodrugs and Derivatives of Taxol with Antitumor
`Activity J Med Chem 35145151 1992
`Norton et al Abstracts of the 2nd National Cancer Institute
`Taxus Sep 2324 1992
`Workshop on Taxol
`Wani et al Plant Antitumor Agents VI The Isolation and
`Structure of Toxol a Novel Antileukemic and Antitumor
`from Taxus brevifolia12 J Am Chem Soc
`Agents
`9323252327 1971
`
`WaterSoluble
`
`Abraxis EX2005
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`1PR201701101 1PR201701103 1PR201701104
`Page 2 of 19
`
`

`

`xN o o
`
`00
`
`1
`
`S11
`
`Waled
`
`syz
`
`lamIS
`
`1
`
`ZJo
`
`DaysPostimplant
`
`Figure1
`
`34
`
`33
`
`I
`
`32
`
`31
`
`30
`
`29
`
`28
`
`27
`
`26
`
`5
`
`25
`
`24
`
`23
`
`22
`
`21
`
`20
`
`19
`
`18
`
`17
`
`16
`
`15
`
`14
`
`13
`
`12
`
`++++
`
`I+
`
`500
`
`1TreatmentPeriod
`
`C CI 7
`
`I O
`
`I
`
`<
`
`x 7
`
`50cn
`
`am
`
`>
`
`x
`
`500
`
`10001
`
`1500
`
`2000
`
`2500
`
`3000
`
`3500
`
`4000
`
`MM3
`
`Volume
`
`Tumor
`
`

`

`°SIl
`
`waled
`
`syz
`
`lamIS
`
`Z
`
`Jo
`
`Z
`
`Uri aVZ
`
`c71 11 c71
`
`49
`
`Day
`
`32
`
`Day
`
`25
`
`Day
`
`20
`
`Day
`
`12
`
`Day
`
`10
`
`8D
`
`ay
`
`5D
`
`ay
`
`3D
`
`ay
`
`0D
`
`ay
`
`Day
`
`PreArthr
`
`DaysAfterTreatment
`
`Figure2
`
`0mgkg
`
`PaclitaxelNanoparticles1
`
`5mgkg
`
`PaclitaxelNanoparticles0
`
`2mgkg+
`
`Prednisone0
`
`treatment
`
`Non
`
`PostTreatment
`
`SizeP
`
`re
`
`Paw
`
`40
`
`80
`
`al
`
`00
`
`60
`
`re
`
`100
`
`II
`
`73
`
`Cl
`
`o
`
`o1
`
`20
`
`140
`
`

`

`1
`PROTEIN STABILIZED
`PHARMACOLOGICALLY ACTIVE AGENTS
`METHODS FOR THE PREPARATION
`THEREOF AND METHODS FOR THE USE
`THEREOF
`
`RELATED APPLICATIONS
`is a continuation inpart of US Ser No
`This application
`08412726 filed Mar 29 1995 now issued as US Pat No
`5560933 which is in turn a divisional of US Ser No
`08023698 filed Feb 22 1993 now issued as US Pat No
`5439686 the entire contents of both of which are hereby
`by reference herein in their entirety
`
`incorporated
`
`FIELD OF THE INVENTION
`
`invention relates to methods for the produc
`The present
`for the intravenous administra
`vehicles
`tion of particulate
`
`as well as novel
`
`the
`
`active
`
`dispersible colloidal
`
`tion of pharmacologically active agents
`thereby In a particular aspect
`compositions produced
`invention relates to methods
`for the in vivo delivery of
`substantially water
`insoluble pharmacologically
`agents eg the anticancer drug taxol
`In another aspect
`containing water
`systems
`insoluble
`agents are provided The sus
`pharmacologically active
`pended particles are encased in a polymeric shell formulated
`from a biocompatible polymer and have a diameter of less
`colloidal systems are pre
`than about 1 micron Invention
`pared without
`the use of conventional
`surfactant
`or any
`polymeric core matrix In a presently preferred aspect of the
`there is provided a method
`invention
`of
`for preparation
`extremely small particles which can be sterile filtered 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
`ie drug molecules
`ately bioavailable drug molecules
`which are molecularly bound to a protein and pure drug
`coated with a protein
`
`particles
`
`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
`
`can provide
`controlled release nanoparticles
`a
`Injectable
`preprogrammed 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 specific tissues or organs Tice and Gilley Journal of
`Controlled Release 2343352 1985
`in the blood are
`Microparticles and foreign bodies present
`generally cleared from the circulation by the blood filtering
`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 68 microns in diameter and platelets typically
`13 microns in diameter The microcirculation in most
`
`5916596
`
`2
`
`organs and tissues allows the free passage of these blood
`cells When microthrombii blood clots of size greater
`than
`1015 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
`than 1015 microns in
`the circulation of particles greater
`diameter therefore must be avoided A suspension of par
`ticles less than 78 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
`ed A Rembaum
`MicrospheresBiomedical Applications
`pp 193227 CRC Press 1988 have described the fate of
`particles to be dependent on their size Particles in the size
`few nanometers nm to 100 nm enter
`the
`range of
`and
`lymphatic capillaries following interstitial
`injection
`phagocytosis may occur within the lymph nodes After
`injection particles less than about 2
`intravenousintraarterial
`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
`injection be trapped in the
`7 microns will after
`lung capillaries After
`injection particles are
`trapped in the first capillary bed reached Inhaled particles
`
`intravenous
`
`intraarterial
`
`a
`
`Pharmaceuticals
`
`are trapped by the alveolar macrophages
`that are water insoluble or poorly water
`soluble and sensitive to acid environments
`in the stomach
`cannot be conventionally administered eg by intravenous
`injection or oral administration The parenteral administra
`tion of such pharmaceuticals
`has been achieved by emulsi
`fication of the oil solubilized drug with an aqueous liquid
`such as normal saline in the presence of surfactants or
`stable microemulsions
`emulsion stabilizers to produce
`These emulsions may be injected
`intravenously provided
`the components of the emulsion are pharmacologically inert
`US Pat No 4073943 describes
`the administration of
`water insoluble pharmacologically active agents dissolved
`in oils and emulsified 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
`coated with a phospholipid such as dimyristoyl
`thetic
`phosphatidylcholine having suitable dimensions for intrad
`ermal or intravenous
`An example of a water insoluble drug is taxol a natural
`from the Pacific Yew tree Taxus
`product
`isolated
`et al J Am Chem Soc
`932325
`by Wani
`brewfolia
`1971 Among the antimitotic agents taxol which contains
`carbon skeleton exhibits a unique mode of
`a diterpene
`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
`is the only plant product known
`assembly of tubulin taxol
`tubulin thus
`the depolymerization process of
`the cell replication process
`a naturally occurring diterpenoid has been shown
`Taxol
`and anticancer effects in
`to have significant antineoplastic
`ovarian cancer Taxol has shown excellent
`drug refractory
`antitumor activity in a wide variety of tumor models such as
`leukemias MX 1 mammary
`the B16 melanoma L1210
`tumors and CS 1 colon tumor xenografts Several
`recent
`the new anticancer
`releases have termed
`taxol as
`press
`wonder drug Indeed taxol has recently been approved by
`the Federal Drug Administration for treatment of ovarian
`
`injection
`
`first
`
`to inhibit
`
`preventing
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
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`
`

`

`5916596
`
`4
`
`cancer
`
`The poor aqueous solubility of
`taxol however
`a problem for human administration Indeed the
`presents
`delivery of drugs that are inherently insoluble or poorly
`soluble in an aqueous medium can be seriously impaired if
`currently used
`oral delivery is not effective Accordingly
`taxol
`to solubilize the
`formulations require a cremaphor
`drug The human clinical dose range is 200500 mg This
`dose is dissolved in a 11 solution of ethanolcremaphor and
`diluted to one liter of fluid given intravenously The crema
`phor currently used is polyethoxylated castor oil
`itself did not show exces
`trials taxol
`In phase I clinical
`sive toxic effects but severe allergic reactions were caused
`by the emulsifiers employed to solubilize the drug The
`current
`treatment of the
`regimen of administration involves
`patient with antihistamines and steroids prior to injection of
`the drug to reduce the allergic side effects of the cremaphore
`
`In an effort
`
`several
`
`facilitate
`
`the
`
`to improve the water solubility of taxol
`investigators have modified its chemical
`structure
`impart enhanced water
`with functional
`groups that
`solubility Among them are the sulfonated
`derivatives
`Kingston et al US Pat No 5059699 1991 and amino
`acid esters Mathew et al J Med Chem 35145151
`1992 which show significant biological activity Modifi
`cations to produce a watersoluble derivative
`in an innocuous
`intravenous
`taxol dissolved
`delivery of
`carrier such as normal saline Such modifications however
`add to the cost of drug preparation may induce undesired
`side reactions andor allergic reactions andor may decrease
`the efficiency of the drug
`have been reported in the literature
`Protein microspheres
`agents Micro
`as carriers of pharmacological or diagnostic
`spheres of albumin have been prepared
`by either
`heat
`or chemical crosslinking
`Heat
`denatured
`denaturation
`are produced from an emulsified mixture eg
`microspheres
`to be incorporated and a suitable oil at
`albumin the agent
`100° C and 150° C The micro
`between
`temperatures
`spheres are then washed with a suitable solvent and stored
`Leucuta
`International Journal of Pharmaceutics
`41213217 1988 describe the method of preparation of
`heat denatured microspheres
`The procedure
`for preparing chemically
`treating the emulsion with glutaral
`microspheres
`involves
`dehyde to crosslink the protein followed by washing
`and
`storage Lee et al Science 213233235 1981 and US
`Pat No 4671954 teach this method of preparation
`The above techniques
`
`et al
`
`crosslinked
`
`for
`
`the preparation of protein
`
`as carriers of pharmacologically active agents
`microspheres
`although suitable for the delivery of watersoluble agents
`are incapable of entrapping water insoluble ones This limi
`in the technique of preparation which relies
`tation is inherent
`of the protein compo
`on crosslinking or heat denaturation
`in the aqueous phase of a water in oil emulsion Any
`nent
`aqueous soluble agent dissolved
`in the protein containing
`phase may be entrapped within
`the resultant
`aqueous
`or heat denatured protein matrix but a poorly
`crosslinked
`aqueous soluble or oil soluble agent cannot be incorporated
`into a protein matrix formed by these techniques
`One conventional method for manufacturing drug
`comprises dissolving polylactic
`containing
`nanoparticles
`acid or other biocompatible water
`insoluble polymers in a
`water immiscible solvent such as methylene
`other chlorinated aliphatic or aromatic solvent dissolving
`
`chloride or
`
`in the polymer solution
`the pharmaceutically active agent
`adding a surfactant
`to the oil phase or the aqueous phase
`forming an oil in water emulsion by suitable means and
`the emulsion slowly under vacuum If
`the oil
`evaporating
`
`droplets are sufficiently small and stable during evaporation
`is obtained Since the
`a suspension of the polymer in water
`in the polymer solution it
`drug is
`is possible
`present
`initially
`to obtain by this method a composition in which the drug
`5 molecules
`are entrapped within particles
`composed of a
`and nano
`polymeric matrix The formation of microspheres
`particles by using the solvent evaporation method has been
`researchers see for example Tice and
`reported by several
`Gilley in Journal of Controlled Release 2343352 1985
`10 Bodmeier and McGinity in Int J Pharmaceutics 43179
`1988 Cavalier et al in J Pharm Pharmacol 38249
`1985 and DSouza et al WO 9410980 while using
`various drugs
`Bazile et al in Biomaterials 131093 1992 and Spen
`lehauer et al in Fr Patent 2 660 556 have reported
`the
`formation of nanoparticles
`two biocompatible
`by using
`polymers one eg 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
`20 active agent After emulsification
`and removal of
`the
`are formed in which the drug is
`solvent nanoparticles
`inside the polymeric matrix of the polylactide par
`
`present
`
`15
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`ticles
`
`The properties of the polymer solution from which the
`polymeric matrix is formed are very important
`to obtain the
`proper emulsion in the first stage For example polylactide
`the polymer commonly used in the preparation of injectable
`nanoparticles has a surface activity which causes the rapid
`the dichloromethanewater
`thereof at
`adsorption
`interface
`causing reduced interfacial tension see for example Boury
`et al in Langmuir 111636 1995 which in turn improves
`the emulsification process In addition the same researchers
`found that Bovine Serum Albumin BSA interacts with the
`polylactide and penetrates
`into the polylactide monolayer
`the oil water
`interface Therefore it
`is expected
`present at
`based on the above reference that emulsification during the
`solvent evaporation method is greatly favored
`conventional
`by the presence of the surface active polymer polylactide
`in the nonaqueous organic phase In fact
`the presence of
`polylactide is not only a sufficient condition but
`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 eg toluene or cyclohexane without any polymer
`
`it
`
`dissolved
`
`adding
`solvent
`in the organic
`to the mixture as an emulsifier
`surfactant
`in water emulsion and then evaporating
`obtain dry particles of the drug see for example Sjostrom
`et al in J Dispersion Science and Technology 1589117
`1994 Upon removal of the nonpolar solvent precipitation
`of the drug inside the solvent droplets occurs and submicron
`particles are obtained
`
`a conventional
`forming an oil
`the solvent
`to
`
`It has been found that
`
`the size of the particles is mainly
`size of the emulsion droplets In
`
`55 controlled by the initial
`addition it
`the final particle size is
`is interesting to note that
`reported to decrease with a decrease in the drug concentra
`phase This finding is contrary to the
`tion in the organic
`results reported herein wherein no conventional
`surfactant
`is used for the preparation of nanoparticles In addition it
`noted by the authors of the Sjostrom paper
`the drug
`that
`used cholesteryl acetate
`is surface active in toluene and
`hence may be oriented at
`the oil water
`interface therefore
`of drug at
`the concentration
`increasing the potential
`for precipitation
`Formation of submicron particles has also been achieved
`in J
`by a precipitation process as described by Calvo et al
`
`60
`
`65
`
`is
`
`the interface
`
`is higher
`
`thus
`
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`

`5916596
`
`5
`Pharm Sci 85530 1996 The process is based on dissolv
`ing the drug eg indomethacin and the polymer poly
`chloride and acetone
`caprolactone in methylene
`pouring the solution into an aqueous phase containing
`a
`surfactant Poloxamer 188 to yield submicron size par
`tides 216 nm However
`the process
`is performed at
`at which no emulsion is formed
`
`solvent concentrations
`
`and then
`
`BRIEF DESCRIPTION OF THE INVENTION
`
`Thus it
`
`is an object of this invention to deliver pharma
`cologically active agents eg taxol taxane Taxotere and
`the like in unmodified form in a composition that does not
`cause allergic reactions due to the presence of added emul
`sifiers and solubilizing agents as are currently employed in
`drug delivery
`
`It
`
`invention to deliver
`is a further object of the present
`pharmacologically active agents in a composition of micro
`particles or nanoparticles optionally suspended in a suitable
`biocompatible liquid
`
`It
`
`invention to provide
`is yet another object of the present
`a method for
`the formation of submicron
`
`particles
`
`active agents by a
`of pharmacologically
`nanoparticles
`solvent evaporation technique from an oil in water emulsion
`using proteins as stabilizing agents in the absence
`of any
`surfactants and in the absence of any poly
`conventional
`meric core material
`
`These and other objects of the invention will become
`and claims
`apparent upon review of the specification
`invention we have dis
`In accordance with the present
`covered
`that substantially water
`insoluble pharmacologi
`in the form of micro
`cally active agents can be delivered
`
`of
`
`that
`are suitable
`or nanoparticles
`for parenteral
`particles
`administration in aqueous suspension This mode of delivery
`obviates
`the necessity
`for administration of substantially
`insoluble pharmacologically active agents eg taxol
`water
`in an emulsion containing for example ethanol
`and poly
`castor oil diluted in normal saline see for
`ethoxylated
`example Norton et al in Abstracts of the 2nd National
`Taxus Sep 2324
`Cancer
`Institute Workshop on Taxol
`1992 A disadvantage of such known compositions is their
`propensity to produce allergic side effects
`Thus in accordance with the present
`invention there are
`the formation of nanoparticles
`provided methods
`for
`pharmacologically active agents by a solvent
`evaporation
`under
`from an oil in water emulsion prepared
`technique
`of high shear forces eg sonication high pres
`conditions
`sure homogenization or the like without
`the use of any
`the use of any poly
`surfactants and without
`conventional
`meric core material to form the matrix of the nanoparticle
`Instead proteins eg human serum albumin are employed
`as a stabilizing agent
`The invention further provides a method for the repro
`formation of unusually small nanoparticles
`ducible
`less
`than 200 nm diameter which can be sterile filtered through
`a 022 micron filter This is achieved by addition of a water
`solvent eg ethanol
`to the organic phase and by
`soluble
`the type of organic phase the phase
`carefully selecting
`fraction and the drug concentration
`in the organic phase The
`ability to form nanoparticles
`is filterable by
`of a size that
`022 micron filters is of great
`and significance
`importance
`since formulations which contain
`a significant amount of
`any protein eg 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
`for
`in
`compositions useful
`
`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
`substantially water
`polymeric shell containing
`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 eg human serum albumin
`and are therefore
`immediately bioavailable upon adminis
`tration to a mammal The other portion of the pharmaco
`logically active
`is contained within nanoparticles
`agent
`the phar
`coated by protein The nanoparticles
`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
`is pre bound to a protein
`macologically active agent
`that
`through hydrophobic or ionic interactions prior to admin
`istration
`
`containing
`
`demonstrates both of the above
`The present disclosure
`described modes of bio availability 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 80337
`1993 The high concentration
`of albumin in invention
`particles compared to Taxol provides a significant 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
`due to a combination of
`prolonged periods is facilitated
`electrical repulsion and steric stabilization
`In accordance with the present
`invention there are also
`form which can
`provided submicron particles in powder
`easily be reconstituted
`in water or saline The powder
`removal of water by lyophilization Human
`obtained after
`serum albumin serves as the structural component of inven
`and recon
`tion nanoparticles and also as a cryoprotectant
`stitution aid The preparation of particles filterable through
`a 022 micron filter according
`to the invention method as
`described herein followed by drying or lyophilization pro
`duces a sterile
`solid formulation useful
`for
`intravenous
`
`is
`
`injection
`The invention provides in a particular aspect
`a compo
`sition of anti cancer drugs eg Taxol
`in the form of
`in a liquid dispersion or as a solid which can
`nanoparticles
`for administration Due to specific
`be easily reconstituted
`properties of certain drugs eg 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 eg size of
`about 5 microns to several hundred microns are formed
`few minutes of storage after
`within a
`the preparation
`process The size of such crystals is typically much greater
`than the allowed size for intravenous
`injection
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
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`
`

`

`7
`
`8
`
`5916596
`
`While it
`is recognized that particles produced according to
`the invention can be either crystalline amorphous or a
`mixture thereof
`the drug be
`is generally preferred that
`form This
`in the formulation in an amorphous
`present
`would lead to greater
`ease of dissolution and absorption
`resulting in better bioavailability
`
`it
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG 1 presents the results of intravenous administration
`to tumor bearing mice n=5 in
`of paclitaxel nanoparticles
`each group showing a complete regression of tumor in the
`
`treatment group M compared with a control group receiv
`
`ing saline 111 Virtually uncontrolled tumor growth is seen
`in the control group Dose for the treatment
`group is 20
`mgkg of paclitaxel administered as an intravenous
`bolus for
`five consecutive days
`FIG 2 presents the results of intraperitoneal administra
`in rats that have developed
`tion of paclitaxel nanoparticles
`in their paws following intradermal
`injection of
`collagen Paw volumes are measured and indicate the sever
`ity of the disease The paw volumes are normalized to 100
`at the beginning of treatment Day 0 represents the initiation
`of treatment There are 3 groupscontrol group receiving
`saline n=2 shown as a thin line and labelled in the figure
`a non treatment
`treatment group receiving pacli
`at a dose of 1 mgkg n=4 shown as a
`taxel nanoparticles
`heavy line and labelled in the figure as paclitaxel nanopar
`tides 10 mgkg and a second treatment group receiving
`at a dose of
`combination therapy of paclitaxel nanoparticles
`a dose of 02 mgkg n=4
`05 mgkg and prednisone
`shown as a heavy line and labelled in the figure as pred
`nisone 02 mgkg+paclitaxel nanoparticles 05 mgkg The
`show a dramatic reduction
`two treatment
`in paw
`groups
`volume with time indicating a regression of arthritis while
`the control group showed an increase in paw volume over
`the same period
`
`arthritis
`
`a first
`
`at
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`In accordance with the present
`invention there are pro
`vided methods
`the preparation of substantially water
`for
`in vivo
`insoluble pharmacologically
`active
`agents for
`delivery said method comprising
`subjecting a mixture comprising
`said pharmacologically
`phase containing
`an organic
`active agent dispersed therein and
`aqueous medium containing
`biocompatible polymer
`wherein
`said mixture contains
`no
`
`substantially
`
`surfactants
`in a high pressure homogenizer at a pressure in the range of
`about 3000 up to 30000 psi Optionally the organic andor
`removed from the mixture
`aqueous phases are thereafter
`after having been