United States Patent fl9)
`Grinstatr et al.
`
`1111111111111111111111 111111111 11111111111111 1111111111111111111111111111
`l1S005498421A
`[Il] Patent Number :
`14:Jj Date of Patent:
`
`5,498,421
`*Mar. 12, 1996
`
`[54J COMPOSITION USEFUL FOR IN VIVO
`DELIVERY OF JUOLOGICS AND METHODS
`Ell,{PLOYING SAME
`
`[75)
`
`lnventors: M:ark W. Gri nstaft', Pasadena; P-.drh:k
`Soon-Shiung, Los Angeles, bOlh of
`Calif.; Michael Wong, Champaign, Ill.;
`Paul A. Sandford, Los Angeles, Calif.;
`Kenneth S. Suslick, Champaign, IlL;
`Neil P. Desai, Los Angeles, Calif.
`
`[73) Assignee: Vivorx Pharmaceuticals, Inc., Santa
`Monica, Calif.
`
`[ • J
`
`:'\Iotice:
`
`The lcnn of this paten! shaH not e:dcnd
`beyond Ihe expiration dale of Pal. No
`5,362,478.
`
`[21J App!. No.: 200,235
`
`[221 Filed:
`
`Feb. 22, 1994
`
`Related U.s. Application Data
`
`[631 Continua/iOD_in_part of Scr. N(). 23,698, Feb. 21, 1993, P-dt.
`No. 5,439,686. and a CQntinuation.in_parl of Se •. No.
`35.150, Mar. 26. 1993. Pal. No. 5,362..478.
`
`A61K 37122; A61 K 91127
`Inl, CI."
`[51]
`(521 U.S, Cl,
`............... 4241450; 424/451; 424/455;
`42419.3; 42419.34; 42419.37; 42419.4; 42419.5
`[58) Field or Search
`............. 424/451,45,450
`
`[56]
`
`Reference.~ Cited
`
`U.S. PATENT DOCUMENTS
`
`. ............. 424119
`511976 Speaker et aI ..
`3.959,457
`211978 Wrctlind c\ Dl.
`. ............... 4241358
`4.073.\043
`111981 Widder ~t al
`252/62.53
`4.241,406
`211986 Feinstein
`12S1661
`4.512,203
`..... 4241450
`611981 Goldberg et a!.
`4,611.954
`......... 12&'660
`111988 Feinuein
`4.118.433
`4,189.550 121\988 Hommd el at.
`... 4241493
`................. 42419
`611989 Widder el aI.
`4.&44,882
`51!990 Ilanolucci
`.. 4241439
`4,929.446
`5,059.699 11)f!99 1 Kingston ................................ 5491511
`42414S9
`511992 Geyer CI al
`5.110.606
`........... 42415
`
`5,150,283 1011993 '''''''m .................
`
`... 42414
`6/1994 Li~r$idgc et aI.
`5.3 18.167
`.. .............. 424(9
`5.3152.478 1lI1994 Desai et aI.
`FOREIGN PATENT DOCUMENTS
`211985 European Pat. all'. .
`0I29619AI
`029S941A2 1211988 European Pat. alI ..
`0361611AI
`411990 European Pal. 00" ..
`0391S18A2 1011990 European Pal. Off ..
`041&153A 1
`311991 European Pal. Off
`511991 European Pal. Off ..
`019005081
`02!33OJIlI
`9f1991 European \'al. Off ..
`lfl985 WIPO .
`85100011
`211987 w<PO
`81101035
`88101506
`3119BB w<PO
`88107365 1011988 WIPO
`89103674
`511989 WIPO.
`901\3780 1111990 WU'O
`90113285 1111990 WlPO .
`91"5947 1011991 w'PO
`atHER PUBLICATIONS
`Abuchowsl::i et at. "Al!eration of Immunological I'ropenies
`of Bovinc Serum Albumin by Covalent Attachment of
`Polyethylene Glycol" J. BioI. Chtm.. 252:3578 (1977).
`Burgess et al .• "Potential use of albumin microspheres as a
`drug delivery system. I. Preparation and in vitro release or
`Jnlenwliufl()/
`}uumal uf
`steroids"
`Pha'muL'euli~'s
`19:129_ \36 (19117)
`(List continued on nc:u page.)
`Primary Examiner- Thurman K. Page
`A.rsislant £Xami/U'r- WiHimn E. Benston, Jr.
`AI/orney. Age'll. or Firm-Slephen E. Reiter: Pretty.
`Schroeder, Brueggemann & Clark
`ABSTRACT
`
`(57]
`
`In accorda.1ce with the present invention, there are provided
`compositions useful for the in lIillo delivery of a biologic.
`wherei n the biologic is associated with a polymeric shell
`fonnulated from a biocumpatibh: UliltCrial. The biologic call
`be associated with the polymeric shell itself, andlor the
`biologic, optionally slIspcndedldispersed in a biocompatible
`dispersing agent. can be encascd by thc polymeric shell. In
`anolher aspect, thc biologic associal.ed with polymeric shell
`is administered to a subject. optionally dispersed in a suit·
`able bioculllpatibic liquid.
`
`30 Claims, 3 Drawing Sheets
`
`.':. .: -': .' : : ':'. •
`
`' .. : : .::
`.. ' .
`:,", ..
`
`0---.,
`
`c
`
`I,
`
`Apotex v. Abraxis - IPR20 18-00 152, Ex. 1014, p.O I of34
`
`

`

`5,498,421
`Page 2
`
`OTHER PUaUCATIQNS
`
`Chen et aI., "Comparison of albumin and casein micro(cid:173)
`spheres as a carrier for doxoru bicin" J. Phr;m". Pham'ocol.
`39:978-985 (1987).
`Feins!ein et aI., "Two-Dimensional Contrast Echoeardio(cid:173)
`graphy. I. In Vitro Development and Quantitative Analysis
`of Echo ContraSt Agents" JACC 3(1):14-20 (1984).
`Grinstaff and Susliek, "Nonaqueous liquid Filled Micro(cid:173)
`capsules" Po/ym. Prepr. 32:255-256 (1991).
`Gupta et al., ·'Albumin microsphcres. Ill. SynthC!5is and
`characterization of microsphcrcs containing adriamycin and
`In/u na/ional Journal of Pharmaceutics
`magnetitc"
`43:167- J77 (1988).
`ishizaka et a1., "Preparation of Egg Albumin Microcapsules
`and ,\1icrospheres" Journal of Pharmaceutical Sciences
`70(4):358-363 (/981).
`KIibanov et al .. "Amphipathie polycthylcncglycols effec,
`tively prolong the circulaJ.ion time of liposomes" FEBS
`268(1):235-237 ( 1990).
`KOI:nig and MelLt.e£, "EITl."1;t of Viscosity on thc Sizc of
`Microbllbbles Generated for Use as Echocardiographic Con(cid:173)
`trast Agents" Joumal of CardilWa$CIl!a1" Ultrasonography
`5(1):3-4 (1986).
`
`Lee o::t a1., "Scolm Albumin Be<tds: An Injectahle, Biode(cid:173)
`gradable System for the Sustained Release of Drugs"' Sci·
`enCti 213:233-235 (1981).
`Leucuta et a1., "Albumi n microspheres as a drug delivery
`system for epirubicin: pharmaceutical, pharrnacokinetic and
`biological aspects:' InlernatiolUl! Journal of Phamtaceu/ics
`41:213- 217 (1988) (1992).
`.
`Mathew [sic) e! al., "Synthesis and Evaluation of some
`Water-Soluble Prodrugs !'Jld Derivatives of Tallol wilh
`Antitumor Activity" J. Med. Chern. 35:145- /5 1.
`Molecular Bi05ystems. Illc .. "A1buTlClI-Prcclinical rnves·
`tigator's Package".
`Moseley et a1., '",\1icrobubb!cs : A No,·e1 MR Susceptibility
`Contrast Agent" 10 Annual meeting of Society of Magnetic
`Resonance in Medicine in San Francisco, Calif. Oct. 1991.
`Sllslick and Grinst.alf, "Protein Microencapsulation of Non(cid:173)
`aqueous Liquids" J. Am. Chern. Soc. 112(21):7807- 7809
`(1990).
`Will mott and Harrison, "Characterization of freeze-dried
`albumin microspbcrcs containing
`the anti-canccr drug
`Intemationul Journal of Pharmaceutics
`adriamycin"
`43:1 6 1-166 (1988).
`
`Apotex v. Abraxis - IPR201 8-00152, Ex. 1014, p.02 of34
`
`

`

`U.S. Patent
`
`Mar. 12, 1996
`
`Sheet I of 3
`
`5,498,421
`
`. . . .
`
`.' . .
`. . . . . . . . . .
`. ,,;. ~-+--B
`. . . . . . . . .
`. . ..
`. .
`.
`.
`. . .
`. . . . . . . .
`..-414-..
`.
`. ..
`. . . . . . . . . :. . .
`c
`
`.1
`
`I·
`
`o
`
`FI G.1
`
`Apotex v. Abraxis · IPR20 18·00 152, Ex. 1014, p.03 of34
`
`

`

`U.S. Patent
`
`Mar. 12, 1996
`
`Sheet 2 of 3
`
`5,498,421
`
`FIG.2
`
`o mN P\lJSPIlATES
`
`_._- Blib SOLUTION
`
`--- Btl> MICROBUBBLES
`SMOOTH CURVE
`• BHb MICRIIIllIBlES DATA
`
`•
`
`•
`~ ____ ~~.~:J~.~1r.
`
`_._ ... -_._.-.
`
`•
`
`•
`•• ••
`
`'--,
`' " ,
`
`0.2
`
`0.4
`
`0.8
`
`1.2
`
`1.4
`
`1.6
`
`1.8
`
`2
`
`log(P02 /torrl
`
`Apotex v. Abraxis - IPR201 8-001 52, Ex. 101 4, p.04 of34
`
`

`

`U.S. Patent
`
`Mar. 12, 1996
`
`Sheet 3 of 3
`
`5,498,421
`
`FIG. 3
`
`1.1 ~1,l - BPG
`
`_ .- BIb SOlUTION
`
`- - ~~ICIIOBIJBBLES SNOOTlI
`• BHb MICROBUBBLES DITA
`
`•
`
`•
`•
`
`•
`••
`••
`
`'-
`
`•
`
`•
`•
`
`O.S
`
`••
`,...-.
`•
`.
`•
`/'
`•
`•
`
`".. "
`
`I.S
`tootP02/torrl
`
`14
`
`12
`
`10
`
`c
`
`~
`~
`
`8
`
`~ 8
`~ ;: 6
`
`~
`
`4
`
`2
`
`00
`
`.~---
`
`2.S
`
`Apotex v. Abraxis - IPR20 18-00 152, Ex. 101 4, p.05 of34
`
`

`

`5,498,421
`
`I
`COMPOSmON USEFUL FOR IN VIVO
`DELIVE RY OF BIOLOGICS AND METHODS
`EMPLOYING SAME
`
`RELATED APPLICATIONS
`
`This application is a continuation-in-pan of U.S. SeT. Nos.
`08/023,698. filed Feb. 22. 1993,now issued as U.S. Pal. No.
`5,439,686 and 081035,]50, filed Mar. 26.1993, now issued
`as U.S. Pal. No. 5,362,478, tbe entire contents of which are
`hereby incorporated by reference herein
`
`FIELD OF THE INVE!\'TI0N
`
`The present invention reial.es 10 in vivo delivery of
`biologics. ill one aspttL. biologic is associated with a
`polymer.c shell fonnulated from a biocompatiblc material.
`The biologic can be associated with the polymeric $hell
`i(Self, andlor the hiolngic, optionally ~uspcndcdldispc~cd in
`a hiocompatible dispersing agent. = be encased by the
`polymeric shell. In another aspect. the biologic associated
`with polymeric shell is administered \0 a subject, optionally
`dispersed in a suitablc biocompatiblc Hquid.
`
`BACKGROUND OF THE INVEt-.'T10N
`
`Micropanielcs and foreign bodies present inlhe blood are
`generdlly cleared from Ille cin:uhnion by the 'blood fillering
`organ$', namely the splccn, lungs and liver, The paniculatc
`matter contained in normal whole blood comprises red blood
`et:lls (typically 8 microns in diameter). white blood et:lls 30
`(typically 6-8 microns in diameter), and platelets (typically
`1- 3 microns in diameter). The microcirculation in most
`organs and thsues allows the free passage of these blood
`cells, When microthrombii (blood clots) of si?,e !o!reater Ulan
`10-15 microns are presem ill ciKulatioo, a risk Ilf infarction 3S
`or blockage of thc capillaries results, leading 10 ischemia or
`oxygen deprivation and possible tissue death. Injection inlo
`Ille cireulation of panieles greater than 10-15 microns in
`diameter, Lhercfore. must be avoided. A suspension of par·
`ticles less than 7-8 microns. is howe~er. relatively safe and 40
`has been used for the delivery of phannacologically act;\'e
`agents in the form of liposomes and emulsions, nutritio nal
`agcnts, and contrast media for imaging applications.
`The size of panicles and their mode of delivery deter(cid:173)
`mines their biological behavior. Stnmu et ai, [in Micro- 45
`spheres-Biomedical Applicalians, ed. A. Rembaum, pp
`193-227, eRe Press (1988)] have described the fate of
`panicles to be dependent on their ~ze_ Panicles in thc size
`range of a few nanometers (nm) to 100 nm ellter Lhe
`lymphatic capillaries following interstitial injection, and so
`phagocytosis may occur wiLhin the lymph oodes. Aflcr
`intravenousJintraanerial injection, particles less than about 2
`microns will be rapidly cleared from Ille blood stream by the
`reticuloendothelial system (RE. ... ), also k.nown as Illc mono(cid:173)
`nuclear phagocyte system (MPS). Panicles larger than aoom 5S
`7 microns will, after intravenous injection, be trapped in the
`lung capillaries. Afte r intraarterial injection, particles are
`trap~d in the first capillary b~d rtlCht,d. Inhaled panicles
`arc trapped by the alvcolar macrophages.
`Pharmaceuticals that arc water-insoluble or poorly water- 60
`sohlble and sensi tive to acid cnvironmcllts in Lhe stomach
`cannot be convClItionaily administered (e.g., by intravenous
`injection or oral administration). The parenteral administra(cid:173)
`tion of such pharmaceuticals has been achieved by emulsi(cid:173)
`fication of oil solubilized drog with an aqueous liquid (slICh
`as normal saline) in the presence of surfaclanlS or emulsion
`stabilizen to produce stable microcmulsions. Thesc ernul·
`
`2
`sions may be injected intravenously, provided the compo(cid:173)
`nents of thc cmulsion arc pharmacologically incrt. For
`example, U.s. Pal. No. 4,073.943 describes the administra(cid:173)
`lion of waler-insoluble pharmacologically active agents dis·
`solved in oils and emulsified wilh water in Lhe presence of
`sunaclams such as egg phosphatides. pluronics (copolymers
`of polypropylene glycol and polyethylene glycol), polyg·
`lyceml oleate, etc. per lruemational Publication No.
`W085100011 describes pharmaceutical microdroplets of an
`10 anaesthetic coated with a phospholipid, such as dimyristoyl
`phosphatidy!choline, having suitable dimensions for intrad(cid:173)
`ermal (lr intravenous injecLion.
`Prolein micTQ$pheres have been reported in the literature
`as carriers of pharmacological or diagnostic agcnts. Micro-
`l~ spheres of albumin have been prepared by eithcr heat
`denaturation or chemical crosslinking. Heat denatured
`microspheres arc produced from an emulsified mixture (e.g.,
`albumin, the agent to be incorporated, and a suitable oil) at
`temperatures between 100" C. and 150" C. The micro-
`20 spheres arc then washed with a suitable solvent and stored.
`Leoeuta et al. [Intcmational Journal of Pharmaccuties Vol.
`41 :213-217 (1988)] describe the method of prcparation of
`heat denatured microspbercs.
`'The procedure for prepari ng chemically crosslinked
`25 microsphere5 invol~es treating the emulsion with glutaral(cid:173)
`dehyde to crosslink the protein, foHowed by washing and
`storage. Lee et aI. [Science Vol. 213:233- 235 (1981)] and
`U.S. Pat. No. 4,671.954 teach this method of preparation.
`The above techniques for the prcparation of prOlein
`mierosphcres as carriers of pharmacologically active agcnts,
`although suitable for the delivery of water-soluble agents,
`are incapable of entrapping water-insoluble ones. This limi(cid:173)
`tation is inherent in the technique of prepar.!.tion which relies
`00 crosslinking or heal denaturation of the protein compo(cid:173)
`nent in the aqueous phase of a watcr-in-oil emulsion. Any
`aqueous-solublc agent dissolved in the protein-containing
`aqueous phase may be cntrapped within Ihe resultant
`crosslinked or heat·denatured protein matrix, blll a poorly
`aqllCous-soluble or oil-soluble agelll cannot be incorporated
`into a protein matrix formed by these techniques.
`Thus, thc poor aqueous solubility of many biologies
`presents a problem for human administration. Indeed, the
`delivery of pharmacologically active agents that are iober(cid:173)
`L'lltly insoluble or poorly soluble in aqueous medium can be
`seriously impaired if oral delivery is not effectivc. Aceord-
`ingly, cu=ntly used formulations for the delivery of phar(cid:173)
`macologically activc agents that arc inhcrenlly insoluhle or
`poorty soluble in aqueous medium require the addition of
`agenlS to solubili"le thc pharmacologically active agent.
`Frequentl y, however, sevcrc allergic reactions arc caused by
`the agents (e.g., emulsifiers) cmployed to so!ubili~.e phar(cid:173)
`macologically aClive agents_ Tnu~, a common regim~n of
`administration iovolvcs treatment of the patient with anti(cid:173)
`histamines and steroids prior to injection of the phannaco(cid:173)
`logically active agent to reduce the allergic side effects of the
`agenlS used 10 aid in drog delivery.
`In an effort to improvc thc water solubility of drugs that
`are inherently insoluble or poorly solublc in aqucous
`medium, 5Cveral im'cstigators have chemicall~' modified the
`that impan
`structure of drugs wilh fu nctional groups
`enhanced water-solubility. Among chemical modifications
`described in the an are the preparation of sulfonated deriva·
`tives [Kingston et al" U.S. Pal. No. 5,059,699 (l991)J, and
`6S amino acid eSters [Mathew ct a1" 1. Med. Chern. Vol.
`35:145- 151 (1992)] which show significant biological activ(cid:173)
`ity. Modifications to produce water-soluble derivatives
`
`Apotex v. Abraxis - IPR20 18-00 15 2, Ex. 1014, p.06 of34
`
`

`

`5.498,421
`
`3
`faciiiLatc the intravenous delivery, in aqueous medium (dis(cid:173)
`solved in an innocuous carrier such as Donnal saline). of
`drugs that arc inherently insoluble or poorly soluble. Such
`modifications, however, add to the cost of drug preparation,
`may induce undesired side-reactions andlor allergic reac(cid:173)
`lions, and/or may decrease the efficiency of the drug.
`Among the biologics which are frequently difficult to
`deliver is oxygen. Indeed, the need for clinically safe and
`effective oxygen carrying media for usc as red blood cell
`substituteS ("blood substitutes" or "artificial blood") cannot 10
`be overemphasized. Some of the potential uses of such
`media indudc (a) general transfusion uses, including both
`routine and emergency situations to replace acute blood loss,
`(b) support of organs in vitro prior \0 transplantation or in
`vivo during surgery, (e) enhancing oxygen delivery to 1$
`ischemic tissues and organs in vi\'o, (d) enhancing oxygen
`delivery to poorly vascularized tumors 10 increase tile lrCat(cid:173)
`ment efficacy of radiation therapy or chemotherapy, (e)
`support of organs or animals during experimental investi(cid:173)
`gations, and (I) incn:ascd oxygen transport to living cells in 20
`culture media.
`Blood transfusions are used to supplement the henlOdy(cid:173)
`namic system of patients who sulfer from a variety of
`disorders, including diminished blood volume, or hypov(cid:173)
`olemia (e.g. due to bleeding), a decreased number of blood
`cells (e.g. due to bone marrow destruction), or impaired or
`damaged blood cells (e.g. due to hemolytic anemia). Blood
`transfusions serve not only to increase the intravascular
`volume, but also to supply red blood cells whicll carry
`dissolved oxygen and facilitate oxygen delivery to tissuc.~
`In the case of transfusion of palicnts who have experi(cid:173)
`enced significanl blond loss, careful malching of dOllor and
`recipient blood Iypes often subjects the patient to periods of
`oxygen deprivation which is detrimental. FunhC11l1ore, even 35
`when autologous, patient-donated, red blood cells arc avail(cid:173)
`able through previous phlebotomy and stornge, the oxygen(cid:173)
`carrying capacity and safety of these autologous cells
`declines as a consequence of storage. Consequently, for a
`period of as much as 24 hours after_ transfusion, the patient 40
`may be subjcct to SUb-optimal oxygen delivery. Finally,
`there is the ever-present danger to the patient of viral and/or
`bacterial contamination in all transfusions of whole blood
`and red cells derived therefrom.
`Thus, there is a recognized need for a substance that is
`useful for oxygen transpon and delivery under normal
`envirunmental conditions thai incorporates Ihe folluwing
`features_ Ideally, a substance employed for oxygen transpon
`and delivery will be capable of canying and delivering
`oxygen to devices, organs and tissues such that normal
`oxygen tensions may be maintained in these environments_
`Such a substance will ideally be safe and non-toxic, free uf
`bacterial andlor viral contamination, and non-antigenic and
`non-pyrogenic (i.e . less than 0.25 EUlml). In addition, Ihe
`substance employed for oxygen transpon and delivery will
`have visrosity, colloid and osmotic propcnies comparable to
`bluud. It is also desirable that such a substance will be
`retained in the vascutar system of the patient for a long
`period of time, thus penniuing erythropoiesis and maturn(cid:173)
`lion of the patient's own red blood cells. Funhennorc, it is 60
`desirable thallhe substance employed nOI interfere with or
`hinder erythropoiesis.
`Ourently, a number of intravenous floids are available for
`the treatment of acute hypovolemia, including crystalloids,
`such as lactated Ringer's solution or normal saline, and
`conoidal solutions , such as normal human serum albumin.
`Crystalloids and colloids temporarily correct the volume
`
`4
`deficit, but do not dircctly supplement oxygen delivery to
`tissues. While blood transfUliion is the preferred mode of
`treatment, availability of sufficient quantities of a safe sup(cid:173)
`ply of blood is a perpetual problem.
`Addi tional biologics which are frequentiy inherently
`insoluble or poorly soluble in aqucous medium, and which
`are desirable to administer dissol ved in an innocuous carrier
`such as IIOrmai saline, while promoting a minimum of
`undesired side-reactions andlor allergic reaclions, are diag(cid:173)
`nostic agents such as contrMt agents. Contrast agents are
`desirable in radiological imaging because they enhance the
`visualization of Olgans (i.e., their location, size and cunfor(cid:173)
`mation) and other cellulae structUfCS from the surrounding
`medium. The soft tissues, for example, have similar cell
`composition (i.e., they arc primarily composed of water)
`even though they may have remarkably different biological
`functions (e.g., liver and pancreas).
`The lechniquc of magnetic resonance imaging (MRJ) or
`nuclear magnetic resonance (NMR) imaging reHes on thc
`detectinn of cenain atomic nuclei at an applied magnetic
`ficld strength using radio -frequen~y rndiation. In some
`respects il is similar to X-ray computer tomogrnphy (CT), in
`that it can provide (in some cases) cross-sectional images of
`organs with potentially cxcellcnt soft tissue resolution. In ils
`2S curret1l use, the images constilute a distribution map of
`prolOns in organs and tissues. However, unlike X-ray com(cid:173)
`pUler tomography, MR1 does not use ionizing radiation. MRl
`is, therefore, a safe non-invasive technique for medical
`imaging
`While Ihe phenomenon of NMR was discovered in 1954,
`itis only recently that ithas found use in medical diagnoslics
`as a means of mapping in1.ernal structure. The technique was
`first developed by Lauterbur [Nature 242: 190-191 ( 1973»).
`It is well known that nuclei with lhe appropriate nuclear
`spin align in the direction of the applied magnetic field_ The
`nuclear spin may be aligned in either of IwO ways: with or
`againstlbe external magnetic field. Alignment with the field
`is more stable; white energy must be absorbed to align;n the
`less stable state (i.e. against the applied field). In the case of
`prOlons, Ihese nuclei precess or resonate at a frequency of
`42.6 Mttr. in the prescucc of a I les!a (I tesla=IO"' gauss)
`magnetic field. AI this frequency, a radio-frequency (RF)
`pulse of radiation will excite the nuclei and change their spin
`45 orientalion 10 be aligned against the applied magnetic ficld_
`After the RF pulse, the cxcited nuclei "relax"" or return to
`equilibrium or alignment with the TIli!.gneti~ field. The decay
`of the relaxatio n signal can. be described using two relax(cid:173)
`ation tenns_ T1, the spin-lattice relaxation time or longitu-
`50 dinal relaxalion time, is Ihe time required by the nuclei 10
`return to equilibrium along the direction of the externally
`applied magm.1ic field . The sct:und, T1, or spin-spin relax(cid:173)
`ation time, is associated wi th the dephasing of the initially
`coherent precession of individual proton spins_ The relax-
`5S alion times for various Muids, organs and tissues in different
`species of mammals is well documented.
`One advantage of MRI is lhat different scanning planes
`and slice thicknesscs can be selected without loss of reso(cid:173)
`lution. This permits high qualilY transverse, coronal and
`sagitlal images to be obtained directly. The absence of any
`mechanical moving pans in the MRl equipment promotes a
`high degree of reliability. It is generally belie .. ed that MRJ
`has greater potential than X_ray computer tomography (CT)
`for the selective cxamination of tissuc.~_ Tn cr, the X-ray
`6S allClluation coefficients alone determine the image contrast,
`whereas at leaslthrce separate variables (T l' T ~ and nuclear
`spin density) contribute 10 the magneti~ resonance image.
`
`'"
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`Apotex v. Abraxis - IPR20 18-0015 2. Ex. 1014. p.07 of34
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`5,498,42 1
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`6
`potential for inducing allergic reactions du e to the use of
`emulsifiers and surfactants (e.g_. egg phophatidcs and egg
`yolk lecithin), 4) limitcd deliw:ry capabilities, and 5) water
`solu ble tluorocarbons are quickly diluted in blood after
`intravenous injection.
`
`2S
`
`5
`Due LO subtle physio-chemical dill'L1"CtlCes among organs
`and tissue, MRI may be capable of differentiating tissue
`types and in detecting diseases that may nO! be detccted by
`X-ray or cr. In comparison, CT and X-ray are only sensiti\'c
`\0 differences in clttlron densities in tissues and organs. The
`images obtainable by MRI techniques can also enable a
`physician [0 detect structures smaller than those detcctable
`by cr, due to itS better spatial resolution. Additionally. any
`imaging scan plane can be readily obtained using MRI
`techniques, induding trans\'crse, coronal and sagittal.
`Currently, MR1 is widely used [0 aid in the diagnosis of
`many medical disorders. Examples include joim injuries,
`bune marrow disorders, soft tissue tumors, ml:uiastinal inva(cid:173)
`sion, lymphadcoopaLhy, cavernous hemangioma, hemochro(cid:173)
`matosis, cirrhosis, renal cell carcinoma, uterine leiomyoma, t5
`adenomyosis, endometriosis, breast carcinomas, stenosis,
`coronary artery disease, aortic dissection, lipomatous hyper(cid:173)
`trOphy, atrial seprom. constrictive pericarditis. and the like
`[$Ce, for example, Edelman & WarRCh. Medical Progress
`328:708-716 (1993); Edelman & Warach, New England J. 20
`of Medicine 328:785- 791 (1993)]
`Routinel y employed magnetic resonance images are pres(cid:173)
`ently bas.ed on proton signals arising from the water mol(cid:173)
`ecules ..... itllin cells, Consequently, it is often difficult to
`decipher the images and distinguish individual organs and
`cellular structures. There are two potential means to better
`differentiate proton signals. The first involves using a con(cid:173)
`trast agent that alters the T , or T2 of the water molecules in
`one region compared 10 anoth~r. For example, gadolinium
`diethyJenetriaminepcntaacetic acid (Od-DTPA) shol1ens tile
`proton T , relaxation time of waLer molecules in near prox(cid:173)
`imity thereto, thereby enhancing the obtained images.
`Paramagnetic cations such as, for example, Od, Mn. and
`I'c arc c:<ce!lent MRI contrast ag cnt~, as suggested above.
`Thei r abili ty to shonen the proton T ] relaxation time of thc
`surrounding water enables enhanced MRI images to be
`obtained which otherwise would be unreadable.
`The second route to differentiate individual organs and
`cellular structures is to introduce another nucleus for imag(cid:173)
`ing (Le., an imaging agent). Using this second approach,
`imaging can only occur where the contrast agent has bcen
`delivered. An advantage of this mcthod is the fact that
`imaging is achieved free from interference from the sur(cid:173)
`rounding water. Suitable contrast agents must be bio-com- 4S
`patible (i.e_ non-toxic. chemically stable. not reacti ve with
`tissues) and of limited lifctime before: elimlnation from the
`hod,.
`Although, hydrogen has typically been selected as the
`basis for MRI scanning (because of its abundance in the
`body). this can result in poorly imaged areas due to lack of
`contrast. nlU~ the use of othcr activc MRI nuclci (such as
`fluorine) can. therefore. be advantageous. The usc of ccnain
`perfluorocarbons in various diagnostic imaging tcchnologies
`such as u1lrllsound, magnetic resonance, r.ldiography and
`computer tomograpby has been described in an .article by
`Mallery [see SPIE, 626, XrVIPACS IV, 18-23 (1 986)]. The
`use uf fluorine i~ Ildvantag~ous since fluorine is nUL nllWnllly
`found within the body_
`Prior art suggestions of fluorine-containing compounds
`useful for magnetic resonance imaging for medical diagnos(cid:173)
`tic purposes arc limited to a select group of fluorine(cid:173)
`comaining molecules that are water soluble or can fonn
`emulsions. Accordingly, prior an use of fluorocarbon emul(cid:173)
`sions of aqueous wluble fluorocarbons suffers from numer(cid:173)
`ous drawbacks, for example. 1) the use of unstable emul(cid:173)
`sions, 2) the lack of organ specificity and targeting, 3) the
`
`BRIEF DESCRIPTION OF THE INVENTION
`In accordance with thc present invention, there arc pro-
`to vidcd compositions useful for in vivo delivery of biologics,
`in the fonn of micro particles that are suitable fOT parenteral
`administration in aqueous suspension. Ill vention compu~i_
`tions comprise biologic (as a solid, liquid or gas) as:socialed
`with a polymeric shdL The polymeric shell is a biocompat(cid:173)
`ible material. crosslinked by the pre~ncc of disulfide bonds.
`The polymeric shell associated with biologic is optionally
`suspended in a biocompatib!e medium for administration.
`Usc of invention compositions for the delivery of biologicS
`obviates the ~cessity for administration of biologics in an
`emulsion containing. for examplc, cthanol and polyethoxy(cid:173)
`lated castor oil, diluted in normal salinc (see, for example,
`NOl1oll ct 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 propen-
`sity to produce allergic side effects
`In accordance with another aspect of the present in ven(cid:173)
`tion, it has surprisingly and unexpectedly been discovered
`that insoluble conStructs o f the protein hemog lobin (Hb)
`prepared in accordance with the im·ention reversibly bind
`30 oxygen. Insoluble hemoglobin constructs (IHC) of the
`present invention bind oxygen with oxygen affinities similar
`to those obtained wit.h soluble hemoglobin moleculn in red
`blood celis, or soluble modified hemoglobin molecules thet
`have been de5cribed in the prior art as potential blood
`3S substitutes
`In accordance with yel another aspect of the present
`invention. there are provided meihods for entrapping bio(cid:173)
`logics in a polymeric shell. Still further in accordanQ: with
`the present invention, there are provided means for obtain(cid:173)
`ing local oxygen and temperature data, and for obtaining
`fluorine magnetic resonance images of body organs and
`tissues.
`The delivcry of biologics in the fonn of a micropartieulate
`suspension allows some degree of targeting to organs such
`as the liver, lungs, splccn, lymphatic cireulation, and thc
`like, through the use of panicles of varying SilC, and th."Ough
`adminiSlnltion by different routes. TIle invention method of
`delivery further allows the adminislr.1tion of biolugics, such
`SO as substantially water insoluhle pharmacologically active
`agents, employing a much smaller volume of liquid and
`requiring greatly reduced adminiSiration time relative In
`administration volumes and times required by prior art
`delivcry systems (e.g., intravenous infusion of approxi-
`S5 matcly one to two liters of fl uid over a 24 hour period arc
`required to deliver a typ ical human dose of 20J-400 mg of
`taxol).
`For example. :t suspension of polymeric shells of the
`invention can be administered intravenously, making imag-
`ing of vascularilCd organs (e_! _. liver, spleen. lymph and
`lung) and hone marrow possible_ Organ target specificity is
`achieved as a result of uptake of the micron-siz.cd organ of(cid:173)
`luorine -containing polymeric shells by the reticu loendothe(cid:173)
`lial sy~tem (RES) (also known as the mononuclear phago-
`6S cyte (MNP) systcm). Organs such as the liver and spleen
`play an imponant role in remOI'ing foreign species (e,g.,
`particulate matter) from the bloodstream, and hence are
`
`60
`
`40
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`Apotex v. Abraxis - IPR20 18-00 152, Ex. 1014, p.08 of3 4
`
`

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`5,498,421
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`7
`often referred to as the "blood fillering organs". These
`organs make up a major part oflhe RES. In addition, lymph
`nodes within the lymphatic circulation contain cells of the
`RES. Consequently, imaging of the lymphatic system is
`possible employing micron-sized organoiluorinc-C(Inlaining
`polymeric shells of the present invention. Given orJlly or as
`a suppository, imaging of the stomach and gastrointestinal
`tract can be camed out Such suspensions can also be
`injected into non-vascular spaCl:, such as the cerebro·spinal
`cavity, allowing imaging of such space as welL
`As a further embodiment of the present invention, para(cid:173)
`magnetic cations such as Gd, Mo, Fe, and the like can be
`hound to polyanion.~, such as alginate, and used as an
`cffecti\'c MRl contrast agcnt.
`The present invention overcomes the drawbacks of the IS
`prior art by providing J) injectable suspensions of polymeric
`shells containing biologie, 2) biologics in a form having
`enhanced stability eompared to ~imple emulsions, 3) organ
`targeting specificity (e.g., liver, spleen, lung, and the like)
`due to uplilkl: of the polymeric shell~ of the ioventioo by the
`RES OJ MNP system, 4) e mulsifier·frcc system, thereby
`avoiding agents that may potentially cause a1lergie reac(cid:173)
`tions, and 5) the ability to inject relatively small doses of
`biologie and still achieve good response because the bio·
`logic-containing polymeric sheHs of the invention can be
`targeted \0 a specific organ.
`
`"
`
`8
`a liquid, optionally dispersed in a biocompatible dis(cid:173)
`persing agent, substantially completely contained
`wilhin a polymeric shell,
`a gas, optionally dispersed in a biocompatible dispcl1i(cid:173)
`ing agent, substantially completely contained within
`a pul ymeric shell,
`a gas associated with a polymeric shell, or mixtores of
`any two or more thereof,
`whcrein the largest cross-sectional dimension of said shell
`is no greatC!" than ahout 10 microns,
`wherein said polymeric shell comprises a biorompatible
`matcrial which i~ substantially crosslinked b)· way of
`disulfide bonds, and
`wherein the exterior of said polymeric shell is optionally
`modified by a suitab lc agent, wherein said agent is
`linked to said polymeric shell through an optional
`covalent linkage.
`As used herein, the term "in vivo ddive ry" refers to
`delivery