United States Patent [191
`Grinsta?‘ et al.
`
`llllllllllllllIIIlllllllllllllgl?ugglgllllllllIllllllllllllllllllllll
`
`[11] Patent Number:
`[45] Date of Patent:
`
`5,498,421
`*Mar. 12, 1996
`
`[54] COMPOSITION USEFUL FOR IN VIVO
`DELIVERY OF BIOLOGICS AND lVIETHODS
`EMPLOYING SAME
`
`[75] Inventors: Mark W. Grinsta?‘, Pasadena; Patrick
`Soon-Shiong, Los Angeles, both of
`Calif.; Michael Wong, Charnpaign, 111.;
`Paul A. Sandford, Los Angeles, Calif.;
`Kenneth S. Suslick, Champaign, 111.;
`Neil P. Desai, Los Angeles, Calif.
`
`[73] Assignee: Vivorx Pharmaceuticals, Inc., Santa
`Monica, Calif.
`
`[*] Notice:
`
`The term of this patent shall not extend
`beyond the expiration date of Pat. No.
`5,362,478.
`
`[21] Appl. No.: 200,235
`[22] Filed:
`Feb. 22, 1994
`
`Related U.S. Application Data
`
`[63] Continuation-impart of Ser. No. 23,698, Feb. 22, 1993, Pat.
`No. 5,439,686, and a continuation-in-part of Ser. No.
`35,150, Mar. 26, 1993, Pat. No. 5,362,478.
`
`[51] Int. Cl.6 ......................... .. A61K 37/22; A61K 9/127
`[52] U.S. Cl. ........................ .. 424/450; 424/451; 424/455;
`424/93; 424/9.34; 424/9.37; 424/9.4; 424/9.5
`[58] Field of Search ............................. .. 424/451, 45, 450
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3,959,457
`
`5/1976 Speaker et al. ......................... .. 424/19
`
`
`
`4,073,943 4,247,406
`
`
`
`2/ 1978 Wretlind et al. l/1981 Widder et a1. ..................... .. 252/6253
`
`2/1986 Feinstein ............................... .. 128/661
`4,572,203
`4,671,954 6/1987 Goldberg et al.
`424/450
`
`4,718,433
`
`1/1988 Feinstein . . . . . . . . . . . . .
`
`. . . .. 128/660
`
`4,789,550 12/1988 Hommel et al.
`4,844,882
`6/1989 Widder et al. ..
`4,929,446
`5/1990 Bartolucci
`
`. 424/493
`....... .. 424/9
`424/439
`
`5,059,699 10/1991 Kingston . . . . .
`
`. . . .. 549/511
`
`424/489
`5/1992 Geyer et al.
`5,110,606
`5,250,283 10/1993 Barnhart .................................... .. 424/5
`
`6/1994 Liversidge et al. ....................... .. 424/4
`5,318,767
`5,362,478 11/1994 Desai et al. ............................... .. 424/9
`FOREIGN PATENT DOCUMENTS
`
`0129619A1 2/1985 European Pat. Off. .
`0295941A2 12/1988
`European Pat. Oif. .
`0361677Al 4/1990
`European Pat. Off. .
`0391518A2 10/1990
`European Pat. Off. .
`0418153A1 3/1991
`European Pat. Oil". .
`O190050B1
`5/1991
`European Pat. Off. .
`O213303B1 9/1991
`European Pat. 01f. .
`85/00011
`1/1985
`WIPO .
`87/01035
`2/1987
`88/01506
`3/1988
`88/07365 10/1988
`89/03674 5/1989
`90/13780 11/1990
`WIPO .
`90/13285 11/1990 WIPO.
`91/15947 10/1991 WIPO.
`
`WIPO .
`
`WIPO .
`
`WIPO .
`
`WIPO .
`
`OTHER PUBLICATIONS
`Abuchowski et al., “Alteration of Immunological Properties
`of Bovine Serum Albumin by Covalent Attachment of
`Polyethylene Glycol” J. Biol. Chem. 252:3578 (1977).
`Burgess et al., “Potential use of albumin microspheres as a
`drug delivery system. 1. Preparation and in vitro release of
`steroids”
`International
`Journal of Pharmaceutics
`39:129—l36 (1987).
`(List continued on next page.)
`Primary Examiner—Thurman K. Page
`Assistant Examiner—Willian1 E. Benston, Jr.
`Attorney, Agent, or Firm—Stephen E. Reiter; Pretty,
`Schroeder, Brueggemann & Clark
`[57]
`ABSTRACT
`
`In accordance with the present invention, there are provided
`compositions useful for the in vivo delivery of a biologic,
`wherein the biologic is associated with a polymeric shell
`formulated from a biocompatible material. The biologic can
`be associated with the polymeric shell itself, and/or the
`biologic, optionally suspended/dispersed in a biocompatible
`dispersing agent, can be encased by the polymeric shell. In
`another aspect, the biologic associated with polymeric shell
`is administered to a subject, optionally dispersed in a suit
`able biocompatible liquid.
`
`30 Claims, 3 Drawing Sheets
`
`CIPLA EXHIBIT 1014
`Page 1 of 34
`
`

`

`5,498,421
`Page 2
`
`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 Contrast Echocardio
`graphy. I. In Vitro Development and Quantitative Analysis
`of Echo Contrast Agents” JACC 3(1):14——20 (1984).
`Grinstaff and Suslick, “Nonaqueous Liquid Filled Micro
`capsules” 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 (I981).
`Klibanov et al., “Amphipathic polyethyleneglycols elfec
`tively prolong the circulation time of liposomes” FEBS
`268(1):235—237 (1990).
`Koenig and Meltzer, “Effect of Viscosity on the Size of
`Microbubbles Generated for Use as Echocardiographic Con
`trast Agents” Journal of Cardiovascular Ultrasonography
`5(I).-3—4 (I986).
`
`Lee et al., “Serum Albumin Beads: An Injectable, Biode
`gradable System for the Sustained Release of Drugs” Sci
`ence 2]3:233—235 (I981).
`Leucuta et al., “Albumin microspheres as a drug delivery
`system for epirubicin: pharmaceutical, phannacokinetic and
`biological aspects,” International Journal of Pharmaceutics
`4I:2]3—2Z7 (I988) (I992).
`'
`Mathew [sic] et al., “Synthesis and Evaluation of some
`Water-Soluble Prodrugs and Derivatives of Taxol with
`Antitumor Activity” J. Med. Chem. 35:145—I5I.
`Molecular Biosystems, Inc., “Albunex—Preclinical Inves
`tigator’s Package”.
`Moseley et al., “Microbubbles: A Novel MR Susceptibility
`Contrast Agent” 10 Annual meeting of Society of Magnetic
`Resonance in Medicine in San Francisco, Calif. Oct. 1991.
`Suslick and Grinstaif, “Protein Microencapsulation of Non—
`aqueous Liquids” J. Am. Chem. Soc. 112(2] ):7807—7809
`( I 990).
`Willmott and Harrison, “Characterization of freeze—dried
`albumin microspheres containing the anti~cancer drug
`adriamycin” International Journal of Pharmaceutics
`43:161-166 (1988).
`
`CIPLA EXHIBIT 1014
`Page 2 of 34
`
`

`

`US Patent
`
`Mar. 12,1996
`
`Sheet 1 0f 3
`
`5,498,421
`
`CIPLA EXHIBIT 1014
`Page 3 of 34
`
`

`

`US. Patent
`
`Mar. 12, 1996
`
`Sheet 2 of 3
`
`5,498,421
`
`[0 ~
`
`9 -
`
`8-
`
`7-
`
`._
`5
`a;
`
`U
`
`e
`
`s
`
`a 4
`
`3r
`
`2
`
`i —
`
`0
`0
`
`0 mM PPOSPHATES
`
`----- BHb SOLUTION
`
`—I- Bib MICROBUBBLES
`SMOOTH CURVE
`I BHb HICROBUBBLES DATA
`
`I
`
`‘\_‘_
`
`l
`0 2
`
`I
`0 4
`
`l
`0.6
`
`J
`0.8
`
`l
`I
`I 2
`I
`log(P02/torr)
`
`l
`I 4
`
`I
`l 6
`
`l
`i 8
`
`I
`2
`
`CIPLA EXHIBIT 1014
`Page 4 of 34
`
`

`

`US. Patent
`
`Mar. 12, 1996
`
`Sheet 3 0f 3
`
`5,498,421
`
`FIG.3
`
`---- BHb SOLUTlON
`
`4- BH) NICROBUBBLES SMOOTH
`CURVE
`I BHb NICROBUBBLES DATA
`
`14
`
`
`
`HILL COEFF (n )
`
`CIPLA EXHIBIT 1014
`Page 5 of 34
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`

`

`5,498,421
`
`1
`COMPOSITION USEFUL FOR IN VIVO
`DELIVERY OF BIOLOGICS AND lVIETHODS
`EMPLOYING SAME
`
`RELATED APPLICATIONS
`
`This application is a continuation-in-part of U.S. Ser. Nos.
`08/023,698, ?led Feb. 22, l993,now issued as U.S. Pat. No.
`5,439,686 and 08/035,150, ?led Mar. 26, 1993, now issued
`as U.S. Pat. No. 5,362,478, the entire contents of which are
`hereby incorporated by reference herein.
`
`FIELD OF THE INVENTION
`
`The present invention relates to in vivo delivery of
`biologics. In one aspect, biologic is associated with a
`polymeric shell formulated from a biocompatible material.
`The biologic can be associated with the polymeric shell
`itself, and/or the biologic, optionally suspended/dispersed in
`a biocompatible dispersing agent, can be encased by the
`polymeric shell. In another aspect, the biologic associated
`with polymeric shell is administered to a subject, optionally
`dispersed in a suitable biocompatible liquid.
`
`20
`
`BACKGROUND OF THE INVENTION
`
`2
`sions may be injected intravenously, provided the compo
`nents of the emulsion are pharmacologically inert. For
`example, U.S. Pat. No. 4,073,943 describes the administra
`tion of water~insoluble pharmacologically active agents dis
`solved in oils and emulsi?ed with water in the presence of
`surfactants such as egg phosphatides, pluronics (copolymers
`of polypropylene glycol and polyethylene glycol), polyg
`lycerol oleate, etc. PCT International Publication No.
`WO85/000ll describes pharmaceutical microdroplets of an
`anaesthetic coated with a phospholipid, such as dimyristoyl
`phosphatidylcholine, having suitable dimensions for intrad
`errnal or intravenous injection.
`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 Phannaceutics Vol.
`41:213-217 (1988)] describe the method of preparation of
`heat denatured microspheres.
`The procedure for preparing chemically crosslinked
`microspheres involves treating the emulsion with glutaral
`dehyde to crosslink the protein, followed by washing and
`storage. Lee et al. [Science Vol. 213:233—235 (1981)] and
`U.S. 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.
`Thus, the poor aqueous solubility of many biologics
`presents a problem for human administration. Indeed, the
`delivery of pharmacologically active agents that are inher
`ently insoluble or poorly soluble in aqueous medium can be
`seriously impaired if oral delivery is not effective. Accord
`ingly, currently used formulations for the delivery of phar
`macologically active agents that are inherently insoluble or
`poorly soluble in aqueous medium require the addition of
`agents to solubilize the pharmacologically active agent.
`Frequently, however, severe allergic reactions are caused by
`the agents (e.g., emulsi?ers) employed to solubilize phar
`macologically active agents. Thus, a common regimen of
`administration involves treatment of the patient with anti
`histamines and steroids prior to injection of the phannaco
`logically active agent to reduce the allergic side effects of the
`agents used to aid in drug delivery.
`In an effort to improve the water solubility of drugs that
`are inherently insoluble or poorly soluble in aqueous
`medium, several investigators have chemically modi?ed the
`structure of drugs with functional groups that impart
`enhanced water-solubility. Among chemical modi?cations
`described in the art are the preparation of sulfonated deriva
`tives [Kingston et al., U.S. Pat. No. 5,059,699 (1991)], and
`amino acid esters [Mathew et al., J. Med. Chem. Vol.
`35 :l45—l5l ( 1992)] which show signi?cant biological activ
`ity. Modi?cations to produce water-soluble derivatives
`
`Microparticles and foreign bodies present in the blood are
`generally cleared from the circulation by the ‘blood ?ltering
`organs’, namely the spleen, lungs and liver. The particulate
`matter contained in normal whole blood comprises red blood
`cells (typically 8 microns in diameter), white blood cells
`(typically 6—8 microns in diameter), and platelets (typically
`l—3 microns in diameter). The rnicrocirculation in most
`organs and tissues allows the free passage of these blood
`cells. When rnicrothrombii (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 Micro
`spheres-Biomedical Applications, ed. A. Rembaum, pp
`193-227, CRC Press (1988)] have described the fate of
`particles to be dependent on their size. Particles in the size
`range of a few nanometers (nm) to 100 nm enter the
`lymphatic capillaries following interstitial injection, and
`phagocytosis may occur within the lymph nodes. After
`intravenous/intraarterial injection, particles less than about 2
`microns will be rapidly cleared from the blood stream by the
`reticuloendothelial system (RES), also known as the 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 oil solubilized drug with an aqueous liquid (such
`as normal saline) in the presence of surfactants or emulsion
`stabilizers to produce stable rnicroemulsions. These emul
`
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`5,498,421
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`3
`facilitate the intravenous delivery, in aqueous medium (dis
`solved in an innocuous carrier such as normal saline), of
`drugs that are inherently insoluble or poorly soluble. Such
`modi?cations, however, add to the cost of drug preparation,
`may induce undesired side-reactions and/or allergic reac
`tions, and/or may decrease the e?iciency of the drug.
`Among the biologics which are frequently dif?cult to
`deliver is oxygen. Indeed, the need for clinically safe and
`effective oxygen carrying media for use as red blood cell
`substitutes (“blood substitutes” or “arti?cial blood”) cannot
`be overemphasized. Some of the potential uses of such
`media include (a) general transfusion uses, including both
`routine and emergency situations to replace acute blood loss,
`(b) support of organs in vitro prior to transplantation or in
`vivo during surgery, (0) enhancing oxygen delivery to
`ischemic tissues and organs in vivo, (d) enhancing oxygen
`delivery to poorly vascularized tumors to increase the treat
`ment e?icacy of radiation therapy or chemotherapy, (e)
`support of organs or animals during experimental investi
`gations, and (f) increased oxygen transport to living cells in
`culture media.
`Blood transfusions are used to supplement the hemody
`namic system of patients who suffer from a variety of
`disorders, including diminished blood volume, or hypov
`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 which carry
`dissolved oxygen and facilitate oxygen delivery to tissues.
`In the case of transfusion of patients who have experi
`enced signi?cant blood loss, careful matching of donor and
`recipient blood types often subjects the patient to periods of
`oxygen deprivation which is detrimental. Furthermore, even
`when autologous, patient-donated, red blood cells are avail
`able through previous phlebotomy and storage, the oxygen
`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
`may be subject 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 transport and delivery under normal
`environmental conditions that incorporates the following
`features. Ideally, a substance employed for oxygen transport
`and delivery will be capable of carrying 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 of
`bacterial and/or viral contamination, and non-antigenic and
`non-pyrogenic (i.e. less than 0.25 EU/ml). In addition, the
`substance employed for oxygen transport and delivery will
`have viscosity, colloid and osmotic properties comparable to
`blood. It is also desirable that such a substance will be
`retained in the vascular system of the patient for a long
`period of time, thus permitting erythropoiesis and matura
`tion of the patient’s own red blood cells. Furthermore, it is
`desirable that the substance employed not interfere with or
`hinder erythropoiesis.
`Currently, a number of intravenous ?uids are available for
`the treatment of acute hypovolemia, including crystalloids,
`such as lactated Ringer’s solution or normal saline, and
`colloidal solutions, such as normal human serum albumin.
`Crystalloids and colloids temporarily correct the volume
`
`4
`de?cit, but do not directly supplement oxygen delivery to
`tissues. While blood transfusion is the preferred mode of
`treatment, availability of sufficient quantities of a safe sup
`ply of blood is a perpetual problem.
`Additional biologics which are frequently inherently
`insoluble or poorly soluble in aqueous medium, and which
`are desirable to administer dissolved in an innocuous carrier
`such as normal saline, while promoting a minimum of
`undesired side-reactions and/or allergic reactions, are diag
`nostic agents such as contrast agents. Contrast agents are
`desirable in radiological imaging because they enhance the
`visualization of organs (i.e., their location, size and confor
`mation) and other cellular structures from the surrounding
`medium. The soft tissues, for example, have similar cell
`composition (i.e., they are primarily composed of water)
`even though they may have remarkably dilferent biological
`functions (e.g., liver and pancreas).
`The technique of magnetic resonance imaging (MRI) or
`nuclear magnetic resonance (NMR) imaging relies on the
`detection of certain atomic nuclei at an applied magnetic
`?eld strength using radio-frequency radiation. In some
`respects it is similar to X-ray computer tomography (CT), in
`that it can provide (in some cases) cross-sectional images of
`organs with potentially excellent soft tissue resolution. In its
`current use, the images constitute a distribution map of
`protons in organs and tissues. However, unlike X-ray com
`puter tomography, MRI does not use ionizing radiation. MRI
`is, therefore, a safe non-invasive technique for medical
`imaging.
`While the phenomenon of NMR was discovered in 1954,
`it is only recently that it has found use in medical diagnostics
`as a means of mapping internal structure. The technique was
`?rst developed by Lauterbur [Nature 242:190-191 (1973)].
`It is well known that nuclei with the appropriate nuclear
`spin align in the direction of the applied magnetic ?eld. The
`nuclear spin may be aligned in either of two ways: with or
`against the external magnetic ?eld. Alignment with the ?eld
`is more stable; while energy must be absorbed to align in the
`less stable state (i.e. against the applied ?eld). In the case of
`protons, these nuclei precess or resonate at a frequency of
`42.6 MHz in the presence of a l tesla (l tesla:l04 gauss)
`magnetic ?eld. At this frequency, a radio-frequency (RF)
`pulse of radiation will excite the nuclei and change their spin
`orientation to be aligned against the applied magnetic ?eld.
`After the RF pulse, the excited nuclei “relax” or return to
`equilibrium or alignment with the magnetic ?eld. The decay
`of the relaxation signal can be described using two relax
`ation terms. T1, the spin-lattice relaxation time or longitu
`dinal relaxation time, is the time required by the nuclei to
`return to equilibrium along the direction of the externally
`applied magnetic ?eld. The second, T2, or spin-spin relax
`ation time, is associated with the dephasing of the initially
`coherent precession of individual proton spins. The relax
`ation times for various ?uids, organs and tissues in different
`species of mammals is well documented.
`One advantage of MRI is that di?erent scanning planes
`and slice thicknesses can be selected without loss of reso~
`lution. This pemiits high quality transverse, coronal and
`sagittal images to be obtained directly. The absence of any
`mechanical moving parts in the MRI equipment promotes a
`high degree of reliability. It is generally believed that MRI
`has greater potential than X-ray computer tomography (CT)
`for the selective examination of tissues. In CT, the X-ray
`attenuation coe?icients alone determine the image contrast,
`whereas at least three separate variables (T 1, T2, and nuclear
`spin density) contribute to the magnetic resonance image.
`
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`5,498,421
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`5
`Due to subtle physio-chemical di?erences among organs
`and tissue, MRI may be capable of differentiating tissue
`types and in detecting diseases that may not be detected by
`X-ray or CT. In comparison, CT and X-ray are only sensitive
`to differences in electron densities in tissues and organs. The
`images obtainable by MRI techniques can also enable a
`physician to detect structures smaller than those detectable
`by CT, due to its better spatial resolution. Additionally, any
`imaging scan plane can be readily obtained using MRI
`techniques, including transverse, coronal and sagittal.
`Currently, MRI is widely used to aid in the diagnosis of
`many medical disorders. Examples include joint injuries,
`bone marrow disorders, soft tissue tumors, mediastinal inva
`sion, lymphadenopathy, cavernous hemangioma, hemochro
`matosis, cirrhosis, renal cell carcinoma, uterine leiomyoma,
`adenomyosis, endometriosis, breast carcinomas, stenosis,
`coronary artery disease, aortic dissection, lipomatous hyper
`trophy, atrial septum, constrictive pericarditis, and the like
`[see, for example, Edelman & Warach, Medical Progress
`328:708-7l6 (1993); Edelman & Warach, New England I.
`of Medicine 328:785-791 (1993)].
`Routinely employed magnetic resonance images are pres
`ently based on proton signals arising from the water mol
`ecules within cells. Consequently, it is often di?icult to
`decipher the images and distinguish individual organs and
`cellular structures. There are two potential means to better
`differentiate proton signals. The ?rst involves using a con
`trast agent that alters the T1 or T2 of the water molecules in
`one region compared to another. For example, gadolinium
`diethylenetriarninepentaacetic acid (Gd-DTPA) shortens the
`proton Tl relaxation time of water molecules in near prox
`imity thereto, thereby enhancing the obtained images.
`Paramagnetic cations such as, for example, Gd, Mn, and
`Fe are excellent MRI contrast agents, as suggested above.
`Their ability to shorten the proton T1 relaxation time of the
`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—
`ing (i.e., an imaging agent). Using this second approach,
`imaging can only occur where the contrast agent has been
`delivered. An advantage of this method is the fact that
`imaging is achieved free from interference from the sur
`rounding water. Suitable contrast agents must be bio-com~
`patible (i.e. non-toxic, chemically stable, not reactive with
`tissues) and of limited lifetime before elimination from the
`body.
`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. Thus the use of other active MRI nuclei (such as
`?uorine) can, therefore, be advantageous. The use of certain
`per?uorocarbons in various diagnostic imaging technologies
`such as ultrasound, magnetic resonance, radiography and
`computer tomography has been described in an article by
`Mattery [see SPIE, 626, XlV/PACS IV, 18-23 (1986)]. The
`use of ?uorine is advantageous since ?uorine is not naturally
`found within the body.
`Prior art suggestions of ?uorine-containing compounds
`useful for magnetic resonance imaging for medical diagnos
`tic purposes are limited to a select group of ?uorine
`containing molecules that are water soluble or can form
`emulsions. Accordingly, prior art use of ?uorocarbon emul
`sions of aqueous soluble ?uorocarbons su?fers from numer
`ous drawbacks, for example, 1) the use of unstable emul
`sions, 2) the lack of organ speci?city and targeting, 3) the
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`potential for inducing allergic reactions due to the use of
`emulsi?ers and surfactants (e.g., egg phophatides and egg
`yolk lecithin), 4) limited delivery capabilities, and 5) water
`soluble ?uorocarbons are quickly diluted in blood after
`intravenous injection.
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`BRIEF DESCRIPTION OF THE INVENTION
`
`In accordance with the present invention, there are pro—
`vided compositions useful for in vivo delivery of biologics,
`in the form of microparticles that are suitable for parenteral
`administration in aqueous suspension. Invention composi
`tions comprise biologic (as a solid, liquid or gas) associated
`with a polymeric shell. The polymeric shell is a biocompat
`ible material, crosslinked by the presence of disul?de bonds.
`The polymeric shell associated with biologic is optionally
`suspended in a biocompatible medium for administration.
`Use of invention compositions for the delivery of biologics
`obviates the necessity for administration of biologics in an
`emulsion containing, for example, ethanol and polyethoxy
`lated 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 propen—
`sity to produce allergic side effects.
`In accordance with another aspect of the present inven—
`tion, it has surprisingly and unexpectedly been discovered
`that insoluble constructs of the protein hemoglobin (Hb)
`prepared in accordance with the invention reversibly bind
`oxygen. Insoluble hemoglobin constructs (IHC) of the
`present invention bind oxygen with oxygen a?inities similar
`to those obtained with soluble hemoglobin molecules in red
`blood cells, or soluble modi?ed hemoglobin molecules that
`have been described in the prior art as potential blood
`substitutes.
`In accordance with yet another aspect of the present
`invention, there are provided methods for entrapping bio—
`logics in a polymeric shell. Still further in accordance with
`the present invention, there are provided means for obtain
`ing local oxygen and temperature data, and for obtaining
`?uorine magnetic resonance images of body organs and
`tissues.
`The delivery of biologics in the form of a rnicroparticulate
`suspension allows some degree of targeting to organs such
`as the liver, lungs, spleen, lymphatic circulation, and the
`like, through the use of particles of varying size, and through
`administration by di?erent routes. The invention method of
`delivery further allows the administration of biologics, such
`as substantially water insoluble pharmacologically active
`agents, employing a much smaller volume of liquid and
`requiring greatly reduced administration time relative to
`administration volumes and times required by prior art
`delivery systems (e.g., intravenous infusion of approxi
`mately one to two liters of ?uid over a 24 hour period are
`required to deliver a typical human dose of 200-400 mg of
`taxol).
`For example, a suspension of polymeric shells of the
`invention can be administered intravenously, making imag
`ing of vascularized organs (e.g., liver, spleen, lymph and
`lung) and bone marrow possible. Organ target speci?city is
`achieved as a result of uptake of the micron-sized organof
`luorine-containing polymeric shells by the reticuloendothe
`lial system (RES) (also known as the mononuclear phago
`cyte (MNP) system). Organs such as the liver and spleen
`play an important role in removing foreign species (e.g.,
`particulate matter) from the bloodstream, and hence are
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`often referred to as the “blood ?ltering organs”. These
`organs make up a major part of the 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 organo?uorine-containing
`polymeric shells of the present invention. Given orally or as
`a suppository, imaging of the stomach and gastrointestinal
`tract can be carried out. Such suspensions can also be
`injected into non-vascular space, such as the cerebro-spinal
`cavity, allowing imaging of such space as well.
`As a further embodiment of the present invention, para
`magnetic cations such as Gd, Mn, Fe, and the like can be
`bound to polyanions, such as alginate, and used as an
`e?°ective MRI contrast agent.
`The present invention overcomes the drawbacks of the
`prior art by providing 1) injectable suspensions of polymeric
`shells containing biologic, 2) biologics in a form having
`enhanced stability compared to simple emulsions, 3) organ
`targeting speci?city (e.g., liver, spleen, lung, and the like)
`due to uptake of the polymeric shells of the invention by the
`RES or MNP system, 4) emulsi?er-free system, thereby
`avoiding agents that may potentially cause allergic reac
`tions, and 5) the ability to inject relatively small doses of
`biologic and still achieve good response because the bio
`logic-containing polymeric shells of the invention can be
`targeted to a speci?c organ.
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`BRIEF DESCRIPTION OF THE FIGURES
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`FIG. 1 shows a schematic of a polymeric shell prepared
`in accordance with the present invention. In the Figure, A
`refers to the insoluble disul?de crosslinked polymeric shell,
`B refers to the interior of the polymeric shell, which can
`contain oxygen or other gas, a ?uorocarbon containing
`dissolved oxygen, a biocompatible oil having biologic dis
`solved therein, a water'in-oil emulsion containing biologic
`dissolved in aqueous media, a suspension of solid particles
`dispersed in a liquid, and the like, C designates the thickness
`of the polymeric shell, typically about 5-50 nanometers, and
`D refers to the diameter of the polymeric shell, typically in
`the range of about 0.1 up to 20 um.
`FIG. 2 presents oxygen binding curves (Le, a graph of
`Hill coe?icient (n) as a function of oxygen partial pressure)
`for a solution of stroma-free hemoglobin (the dashed line
`curve) and a solution containing insolubilized hemoglobin
`constructs of the present invention (the solid line curve).
`Actual data points with the insolubilized hemoglobin con
`structs of the present invention are shown as solid boxes.
`FIG. 3 presents oxygen binding curves for a solution of
`stroma-free hemoglobin (the dashed line curve) and a solu
`tion containing insolubilized hemoglobin constructs of the
`present invention (the solid line curve) following treatment
`with 1.7 mM of the allosteric effector, 2,3-bisphosphoglyc
`erate (2,3-BPG). Actual data points with the insolubilized
`hemoglobin constructs of the present invention are shown as
`solid boxes.
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`DETAILED DESCRIPTION OF THE
`INVENTION
`
`In accordance with the present invention, there are pro
`vided compositions for in vivo delivery of a biologic,
`wherein said biologic is selected from:
`a solid, optionally dispersed in a biocompatible dis
`persing agent, substantially completely contained
`within a polymeric shell,
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`a liquid, optionally dispersed in a biocompatible dis
`p