WO 94/18954
`
`wsa cS
`
`(74) Agent: REITER, Stephen, E.; Pretty, Schroeder, Brueggemann
`& Clark, 444 South Flower Street, Los Angeles, CA 90071
`(US).
`
`(81) Designated States: AT, AU, BB, BG, BR, BY, CA, CH, CN,
`CZ, DE, DK, ES, FI, GB, HU, JP, KP, KR, KZ
`LV, MG, MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD,
`SE, SK, UA, US, UZ, VN, European patent (AT, B
`DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE),
`OAPIpatent (BF, BJ, CF, CG
`NE, SN, TD, TG).
`
`Published
`With international search report.
`
`
`
`(30) Priority Data:
`08/023,698
`08/035,150
`
`22 February 1993 (22.02.93)
`26 March 1993 (26.03.93)
`
`US
`Us
`
`(60) Parent Applications or Grants
`(63) Related by Continuation
`US
`Filed on
`US
`Filed on
`
`08/023,698 (CIP)
`22 February 1993 (22.02.93)
`08/035,150 (CIP)
`26 March 1993 (26.03.93)
`
`CONSOLIDATED, LIMITED [CH/CH]; 37, avenue de
`Rumini, CH-1002 Lausanne (CH).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): GRINSTAFF, Mark, W.
`[US/US]; 330 South Mentor, #231, Pasadena, CA 91106
`(US). SOON-SHIONG,Patrick [US/US]; 11755 Chenault
`Street, Los Angeles, CA 90049 (US). WONG, Michael
`[US/US]; 601 Crescent, #16, Champagne, IL 61820 (US).
`
`(57) Abstract
`
`les Title: METHODS FORJNVIVODELIVERY OFBIOLOGICS AND COMPOSITIONS USEFULTHEREFOR.
`
`
`
`PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`
`
`(11) International Publication Number:
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`
`(51) International Patent Classification 5 :
`
`A61K 9/48
`
`(43) International Publication Date:
`1 September 1994 (01.09.94)
`
`
`
`SANDFORD,Paul, A. [US/US]; 2822 Overland Avenue,
`(21) International Application Number:
`PCT/US94/01985
`Los Angeles, CA 90064 (US). SUSLICK, Kenneth, S.
`'.|(22) International Filing Date:
`[US/US]; 63 Chestnut Court, Champagne, IL 61821 (US).
`22 February 1994 (22.02.94)
`DESAI, Neil, P.
`[IN/US]; 847 Alandele Avenue, Los
`Angeles, CA 90036 (US).
`
` (71) Applicant (for all designated States except US): CLOVER
`
`
`
`In accordance with the present invention, there
`are provided compositions useful for the in vivo de-
`livery of a biologic, wherein the biologic is associated
`with a polymeric shell formulated from a biocompatible
`material. The biologic can be associated with the poly-
`meric shell itself, and/or the biologic, optionally sus-
`pended/dispersed in a biocompatible dispersing agent,
`can be encased by the polymeric shell. In another as-
`pect, the biologic associated with polymeric shell is ad-
`ministered to a subject, optionally dispersed in a suitable
`biocompatible liquid.
`.
`
`
`
`
`
`
`
`
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`
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`Mongolia Poland
`
`BEZSSSERFREFERSIAG
`
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovenia
`Slovakia
`Senegal
`Chad
`Togo
`Tajikistan
`Trinidad and Tobago
`Ukraine
`United States of America
`Uzbekistan
`Viet Nam
`
`AT
`AU
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`cI
`CM
`CN
`cs
`Cz
`DE
`DK
`ES
`FI
`FR
`GA
`
`Belarus
`
`Central African Republic
`Congo
`Switzerland
`Cdte d'Ivoire
`Cameroon
`China
`Czechoslovakia
`Czech Republic
`Germany
`Denmark
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT onthe front pages of pamphlets publishing international
`applications under the PCT.
`
`82aS
`
`United Kingdom
`Georgia
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Kenya
`Kyrgystan
`Democratic People’s Republic
`of Korea
`Republic of Korea
`
`Republic of Moldova
`Madagascar
`Mali
`
`4%
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`WO 94/18954
`
`PCT/US94/01985
`
`METHODS FOR IN VIVO DELIVERY OF BIOLOGICS AND
`COMPOSITIONS USEFUL THEREFOR
`
`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.
`
`BACKGROUND OF THE INVENTION
`
`Microparticles and foreign bodies present in the
`blood are 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 6-8 microns
`in
`diameter),
`and platelets
`(typically 1-3 microns
`in
`diameter). The microcirculation in most organs and tissues
`allows
`the free passage of
`these blood cells.
`When
`microthrombii
`(blood clots) of size greater than 10-15
`microns are present in circulation, a risk of infarction or
`blockage of the capillaries results, leading to ischemia or
`oxygen deprivation and possible tissue death.
`Injection
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`PCT/US94/01985
`
`2
`
`into the circulation of particles greater
`
`than 10-15
`
`microns
`in diameter,
`therefore, must be
`avoided.
`A
`suspension of particles 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
`determines their biological behavior.
`Strand et al.
`[in
`Microspheres-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 mononuclear 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 first
`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 administration of such pharmaceuticals has been
`achieved by emulsification of oil solubilized drug with an
`aqueous. liquid (such as normal saline)
`in the presence of
`surfactants or emulsion stabilizers to produce
`stable
`microemulsions.
`These
`emulsions may
`be
`injected
`intravenously, provided the components of the emulsion are
`pharmacologically inert.
`For
`example,
`US Patent No.
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`WO 94/18954
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`PCT/US94/01985
`
`3
`
`4,073,943 describes the administration of water-insoluble
`
`pharmacologically active agents dissolved in oils and
`emulsified with water in the presence of surfactants such
`as egg phosphatides, pluronics (copolymers of polypropylene
`glycol and polyethylene glycol), polyglycerol oleate, etc.
`PCT International Publication No. WO85/00011 describes
`pharmaceutical microdroplets of an anaesthetic: coated with
`a phospholipid,
`such as dimyristoyl phosphatidylcholine,
`having suitable dimensions for intradermal or intravenous
`injection.
`
`Protein microspheres have been reported in the
`literature as carriers of pharmacological or diagnostic
`agents. Microspheres of albumin have been prepared by
`either heat denaturation or chemical crosslinking. Heat
`denatured microspheres are 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 microspheres are then washed with a suitable solvent
`
`[International Journal of
`Leucuta et al.
`and stored.
`Pharmaceutics Vol. 41:213-217 (1988)] describe the method
`of preparation of heat denatured microspheres.
`
`chemically
`preparing
`for
`procedure
`The
`crosslinked microspheres involves treating the emulsion
`with glutaraldehyde to crosslink the protein,
`followed by
`washing and storage. Lee et al.
`[Science Vol. 213:233-235
`(1981)] and U.S. Patent No. 4,671,954 teach this method of
`
`preparation.
`
`the preparation of
`for
`techniques
`above
`The
`as carriers of pharmacologically
`protein microspheres
`active agents, although suitable for the delivery of water-
`soluble agents, are incapable of entrapping water-insoluble
`ones.
`This limitation is inherent
`in the technique of
`preparation which
`relies
`on
`crosslinking
`or
`heat
`denaturation of the protein component in the aqueous phase
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`PCT/US94/01985
`
`4
`
`Any aqueous-soluble agent
`a water-in-oil emulsion.
`of
`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.
`
`solubility of many
`aqueous
`poor
`the
`Thus,
`biologics presents a problem for human administration.
`Indeed,
`the delivery of pharmacologically active agents
`that are inherently insoluble or poorly soluble in aqueous
`medium can be seriously impaired if oral delivery is not
`effective. Accordingly, currently used formulations for
`the delivery of pharmacologically 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.,
`emulsifiers)
`employed
`to solubilize pharmacologically
`active agents.
`Thus, a common regimen of administration
`involves treatment of the patient with antihistamines and
`steroids prior to injection of the pharmacologically 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
`a@rugs that are inherently insoluble or poorly soluble in
`aqueous medium,
`several
`investigators have
`chemically
`modified the structure of drugs with functional groups that
`impart
`enhanced water-solubility.
`Among
`chemical
`modifications described in the art are the preparation of
`sulfonated derivatives
`[Kingston et al., U.S. Patent
`5,059,699 (1991)], and amino acid esters [Mathew et al., J.
`Med. Chem. Vol. 35:145-151 (1992)] which show significant
`biological activity. Modifications
`to produce water-
`soluble derivatives facilitate the intravenous delivery, in
`aqueous medium (dissolved in an innocuous carrier such as
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`WO 94/18954
`
`PCT/US94/01985
`
`5
`
`normal saline), of drugs that are inherently insoluble or
`poorly soluble.
`Such modifications, however, add to the
`cost of drug preparation, may
`induce undesired side-
`reactions and/or allergic reactions, and/or may decrease
`the efficiency of the drug.
`
`frequently
`are
`biologics which
`the
`Among
`is oxygen.
`Indeed,
`the need for
`to deliver
`difficult
`clinically safe and effective oxygen carrying media for use
`as
`red blood cell substitutes
`("blood substitutes" or
`"artificial 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,
`(c) enhancing oxygen delivery to ischemic tissues
`and organs in vivo,
`(d) enhancing oxygen delivery to poorly
`vascularized tumors to increase the treatment efficacy of
`radiation therapy or chemotherapy,
`(e) support of organs or
`animals
`during
`experimental
`investigations,
`and
`(f)
`increased oxygen transport
`to living cells in culture
`media.
`
`the
`Blood transfusions are used to supplement
`hemodynamic system of patients who suffer froma variety of
`disorders,
`including
`diminishe@ blood
` volune,
`or
`hypovolemia (e.g. due to bleeding), a decreased number of
`blood celis (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
`experienced significant blood loss, careful matching of
`donor and recipient blood types often subjects the patient
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`WO 94/18954
`
`PCT/US94/01985
`
`6
`
`to periods of oxygen deprivation which is detrimental.
`Furthermore,
`even when autologous, patient-donated,
`red
`blood cells are available 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
`is useful
`for oxygen transport and delivery under
`that
`normal
`environmental
`conditions
`that
`incorporates
`the
`
`a substance employed for
`Ideally,
`following features.
`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 maturation of
`the
`patient's
`own
`red blood cells.
`Furthermore,
`it
`is
`desirable that the substance employed not interfere with or
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`hinder erythropoiesis.
`
`Currently,
`
`a number of
`
`intravenous fluids 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
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`PCT/US94/01985
`
`7
`
`volume deficit, but do not directly supplement oxygen
`delivery to tissues. While blood transfusion is the
`
`preferred mode of
`quantities of
`a
`problen.
`
`treatment, availability of sufficient
`safe supply of blood is a perpetual
`
`frequently
`are
`biologics which
`Additional
`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 diagnostic 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 conformation) 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 different 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
`field 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 computer 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
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`
`8
`
`technique was
`
`first
`
`developed
`
`by
`
`lLauterbur
`
`[Nature
`
`242:190-191 (1973)].
`
`It is well known that nuclei with the appropriate
`nuclear spin align in the direction of the applied magnetic
`field.
`The nuclear spin may be aligned in either of two
`ways: with or
`against
`the
`external magnetic
`field.
`Alignment with the field is more stable; while energy must
`be absorbed to align in the less stable state (i.e. against
`
`the applied field).
`In the case of protons,
`these nuclei
`precess or resonate at a frequency of 42.6 MHz
`in the
`presence of a 1 tesla (1 tesla = 10° gauss) magnetic field.
`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
`
`the excited nuclei "relax" or
`field. After the RF pulse,
`return to equilibrium or alignment with the magnetic field.
`The decay of the relaxation signal can be described using
`two relaxation terms. T,, the spin-lattice relaxation time
`or longitudinal relaxation time,
`is the time required by
`the nuclei to return to equilibrium along the direction of
`the externally applied magnetic field.
`The second, T,, or
`spin-spin relaxation time, is associated with the dephasing
`of the initially coherent precession of individual proton
`spins. The relaxation times for various fluids, organs and
`tissues in different species of mammals is well documented.
`
`One advantage of MRI is that different scanning
`planes and slice thicknesses can be selected without loss
`
`This permits high quality transverse,
`resolution.
`of
`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 coefficients alone determine
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`
`image contrast, whereas at
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`three separate
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`9
`
`variables (T,, T,, and nuclear spin density) contribute to
`the magnetic resonance image.
`
`Due to subtle physio-chemical differences 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.
`
`is widely used to aid in the
`Currently, MRI
`diagnosis of many medical disorders.
`Examples
`include
`joint injuries, bone marrow disorders, soft tissue tumors,
`mediastinal
`invasion,
`lymphadenopathy,
`cavernous
`hemangioma,
`hemochromatosis,
`cirrhosis,
`renal
`cell
`carcinoma, uterine leiomyoma, adenomyosis, endometriosis,
`breast
`carcinomas,
`stenosis,
`coronary artery disease,
`aortic dissection,
`lipomatous hypertrophy, atrial septum,
`constrictive pericarditis, and the like {see, for example,
`Edelman & Warach, Medical Progress 328:708-716 (1993);
`Edelman & Warach, New England J. of Medicine 328:785-791
`(1993)}.
`
`Routinely employed magnetic resonance images are
`presently based on proton signals arising from the water
`molecules within 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 contrast agent that alters the T, or T, of
`the water molecules in one region compared to another. For
`example,
`gadolinium diethylenetriaminepentaacetic
`acid
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`
`10
`
`(Gd-DTPA) shortens the proton T, relaxation time of water
`molecules in near proximity thereto,
`thereby enhancing the
`
`obtained images.
`
`for example, Gd,
`Paramagnetic cations such as,
`Mn, and Fe are excellent MRI contrast agents, as suggested
`above. Their ability to shorten the proton T, relaxation
`time of the surrounding water enables enhanced MRI
`images
`to be obtained which otherwise would be unreadable.
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`
`The
`
`second route to differentiate individual
`
`organs and cellular structures is to introduce another
`nucleus for imaging (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 surrounding water. Suitable contrast agents must
`be bio-compatible (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
`
`The use of
`therefore, be advantageous.
`as fluorine) can,
`certain perfluorocarbons
`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, XIV/PACS IV, 18-23
`(1986) ].
`The use of
`fluorine is advantageous’
`since
`fluorine is not naturally found within the body.
`
`fluorine-containing
`suggestions of
`Prior art
`compounds useful for magnetic resonance imaging for medical
`diagnostic purposes are limited to a select group of
`fluorine-containing molecules that are water soluble or can
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`form emulsions. Accordingly, prior art use of fluorocarbon
`emulsions of aqueous soluble fluorocarbons suffers from
`
`the use of unstable
`1)
`for example,
`numesous drawbacks,
`emulsions, 2)
`the lack of organ specificity and targeting,
`3) the potential for inducing allergic reactions due to the
`use of emulsifiers and surfactants (e.g., egg phophatides
`and egg yolk lecithin), 4)
`limited delivery capabilities,
`and 5) water soluble fluorocarbons are quickly diluted in
`blood after intravenous injection.
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`10
`
`BRIEF DESCRIPTION OF THE INVENTION
`
`15
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`
`there
`In accordance with the present invention,
`are provided compositions useful for in vivo delivery of
`biologics,
`in the form of microparticles that are suitable
`for parenteral
`administration in aqueous
`suspension.
`Invention compositions. comprise biologic
`(as
`a_
`solid,
`liquid or gas) associated with a polymeric shell.
`The
`polymeric shell is a biocompatible material, crosslinked by
`the presence of disulfide 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 polyethoxylated castor
`oil, diluted in normal saline (see, for example, Norton et
`al.,
`in Abstracts of
`the 2nd National Cancer Institute
`Workshop on Taxol
`& Taxus, September 23-24, 1992).
`A
`disadvantage of such known compositions is their propensity
`to produce allergic side effects.
`
`In accordance with another aspect of the present
`it has
`surprisingly and unexpectedly been
`invention,
`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
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`affinities
`
`similar
`
`to
`
`those
`
`obtained with
`
`soluble
`
`hemoglobin molecules or_solublein red blood cells,
`
`
`modified 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 biologics in a polymeric shell. Still further
`in accordance with the present
`invention,
`there are
`provided means for obtaining local oxygen and temperature
`data, and for obtaining fluorine magnetic resonance images
`of body organs and tissues.
`
`a
`in the form of
`The delivery of biologics
`microparticulate 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 different
`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 approximately one to two
`liters of fluid 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
`imaging of vascularized organs (e.g., liver, spleen,
`lymph
`and
`lung)
`and
`bone marrow possible.
`Organ
`target
`specificity is achieved as
`a result of uptake of
`the
`micron-sized organofluorine-containing polymeric shells by
`the reticuloendothelial system (RES)
`(also known as the
`mononuclear phagocyte (MNP) system). Organs such as the
`liver and spleen play an important role in removing foreign
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`from the bloodstrean,
`(e.g., particulate matter)
`Species
`and hence are often referred to as the "blood filtering
`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
`organofluorine-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.
`
`10
`
`As a further embodiment of the present invention,
`paramagnetic cations such as Gd, Mn, Fe, and the like can
`be bound to polyanions, such as alginate, and used as an
`effective 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 specificity (e.g.,
`liver,
`spleen,
`lung, and the like) due to uptake of the polymeric
`shells of
`the
`invention by
`the RES or MNP
`systen,
`4) emulsifier-free system, thereby avoiding agents that may
`potentially cause allergic reactions, and 5) the ability to
`inject relatively small doses of biologic and still achieve
`good response because the biologic-containing polymeric
`shells of
`the invention can be targeted to a specific
`organ.
`
`BRIEF DESCRIPTION OF THE FIGURES
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`Figure 1 shows a schematic of a polymeric shell
`prepared in accordance with the present invention.
`In the
`Figure, A refers to the insoluble disulfide crosslinked
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`polymeric shell, B refers to the interior of the polymeric
`shell, which
`can
`contain
`oxygen
`or
`other
`gas,
`a
`fluorocarbon containing dissolved oxygen, a biocompatible
`oil having biologic dissolved therein,
`a water-in-oil
`emulsion containing biologic dissolved in aqueous media, a
`suspension of solid particles dispersed in a liquid, and
`the like,
`Cc 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
`
`10
`
`about 0.1 up to 20 pm.
`
`Figure 2 presents oxygen binding curves (i.e., a
`graph of Hill coefficient
`(n)
`as a function of oxygen
`partial pressure) for a solution of stroma-free hemoglobin
`(the
`dashed
`line
`curve)
`and
`a_=e
`solution containing
`insolubilized
`hemoglobin
`constructs
`of
`the
`present
`invention (the solid line curve). Actual data points with
`the insolubilized hemoglobin constructs of
`the present
`invention are shown as solid boxes.
`
`Figure 3 presents oxygen binding curves 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)
`following treatment with 1.7mM of the allosteric effector,
`2,3-bisphosphoglycerate (2,3-BPG). Actual data points with
`the insolubilized hemoglobin constructs of
`the present
`invention are shown as solid boxes.
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`15
`
`20
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`
`DETAILED DESCRIPTION OF THE INVENTION
`
`there
`In accordance with the present invention,
`are provided compositions
`for
`in vivo delivery of
`a
`
`30
`
`biologic,
`
`wherein said biologic is selected from:
`a
`solid,
`optionally
`dispersed
`biocompatible
`dispersing
`
`a
`in
`agent,
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`a
`
`a
`
`5
`
`10
`
`15
`
`20
`
`completely
`substantially
`within a polymeric shell,
`liquid,
`optionally
`dispers2d
`biocompatible
`dispersing
`substantially
`completely
`within a polymeric shell,
`gas,
`optionally
`dispersed
`biocompatible
`dispersing
`substantially
`completely
`within a polymeric shell,
`a gas associated with a polymeric shell, or
`mixtures of any two or more thereof,
`wherein the largest cross-sectional dimension of
`said shell
`is no greater
`than about
`10
`microns,
`shell
`polymeric
`wherein
`said
`biocompatible material
`substantially
`crosslinked
`disulfide bonds, and
`wherein the exterior of said polymeric shell is
`optionally modified by a suitable agent,
`
`wherein linked to_saidsaid agent is
`
`
`polymeric shell through an optional covalent
`linkage.
`
`contained
`
`a
`in
`agent,
`contained
`
`a
`in
`agent,
`contained
`
`comprises
`which
`by
`way
`
`a
`is
`of
`
`25
`
`the term "in vivo delivery"
`As used herein,
`refers to delivery of
`a biologic by
`such routes of
`administration
`as
`oral,
`intravenous,
`subcutaneous,
`intraperitoneal, intrathecal,
`intramuscular, intracranial,
`inhalational,
`topical,
`transdermal, suppository (rectal),
`30 pessary (vaginal), and the like.
`
`the term "biologic" refers to
`As used herein,
`.
`pharmaceutically active agents (such as analgesic agents,
`anesthetic agents,
`anti-asthamatic agents, antibiotics,
`anti-depressant agents, anti-diabetic agents, anti-fungal
`agents, anti-hypertensive agents, anti-inflammatory agents,
`
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`anti-neoplastic agents, anxiolytic agents, enzymatically
`active agents, nucleic acid constructs,
`immunostimulating
`agents,
`immunosuppressive ayents, physiologically active
`gases, vaccines, and the like), diagnostic agents (such as
`ultrasound contrast
`agents,
`radiocontrast
`agents,
`or
`magnetic contrast agents), agents of nutritional value, and
`the like.
`
`As used herein,
`
`the term "micron" refers to a
`
`unit of measure of one one-thousandth of a millimeter.
`
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`A number of biocompatible materials may be
`employed in the practice of the present in