`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 5 :
`
`(11) International Publication Number:
`
`WO 94/18954
`
`A61K 9/48
`
`(43) International Publication Date:
`
`1 September 1994 (01.09.94)
`
`(21) International Application Number:
`
`PCT/U894/01985
`
`(22) International Filing Date:
`
`22 February 1994 (22.02.94)
`
`(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)
`
`(71) Applicant (for all designated States except US): CLOVER
`CONSOLIDATED, LIMITED [CH/CH]; 37, avenue de
`Rumini, CIT-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).
`
`SANDFORD, Paul, A. [US/US]; 2822 Overland Avenue,
`Los Angeles, CA 90064 (US). SUSLICK, Kenneth, S.
`[US/US]; 63 Chestnut Court, Champagne, IL 61821 (US).
`DESAI, Neil, P.
`[IN/US]; 847 Alandele Avenue, has
`Angeles, CA 90036 (US).
`
`(74) Agent: REITER, Stephen, E.; Pretty, Schroeder, Brueggemann
`& Clark, 444 South Flower Street, Ins Angeles, CA 90071
`(US).
`
`CZ, DE, DK, ES, FI, GB, HU, JP,
`LV, MG, MN, MW, NL, NO, NZ,
`SE, SK, UA, US, UZ, VN, Euro
`DE, DK, ES, FR, GB, GR,
`OAPI patent (BF, BJ, CF, C
`NE, SN, TD, TG).
`
`Published
`With international search report.
`
`
`
`
`
`
`
`
`
` I7(54) Title: METHODS FOR IN VIVO DELIVERY OF BIOLOGICS AND COMPOSITIONS USEFUL THEREOR
`
`(57) Abstract
`
`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 biocompan'ble
`material. The biologic can be associated with the poly-
`meric shell itself, and/or the biologic, optionally sus-
`pended/dispersed in a biooompatible 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
`biooompafible liquid.
`'
`
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`
`
`
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Contra] African chublic
`Congo
`Switza’land
`com d’Ivoim
`Camemon
`(Inna
`Czechoslovakia
`Czech Republic
`
`.
`
`EEERRESQQEQQSQgfii'aESEEEHEfi
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCI' on the front pages of pamphlets publishing international
`applications under the PCI'.
`
`United Kingdom
`Georgia
`Guinea
`Greece
`
`-
`
`Democratic People's Republic
`of Kom
`Rqaublic of Kmea
`Kazaklwan
`Liechtenstein
`Sri Lanka
`Luxembourg
`Latvia
`Monaco
`uninc of Moldova
`
`Poland
`Portugal
`Romania
`Russian Fedaadon
`Sudan
`Swodan
`Slovenia
`Slovakia
`Senegal
`Chad
`Togo
`Tajikistan
`'h-inidad and Tobago
`Ukraine
`United States of Main
`Uzbekistan
`Viet Nam
`
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`W0 94/18954
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`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
`
`diameter),
`
`and platelets
`
`(typically 1-3 microns
`
`in
`
`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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`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
`
`After
`phagocytosis may occur within the lymph nodes.
`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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`
`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. W085/00011 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
`
`and stored.
`
`Leucuta et al.
`
`[International Journal of
`
`Pharmaceutics Vol. 51:213-217 (1988)] describe the method
`
`of preparation of heat denatured microspheres.
`
`The
`
`procedure
`
`for
`
`preparing
`
`chemically
`
`crosslinked. microspheres involves treating the emulsion
`
`with glutaraldehyde to crosslink the protein,
`
`followed by
`
`washing and storage. Lee et al.
`
`[Science Vol. g;;:233-235
`
`(1981)] and U.S. Patent 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 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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`
`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 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 laddition
`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
`
`drugs 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.
`
`;§: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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`
`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.
`
`
`
`Among
`
`the
`
`biologics which
`
`are
`
`frequently
`
`difficult
`
`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
`
`"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
`media.
`
`to living cells in culture
`
`Blood transfusions are used to supplement
`
`the
`
`hemodynamic system of patients who suffer from a variety of
`
`disorders,
`
`including
`
`diminished
`
`blood
`
`volume,
`
`or
`
`hypovolemia (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
`
`experienced significant blood loss, careful matching of
`
`donor and recipient blood types often subjects the patient
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`
`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
`
`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 maturation 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 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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`
`volume deficit, 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 supply 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 disso1ved 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,
`
`fdr 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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`technique was
`
`first
`
`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
`
`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 = 104 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
`
`field. After the RF pulse,
`
`the excited nuclei "relax" or
`
`return to equilibrium or alignment with the magnetic field.
`
`The decay of the relaxation signal can be described using
`
`two relaxation terms.
`
`TU 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, T2, 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
`
`of
`resolution.
`This permits 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 coefficients alone determine
`
`the
`
`image contrast, whereas at
`
`least
`
`three separate
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`variables (T1, T2, 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
`
`ismaller 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
`
`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 ;;§:708-716 (1993);
`
`Edelman & Warach, New England J. of Medicine §g§: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 T1 or T2 of
`the water molecules in one region compared to another. For
`
`example,
`
`gadolinium giethylenetriaminepentaacetic
`
`acid
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`(Gd-DTPA) shortens the proton T1 relaxation time of water
`
`molecules in near proximity 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 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
`
`as fluorine) can,
`
`therefore, be advantageous.
`
`The use of
`
`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.
`
`Prior art
`
`suggestions of
`
`fluorine—containing
`
`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
`
`numerous drawbacks,
`
`for example,
`
`1)
`
`the use of unstable
`
`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.
`
`BRIEF DESCRIPTION OF THE INVENTION
`
`
`
`In accordance with the present invention,
`
`there
`
`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 compositionsx 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
`
`the 2nd National Cancer Institute
`in Abstracts of
`al.,
`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
`
`invention,
`
`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
`
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`
`affinities
`
`similar
`
`to
`
`those
`
`obtained with
`
`soluble
`
`hemoglobin molecules
`
`in red blood cells,
`
`or
`
`soluble
`
`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.
`
`The delivery of biologics
`
`in the form of
`
`a
`
`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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`
`species (e.g., particulate matter)
`
`from the bloodstream,
`
`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.
`
`
`
`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
`emulsions,
`3) organ targeting specificity (e.g.,
`
`simple
`liver,
`
`spleen,
`
`lung, and the like) due to uptake of the polymeric
`
`shells of
`
`the
`
`invention by
`
`the RES or MNP
`
`system,
`
`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
`organ.
`
`the invention can be targeted.
`
`to a specific
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`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,
`
`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.
`
`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
`
`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.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`
`
`In accordance with the present inVention,
`
`there
`
`are provided compositions
`
`for
`
`in vivo delivery of
`
`a
`
`biologic,
`
`wherein said biologic is selected from:
`
`a
`
`solid,
`
`optionally
`
`dispersed
`
`in
`
`a
`
`biocompatible
`
`dispersing
`
`agent,
`
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`
`substantially
`
`completely
`
`contained
`
`within a polymeric shell,
`
`liquid,
`
`optionally
`
`dispersed
`
`in
`
`a
`
`biocompatible
`
`dispersing
`
`agent,
`
`substantially
`
`completely
`
`contained
`
`within a polymeric shell,
`
`gas,
`
`optionally
`
`dispersed
`
`in
`
`a
`
`biocompatible
`
`dispersing
`
`agent,
`
`substantially
`
`completely
`
`contained
`
`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,
`
`wherein
`
`said
`
`polymeric
`
`shell
`
`comprises
`
`biocompatible material
`
`which
`
`substantially
`
`crosslinked
`
`by
`
`way
`
`disulfide bonds, and
`
`a
`
`is
`
`of
`
`wherein the exterior of said polymeric shell is
`
`optionally’ modified by a suitable agent,
`
`wherein
`
`said agent
`
`is
`
`linked to said
`
`polymeric shell through an optional covalent
`
`linkage.
`
`
`
`As used herein,
`
`the term "in vivo delivery"
`
`refers to delivery of
`
`a biologic by
`
`such routes of
`
`administration
`
`as
`
`oral,
`
`intravenous,
`
`subcutaneous,
`
`intraperitoneal, intrathecal,
`
`intramuscular, intracranial,
`
`inhalational,
`
`topical,
`
`transdermal, suppository (rectal),
`
`pessary (vaginal), and the like.
`
`As used herein,
`
`the term "biologic" refers to
`
`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 agents, physiologically active
`
`gases, vaccines, and the like), diagnostic agents (such as
`
`ultra