EP0973553A2 - Vehicule du pool sanguin pour agents d'imagerie lipophiles - Google Patents

Vehicule du pool sanguin pour agents d'imagerie lipophiles

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Publication number
EP0973553A2
EP0973553A2 EP98918097A EP98918097A EP0973553A2 EP 0973553 A2 EP0973553 A2 EP 0973553A2 EP 98918097 A EP98918097 A EP 98918097A EP 98918097 A EP98918097 A EP 98918097A EP 0973553 A2 EP0973553 A2 EP 0973553A2
Authority
EP
European Patent Office
Prior art keywords
oil
water emulsion
emulsion
lipophilic
water
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98918097A
Other languages
German (de)
English (en)
Inventor
Raymond E. Counsell
Marc A. Longino
Jamey P. Weichert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Michigan System
University of Michigan Ann Arbor
Original Assignee
University of Michigan System
University of Michigan Ann Arbor
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Filing date
Publication date
Application filed by University of Michigan System, University of Michigan Ann Arbor filed Critical University of Michigan System
Publication of EP0973553A2 publication Critical patent/EP0973553A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A61K49/0433—X-ray contrast preparations containing an organic halogenated X-ray contrast-enhancing agent
    • A61K49/0447—Physical forms of mixtures of two different X-ray contrast-enhancing agents, containing at least one X-ray contrast-enhancing agent which is a halogenated organic compound
    • A61K49/0461—Dispersions, colloids, emulsions or suspensions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0404—Lipids, e.g. triglycerides; Polycationic carriers
    • A61K51/0406—Amines, polyamines, e.g. spermine, spermidine, amino acids, (bis)guanidines

Definitions

  • This invention relates generally to an oil-in-water emulsion, and more particularly, to an oil-in-water emulsion that functions as a blood-pool selective carrier or delivery vehicle for lipophilic imaging agents, or lipid-soluble derivatives of water- soluble, imaging agents incorporated therein.
  • CT scanning must be accomplished within 30 seconds of administration while the agent is still in the circulation phase. Intravascular contrast is rapidly lost as the agent diffuses into the extravascular space and distributes nonspecifically throughout the body. There is, therefore, a need for a delivery vehicle for CT scanning that can be administered less invasively and that will prolong the presence of the agent in the blood.
  • liposomal oil-in- water emulsions have been developed wherein the inclusion of polyethylene glycoi (PEG) or a PEG derivative of a phospholipid, was found to reduce RES uptake and clearance of parenterally administered delivery vehicles and to prolong the blood half life of the vehicles.
  • PEG polyethylene glycoi
  • liposomes and lipoproteins share some common structural lipid components and have considerable overlap in particle size, there remain significant differences in particle structure and in the mechanism of sequestration of the two particle types by their respective target tissues.
  • Liposomes which are artificially prepared lipid vesicles formed by single or multiple polar lipid bilayers, consisting primarily of phospholipids and cholesterol, enclosing aqueous compartments are particulate in nature, and hence, have potential for delivering agents contained therein to the RES. Investigators have attempted to load liposomes with both ionic and non-ionic water-soluble urographic contrast media.
  • Lipoproteins are naturally-occurring, oil-in-water emulsions composed of a monolayer of polar (amphiphilic) lipids that surround a neutral lipid core made up of cholesteryl esters and triglycerides.
  • a variety of apolipoproteins associate with the polar monolayer of these lipid-transport particles.
  • Each of the apolipoproteins plays a role as a recognition factor for tissue-selective, receptor-mediated uptake or in enzyme-mediated metabolism of the various classes of lipoproteins.
  • Liposomes which lack these specific surface recognition proteins, are rapidly sequestered by macrophages of the RES in the lungs, liver (Kupffer cells), spleen, and bone marrow. Liposomal biodistribution can be modulated somewhat by alteration of the surface charge, particle size, and chemical modification of surface components, although a significant portion of the modified liposomal material is still sequestered by the macrophages.
  • a problem with RES-mediated particulates, such as the aforementioned liposomes is toxicity. Large imaging doses of particulate contrast agents have been associated with engorgement of the Kupffer cells of the liver resulting in sinusoidal congestion and consequent activation of macrophages which release toxic mediators.
  • an object of this invention to provide a delivery vehicle, specifically a blood-pool selective, surface- modified, oil-in-water emulsion, for transport of lipophilic agents, or lipophilic derivatives of water soluble agents, such as radiologic contrast agents.
  • this invention is a surface- modified synthetic oil-in-water lipid emulsion, resembling endogenous lipoproteins, in order to take advantage of the natural lipid transport system of a living being.
  • the surface-modified oil-in-water emulsion of the present invention have been modified with derivatized polyethylene glycoi or polyethylene glycoi derivatives of phospholipids to prolong retention time in the blood, possibly by interfering with the association of the emulsion particles with apolipoproteins and/or opsonins which are responsible for mediating cellular uptake and circulatory elimination of the vehicle.
  • Lipophilic agents or lipophilic derivatives of water-soluble agents which are diagnostically, therapeutically, or biologically active or inactive, inserted into the lipid core of the emulsion are retained in the blood.
  • the mean oil phase particle size is between 50 and 150 nm (number weighted), with a narrow size distribution (50 to 250 nm) wherein no more than 2% of the particles have a diameter that falls outside of the range (i.e. , being greater than 250 nm).
  • the emulsion should have no detectable particles with a diameter greater than 1 ⁇ m. Moreover, the emulsion should not be contaminated with liposomes.
  • the synthetic oil-in-water emulsion of the present invention has the general formula:
  • the types of agents that can be administered by incorporation into the lipophilic core of the synthetic oil-in-water emulsions of the present invention are lipophilic contrast agents and/or lipophilic derivatives of conventional water-soluble contrast agents.
  • the lipophilic core components comprise up to 50% (w/v) of the emulsion, and preferably between about 10% and 40% (w/v).
  • the lipophilic core may comprise any pharmaceutically acceptable fat or oil of natural, synthetic, or semi-synthetic origin which is a pharmacologically inert nonpolar lipid that will locate in the lipophilic core of the oil-in-water emulsion.
  • the lipophilic core includes lipophilic contrast agents or lipophilic derivatives of water-soluble contrast agents that may be used for diagnostic purposes.
  • exemplary agents include, but are not limited to, halogenated triglycerides, such as iodinated or fluorinated triglycerides; perfluorinated lower alkyls; or aliphatic esters of conventional water-soluble contrast agents, such as aliphatic esters of iopanoic acid, which agents may contain a stable or radioactive isotope of the halogen.
  • halogenated triglycerides such as iodinated or fluorinated triglycerides
  • perfluorinated lower alkyls or aliphatic esters of conventional water-soluble contrast agents, such as aliphatic esters of iopanoic acid, which agents may contain a stable or radioactive isotope of the halogen.
  • contrast agent or “imaging agent” is used herein to denote generically an agent useful for any imaging modality.
  • the lipophilic core includes a mixture of at least one pharmacologically inert (or inactive) oil and a contrast agent in a molar ratio in the range of 0.1 to 3.
  • the ratio of inert oil to contrast agent is from 0.1: 1.0 to 2: 1 , and more preferably 1 : 1.
  • the lipophilicity of each core component is comparable to ensure suitable blending of the lipid components.
  • iodine-containing lipids of the type known in the art, can be used.
  • lipids include iodinated fatty acids in the form of glycerol or alkyl esters.
  • the iodine-containing lipids are synthetic aromatic compounds of known purity that are stabilized against in vivo degradation of the iodine linkage.
  • radioactive or non-radioactive halogenated triglycerides useful in the practice of the invention include, without limitation, iodinated triglycerides of the type described in United States Patent No.
  • Exemplary iodinated triglycerides are 2-oleoylglycerol- l ,3-bis[7-(3-amino-2,4,6- triiodophenyl)heptanoate] (DHOG) and 2-oleoylglycerol-l ,3-bis[4-(3-amino-2,4,6- triiodopheny butanoate] (DBOG), such as disclosed in International Publication No. WO 95/31181 published December 14, 1995, the text of which is incorporated herein by reference.
  • DHOG 2-oleoylglycerol- l ,3-bis[7-(3-amino-2,4,6- triiodophenyl)heptanoate]
  • DBOG 2-oleoylglycerol-l ,3-bis[4-(3-amino-2,4,6- triiodopheny butanoate]
  • 123 1, 125 I, and 131 I are the iodine isotopes most often used with currently available scanning instrumentation.
  • 13I I-radiolabeled triglycerides may be used for therapeutic purposes, as is known in the art.
  • any radioactive isotope of iodine is within the contemplation of the invention.
  • a listing of all iodine isotopes is available, for example, at pages Misc. 47-49 of the Merck Index, 11 th Edition, and at pages 1 1-68 to 11-70 of the Handbook of Chemistry and Physics. 72d Edition, CRC Press, 1991-1992.
  • 127 I is the naturally-occurring stable isotope and is not considered to be "radioactive".
  • specific examples include stable ( 19 F) or radioactive ( 18 F) fluorinated triglycerides that are analogous to the iodinated triglycerides discussed above, illustratively glyceryl-2-oleoyl-l,3-bis(trifluoromethyl)phenyl acetate.
  • fluorine-containing lipids may be esters or triglycerides of perfluoro-t-butyl-containing fatty acid compounds, such as described in United States Patent Numbers 5, 116,599 and 5,234,680, illustratively, 7,7,7-trifluoro- 6,6-bis (trifluoromethyl)-heptanoic acid or 8,8,8-trifluoro-7,7-bis(trifluoromethyl)- octanoic acid.
  • Other examples include the perfluorinated low molecular weight hydrocarbons, useful as ultrasound imaging agents, such as described in United States
  • the contrast agent may comprise brominated compounds, such as brominated ethyl esters of fatty acids or monobrominat- ed perfluorocarbons.
  • brominated compounds such as brominated ethyl esters of fatty acids or monobrominat- ed perfluorocarbons.
  • therapeutic agents specifically radiopharmaceuticals
  • lipophilic core of the synthetic oil-in-water emulsion of the present invention may be included in the lipophilic core of the synthetic oil-in-water emulsion of the present invention.
  • the monolayer surrounding the nonpolar lipophilic core comprises up to about 10% (w/v) of an amphipathic lipid monolayer component, which may be an emulsifier.
  • Phospholipids of natural, synthetic, or semi-synthetic origin are suitable for use in the practice of the invention.
  • Traditional lipid emulsions for delivery of contrast agents use natural phospholipids, such as soy lecithin and egg phosphatidylcholine (e.g. , Intralipid).
  • the emulsion components are synthetic, semi-synthetic, and/or naturally occurring components of known origin, purity and relative concentrations.
  • the improper use of egg lecithins (mixtures of phospholipids) and/or crude oils (cottonseed, poppy seed, and the like), as in typical prior art emulsions, may result in variable and non-reproducible compositions.
  • DOPC dioleoylphosphatidylcholine
  • emulsifier or monolayer surfactant.
  • DOPC is a semi-synthetic, chemically defined phospholipid emulsifier of high purity (available from Avanti Polar Lipids,
  • polyethylene glycol-linked lipids are incorporated into the monolayer.
  • a derivatized polyethylene glycoi such as methoxy polyethylene glycoi (MPEG), having a molecular weight between about 1000 and 6000 and/or polyethylene glycol-derivatized lipids, such as MPEG-linked to phosphatidylethanol- amine or distearoyl phosphatidylethanolamine are preferred.
  • the PEG component should comprise between about 0. 1 and 30 mole percent of the monolayer components for achieving attenuation of retention time of the delivered contrast agent in the blood.
  • the synthetic MPEG-linked phospholipids may contain fatty acyl groups, including but not limited to myristoyl, palmitoyl, steroyl, oleoyl, or combinations thereof.
  • MPEGs can be covalently linked to the phospholipid moiety by succinate, carbamate, or amide linkages, or by other covalent linkages known to those skilled in the art.
  • MPEG-linked phospholipids are available commercially from Matreya, Inc., Pleasant Gap, PA and Avanti Polar Lipids, Inc., Alabaster, AL.
  • Preferred MPEG- modified phospholipids include MPEG-linked phosphatidylethanolamine; MPEG-2000-
  • 1,2-distearoyl and MPEG-2000-l ,2-dioleoyl phosphatidylethanolamine.
  • other polysaccharides can be associated with phospholipids, or other suitable membrane lipid moieties, to modify the surface of the monolayer in order to block association of the emulsion particles with apolipoproteins and/or opsonins, thereby interfering with receptor-mediated uptake and prolonging the residence time of the lipid emulsion in the blood.
  • the composition also contains a sterol, which is preferably cholesterol, in an amount of up to 5% by weight in order to stabilize the emulsion, and preferably in the range of 0.4 to 0.5% (w/v).
  • a sterol which is preferably cholesterol
  • the molar ratio of sterol to emulsifier which may be a natural, synthetic, or semi- synthetic phospholipid, has been found to directly affect the particle diameter and dimensional stability.
  • the preferred molar ratio of sterol to phospholipid for achieving an emulsion of the desired size in the range of 0.05 to 0.70, and more specifically at 0.40 for delivery of iodinated triglycerides.
  • the remainder of the emulsion formulation comprises the bulk or aqueous phase containing up to 5% (w/v) USP glycerol.
  • the aqueous phase is de-ionized water of a grade suitable for parenteral administration.
  • salt NaCl
  • ionic buffers ionic buffers
  • the presence of salt in the formulation has an adverse effect on the ability of the emulsion to survive autoclave sterilization without a significant change in mean particle size as well as on the temporal stability of an autoclaved emulsion.
  • Any ionic species in the formulation adversely impacts the long term stability of the emulsion.
  • Other conventional additives such as antioxidants, buffers, preservatives, viscosity adjusting agents, and the like, may be included in the composition. In particular, up to 1 % w/v of an antioxidant, such as ⁇ -tocopherol, flavinoids, BHT, or BHA, is recommended.
  • an antioxidant such as ⁇ -tocopherol, flavinoids, BHT, or BHA
  • the additive should not adversely affect the physical and biological characteristics of the emulsion, such as particle size, shelf stability, and biodistribution.
  • the techniques used to formulate the oil-in-water emulsions of the present invention are important in achieving small particle diameter, uniform size distribution, lack of liposome contamination, etc.
  • the lipophilic components of the oil-in-water emulsion including nonpolar core lipids, polar lipid emulsifiers, and other lipophilic components, such as contrast agents, are blended together to form a premixed lipid phase.
  • the aqueous components are combined and added to the premixed lipid phase.
  • the premixed lipid phase and aqueous components are homogenized to form a crude oil-in-water emulsion.
  • the crude oil-in-water emulsion is subjected to ultra high energy emulsification to produce a fine oil-in-water emulsion having a mean particle diameter of the oil phase between 50 to 150 nm with greater than 98 % of the particles being less than 250 nm.
  • the fine oil-in-water emulsion is sequentially filtered.
  • the lipid components are initially blended or homogenized with USP glycerol using a high speed mixer, such as a Polytron homogenizer (Kinematica GmbH, Lucerne, Switzerland) or Ultra Turrax (IKA- Works, Cincinnati, OH), operating at 12,500 rpm under a nitrogen atmosphere at 55° C for at least 5 minutes. Then, the aqueous components are added to the anhydrous glycerol-lipid emulsion and emulsified by high speed mixing or homogenization at
  • 25,000 rpm under the same, or similar, conditions to form a crude oil-in-water emulsion.
  • Final processing is accomplished with ultra high energy mixing equipment, such as a MicroFluidizer high pressure homogenizer (Model 1 10S, Microfluidics Corp. , Newton, MA; see, USPN 4,533,254), or equivalent equipment, such as the Emulsiflex (Avestin Inc. , Ottawa, Ontario, Canada) or the Manton-Gaulin (APV Gaulin Rannie, St. Paul, MN), operating in the recycling mode at 35-60° C and 10,000 to 30,000 psi, and preferably at about 14,700-23,000 psi, for up to about 20 minutes.
  • the emulsion is passed sequentially through sterile 0.45 ⁇ m and 0.22 ⁇ m sterile filters. The sequential filtration removes any large particles and offers the potential of end-point sterilization of the product.
  • the temperature for high energy mixing is illustrative, and should be chosen relative to the contrast agent. In other words, the temperature should be greater than or equal to the phase transition temperature or melting point of the contrast agent or emulsifier (phospholipid or MPEG-linked phospholipid). An upper bound, however, is determined by whether the temperature would cause degradation or decomposition of any components in the composition.
  • an ultra high pressure homogenizer ensures small particle size with a narrow size range distribution.
  • Conventional systems for forming emulsions such as homogenizers, sonicators, mills, and shaking systems provide a shearing force on the liquid components whereas the ultra high energy mixing equipment puts the emulsion components under pressure and forces them through small openings to reduce particle size.
  • Size distribution may be measured by submicron laser photon correlation spectroscopy (PCS) on a Nicomp 370 Dynamic Laser Light Scattering Autocorrelator (Nicomp Particle Sizing Systems, Santa Barbara, CA) or similar equipment.
  • a lipid emulsion which is suitable for the practice of the present invention, will have a mean particle diameter less than about 250 nm, and preferably in the range of 50 to 150 nm as measured by Nicomp number weighting analysis.
  • the particles should have a narrow size distribution, with about 98% of the particles being in the 50 to 250 nm. No particles should be detected with a diameter of greater than l ⁇ m.
  • an oil-in-water emulsion of the present invention containing a contrast enhancing agent is administered to a mammal and the mammal is subjected to x-ray computed tomographic imaging after the emulsion has reached stable blood levels, e.g. , 1-30 minutes post-injection and prior to decline in levels (up to about 2 hours).
  • appropriate oil-in-water emulsions, containing contrast agents suitable for other diagnostic modalities such as proton magnetic resonance imaging (MRI), 19 F-MRI, ultrasonography, or scintigraphy may be administered for visualization and/or detection.
  • Fig. 1 is an illustrative preparatory scheme for a series of fluorinated or iodinated triglycerides, specifically 1 ,3-disubstituted triacylglycerols, suitable for use in the practice of the present invention
  • Fig. 2 shows molecular formulae for lipophilic triacylglycerols useful in the practice of the present invention
  • Fig. 4 is a graphical representation of CT density in Hounsfield Units (HU) versus time in minutes in the blood of female New Zealand White rabbits following iv administration of DHOG-PEG or DHOG-LE; and
  • Fig. 5 is a graphical representation of pulmonary artery pressure and heart rate as a function of time (in minutes) post-administration of a contrast-agent containing emulsion of present invention to a pig.
  • the oil phase particle of the present invention has a lipophilic lipid core surrounded by a monolayer consisting of an emulsifier, which may be a phospholipid, a stabilizer, such as cholesterol, the polyethylene glycol-derivatized component.
  • the lipid core contains a pharmacologically inert fat or oil, such as a triglyceride (e.g. , triolein) and/or a lipophilic agent, such as a radiologic contrast agent, or a lipophilic derivative of a water-soluble contrast agent.
  • a pharmacologically inert fat or oil such as a triglyceride (e.g. , triolein) and/or a lipophilic agent, such as a radiologic contrast agent, or a lipophilic derivative of a water-soluble contrast agent.
  • the polar moieties e.g.
  • polar head portions of a phospholipid emulsifier) of the monolayer face outward into the bulk water phase whereas the nonpolar moieties (tails of the phospholipid emulsifier) of the monolayer are oriented toward the lipid core.
  • a purely lipophilic compound to be delivered in accordance with the principles of the invention would reside almost entirely in the core of the lipid particle beneath the monolayer.
  • Exemplary lipophilic contrast agents include, but are not limited to, agents of the type reported by Weichert, et al. (see, for example, Weichert, et al. , J. Medicinal Chemistrv (1986, 29: 1674-82); (1986, 29:2457-65); and (1995, 58:636-46)) as well as other lipid soluble derivatives of traditional water-soluble contrast agents including, but not limited to, aliphatic esters of iopanoic, diatrizoic, and acetrizoic acids as listed in "Principles of Medicinal Chemistry (4 th edition)," edited by William Foye, Chapter 43, R. E. Counsell and J. P. Weichert authors, Williams and Wilkins, 1995.
  • radioactive or non-radioactive polyhalogenated triglycerides particularly suitable for use in the practice of the invention are described in United States Patent No. 4,873,075 issued on October 10, 1989; United States Patent No. 4,957,729 issued on September 18, 1990; and United States Patent No. 5,093,043 issued on March 3, 1992, the disclosures of which are incorporated by reference herein in their entirety.
  • the iodinated arylaliphatic triglyceride analogs of the aforementioned patents have a triglyceride backbone structure that is 1,3-disubstituted or 1,2,3- trisubstituted with a 3-substituted 2,4,6-triiodophenyl aliphatic chain or a monoiodophen- yl aliphatic chain.
  • all of the aliphatic chains, whether on the iodinated moiety or an open position on the triglyceride backbone structure are saturated or unsaturated aliphatic hydrocarbon chains of the type found in naturally-occurring fatty acids.
  • Naturally-occurring fatty acids may include those containing about 4-20 carbons, illustratively palmitic acid (16), palmitoleic acid (16: 1), oleic acid (18: 1), linoleic acid (18:2), arachidonic acid (20:4), etc.
  • glyceryl-2-palmitoyl-l,3-di-(3- amino-2,4,6-triiodophenyl)iopanoate glyceryl-2-palmitoyl- 1 ,3-di-(3-amino-2,4,6- triiodophenyl)dodecanoate
  • glyceryl-2-palmitoyl- l , 3-di-(3-amino-2 , 4 , 6- triiodophenyl)propionoate glyceryl-l,2,3-triiopanoate
  • iodinated triglycerides were synthesized and radioiodinated with I25 I via isotope exchange in a melt of pivalic acid in accordance with a method known in the art.
  • radioiodination of the iodinated triglycerides, or one of the intermediates in their synthesis pathway can be accomplished by a variety of techniques, known to those of skill in the art.
  • Example 1 illustrate some of the many possible lipophilic contrast agents that can be delivered to the blood pool in oil-in-water emulsions made in accordance with the principles of the invention.
  • Example 1 illustrate some of the many possible lipophilic contrast agents that can be delivered to the blood pool in oil-in-water emulsions made in accordance with the principles of the invention.
  • Example 1 illustrate some of the many possible lipophilic contrast agents that can be delivered to the blood pool in oil-in-water emulsions made in accordance with the principles of the invention.
  • a general reaction scheme is shown for iodinated or fluorinated triglycerides, specifically 1 ,3-disubstituted triacylglycerols, or ⁇ -(3-amino- 2,4,6-triiodophenyl)alkanoates, suitable for use in the practice of the present invention
  • Compounds 22 are 2- oleoylglycerol- 1 ,3-bis-[3-amino-2, 4, 6-triiodophenyl)alkanoates] which were synthesized via dicyclohexylcarbodiimide/4-dimethylaminopyridine (DCC/DMAP) coupling of a 2- monoolein (Compounds 21) with 2 equivalents of the corresponding ⁇ -(3-amino-2,4,6- triiodophenyl)alkanoic acid (Compounds 20) as described below.
  • DCC/DMAP dicyclohexylcarbodiimide/4-dimethylaminopyridine
  • Example 2 The iodinated triglycerides of Example 1 were incorporated into the lipid core of an oil-in-water emulsion by formulation techniques in accordance with the invention as set forth more completely in the following examples.
  • Example 2 The iodinated triglycerides of Example 1 were incorporated into the lipid core of an oil-in-water emulsion by formulation techniques in accordance with the invention as set forth more completely in the following examples.
  • Example 2 The iodinated triglycerides of Example 1 were incorporated into the lipid core of an oil-in-water emulsion by formulation techniques in accordance with the invention as set forth more completely in the following examples.
  • Example 2 The iodinated triglycerides of Example 1 were incorporated into the lipid core of an oil-in-water emulsion by formulation techniques in accordance with the invention as set forth more completely in the following examples.
  • Example 2 The iodinated triglycerides of Example 1 were incorporated into the lipid core of
  • Compound 31 which is DHOG
  • Compounds 32 and 33 are formulated into an oil-in-water emulsions ⁇ . accordance with the methods set forth below: Emulsion Example 1 :
  • DHOG (0.7507 g), triolein (0.7509 g), cholesterol (0.0613 g), ⁇ -tocopherol (0.0900 g) and MPEG-DSPE (0.0757 g) are weighed sequentially into a tared tube into which DOPC (0.2850 g) in ethanol solution is introduced. A 5 ml volume of chloroform is added to the tube to dissolve the lipid components. The solvents are removed in vacuo at 37°C on a rotary evaporator, interrupting the process once to rinse down the tube with an additional 1.5 ml of CHC1 3 . After completion of solvent removal, the tube is tared and anhydrous glycerol (0.7530 g) is added to the lipid mixture.
  • the tube is positioned on a Polytron homogenizer for a 5 min preliminary emulsification at 12,500 m under a nitrogen atmosphere at 50-55 °C. A 10 ml aliquot of sterile water is added with continuous mixing, followed by 5 min of emulsification at 25,000 ⁇ m under the same conditions. The volume of the crude emulsion is adjusted to a final volume of 15 ml with additional sterile water. The preparation is transferred to a Model 110-S MicroFluidizer for final emulsification at 18,200 psi for 10 min between 54-55.8°C. The emulsion is then passed sequentially through sterile 0.45 mm and 0.2 mm filter units into a sterile multidose vial. The emulsion is equilibrated at room temperature prior to determining mean particle diameter by submicron laser photon correlation spectroscopy (PCS). Mean particle diameter is 74 nm. Emulsion Example 2:
  • Ethyl iopanoate (0.5044 g), triolein (0.5042 g), cholesterol (0.0410 g), ⁇ - tocopherol (0.0619 g) and MPEG-DSPE (0.0500 g) are weighed into a tared tube into which DOPC (0. 1900 g) in ethanol solution is added. A 4 ml portion of CHC1 3 is added to the tube to dissolve the lipid mixture. The solvents are evaporated in vacuo at 37°C as described in example 1. After evaporation of the solvents, anhydrous glycerol (0.5014 g) is added to the lipid mixture and emulsified for 5 min at 12,500 ⁇ m on the Polytron under nitrogen.
  • a 6 ml aliquot of sterile water is added with continuous mixing and emulsified at 25,000 ⁇ m for 5 min at a temperature of approximately 55°C.
  • the crude emulsion is transferred to the MicroFluidizer after dilution to a final volume of 10 ml with sterile water.
  • the final emulsification is performed at 18,200 psi for 10 min at
  • Emulsion Example 3 n-Butyl iopanoate ((0.5037 g), triolein (0.5025 g), cholesterol (0.0416 g), ⁇ - tocopherol (0.0620 g) and MPEG-DSPE (0.0503 g) are weighed into a tared tube into which DOPC (0.1900 g) in ethanol solution is added. A 5 ml volume of CHC1 3 is added to the tube to dissolve the lipids. The solvents are evaporated in vacuo as described in example 1. After evaporation of the solvents, anhydrous glycerol
  • a 20% blood-pool emulsion is prepared in the following manner.
  • DHOG (1.6507 g), triolein ( 1.6507 g), cholesterol (0.0996 g), ⁇ -tocopherol (0.0994 g) and MPEG-DSPE (0. 1245 g) are weighed sequentially into a tared tube into which DOPC (0.4700 g) in ethanol solution is introduced.
  • DOPC 0.4700 g
  • a 5 ml volume of chloroform is added to the tube to dissolve the lipid components.
  • the solvents are evaporated at 37°C in vacuo as described in example 1. After evaporation of the solvents the tube is tared prior to addition of anhydrous glycerol (0.8266 g) to the lipid mixture.
  • the tube is positioned on the Polytron to emulsify the mixture at 12,500 ⁇ m for 5 min at less than 55 °C under a nitrogen atmosphere.
  • a 10 ml aliquot of sterile water is added with continuous mixing prior to emulsification at 25,000 ⁇ m under the same conditions.
  • the emulsion is transferred to the MicroFluidizer after dilution to a total volume of 16.5 ml with sterile water.
  • Final emulsification is performed at 18,600 psi for 5 min at 42.4-51.4°C.
  • the emulsion is filtered into a sterile multidose vial and equilibrated at room temperature.
  • the mean particle diameter is approximately 66.8 nm as determined by PCS sizing.
  • Emulsion Example 5 A radiolabeled form of the 10% blood-pool emulsion is prepared as follows.
  • DHOG (0.5003 g), triolein (0.5008 g), cholesterol (0.0403 g), ⁇ -tocopherol (0.0604 g) and MPEG-DSPE (0.0501 g) are weighed sequentially into a tared tube into which DOPC (0.1900 g) in ethanol solution is introduced. A 5 ml volume of chloroform is added to the tube to dissolve the lipid components. The solvents are evaporated as described in example . A 0.25 ml aliquot of 125 I-DHOG in CHC1 3 solution is added to the tube which is then rinsed down with additional CHC1 3 (1.2 ml). The chloroform is evaporated as before.
  • the tube is tared and anhydrous glycerol (0.5008 g) is added to the tube which is then positioned on the Polytron.
  • the mixture is emulsified under nitrogen at about 55°C for 5 min at 12,500 ⁇ m.
  • a 6.5 ml aliquot of sterile water is added with continuous mixing and emulsified at 25,000 ⁇ m for 5 min as described above.
  • the emulsion is transferred to the MicroFluidizer 1 10-S for final emulsification at 18,200 psi for 10 min between 54.4-55.5°C.
  • the emulsion is filtered through sterile filter units into a sterile multidose vial.
  • the activity of the emulsion is 15.2 mCi/ml.
  • DHOG-LE radioactive lipid emulsion that does not contain the PEG moiety
  • a radiolabeled form of the 10% DHOG emulsion is prepared as follows. DHOG (0.5004 g), triolein (0.5003 g), cholesterol (0.0472 g) and ⁇ -tocopherol
  • the mixture is emulsified under nitrogen at about 55 °C for 5 min at 12,500 ⁇ m.
  • a 6.5 ml aliquot of sterile water is added with continuous mixing and emulsified at 25,000 ⁇ m for 5 min as described above.
  • the emulsion is transferred to the MicroFluidizer 1 10-S for final emulsification at 18,200 psi for 10 min between 33.5-36.4°C.
  • the emulsion is filtered through sterile filter units into a sterile multidose vial.
  • the activity of the emulsion is 15.7 mCi/ml.
  • Emulsion Example 7 A 30% blood-pool emulsion is prepared in the following manner: DHOG
  • the tube is tared prior to addition of anhydrous glycerol (0.8265 g) to the lipid mixture.
  • the tube is positioned on the Polytron to emulsify the mixture at 12,500 ⁇ m for 5 minutes at less than 55 °C under a nitrogen atmosphere.
  • a 9 ml aliquot of sterile water is added to the anhydrous emulsion with continuous mixing prior to further emulsification at 25,000 ⁇ m for 5 minutes under the same conditions.
  • the emulsion is transferred to the MicroFluidizer after dilution to a total volume of 16.5 ml with sterile water.
  • Radioactive emulsions of the iodinated triglyceride DHOG was prepared by the technique set forth in Emulsion Example 5 (DHOG-PEG) and the corresponding hepatocyte-selective form of Emulsion Example 6 (DHOG-LE).
  • Fig. 4 is a graphical representation of CT density in Hounsfield
  • HU Units (HU) versus time in minutes in the blood of female New Zealand White rabbits following iv administration of DHOG-PEG or DHOG-LE. Blood-pool density enhancement was sustained for DHOG-PEG relative to DHOG-LE. Blood levels remained elevated up to 2 hours post-administration. However, follow-up CT studies, completed 24 hours after the initial administration, revealed that blood levels had dropped essentially to baseline.
  • Emulsion Example 1 which is a 10% emulsion of iodinated DHOG (60 mg I/kg), was administered to a pig.
  • Swine serve as an indicator of particulate-induced pulmonary hypertension because they have an unusually high number of RES cells in their lungs.
  • Fig. 5 is a graphical representation of pulmonary artery pressure and heart rate with respect to time (in minutes) post-injection.
  • a hepatocyte- selective oil-in-water emulsion is co-administered with the blood-pool agent of the present invention.
  • Liver-selective particulate agents hepatocyte-selective or Kupffer cell-selective
  • DHOG-PEG Example 1
  • hepatocyte- selective DHOG-LE Emsion Example 6, non-radioactive form
  • Rabbits (mean weight 2.5 kg) were inoculated with VX2 carcinoma directly into the hepatic parenchyma to produce a total of 8 focal lesions (2- 10 mm). Ten days later, the rabbits were scanned with multiple techniques including noncontrast, helical IV enhanced (600 mg I/kg iohexol), and 24 hours later using the iodinated microparticulate emulsion (200 mg I/kg). Tissue density measurements (HU) were made of liver, lesions, and blood (descending aorta). Tumor mo ⁇ hology was verified by gross pathologic examination.
  • Enhancement of liver tissue was greater for iohexol (+66.8 HU) than for the emulsions (+31.6 HU), but the liver-to-lesion difference favored the emulsions due to the lack of tumor enhancement (31.8 versus 28.8).
  • the lesions were subjectively better delineated with the emulsions due to sharper edge definition.
  • the results set forth in the Table 3 indicate that the combination opacified both the liver cells and the blood to improve both sensitivity and confidence in detection of very small tumors.
  • the receptor-mediated, hepatocyte-selective emulsion DHOG-LE enhances the liver significantly within 15- 30 minutes post-injection.
  • Blood-pool enhancement occurs transiently following iv administration of the hepatocyte-selective formulation and then diminishes rapidly as the agent is sequestered by the liver.
  • the blood-pool agent of the present invention does not enhance the liver for the first two hours following injection.
  • co- administration of a hepatocyte-selective lipid emulsion with the blood-pool lipid selec- tive emulsion of the present invention advantageously results in enhancement of the normal liver parenchyma and hepatic/systemic vasculature without significant tumor enhancement.
  • the oil-in-water emulsions of the present invention are suitable for parenteral administration to a mammalian subject, typically by intravenous administration. However, intramuscular, subcutaneous, intraperitoneal, and other delivery routes are within the contemplation of the invention. Further, the oil-in-water emulsions of the present invention may be administered by other routes, such as oral. It is a specific advantage of the oil-in-water emulsions of the present invention that they may be administered intravenously versus arterially, and in doses small enough, and slow enough, to be well-tolerated. Anticipated dose levels are 20 to 250 mg I/kg body weight.
  • blood-pool imaging not only offers diagnostic potential for virtually all vascular diseases including atherosclerosis and aneurysms, but also has potential to demonstrate organ perfusion defects.
  • new advances in CT scanner technology namely the introduction of ultra fast electron-beam CT scanners, may render this agent directly useful for cardiac angiography without the need for invasive and costly catheterization procedures.
  • animal models selected and used in the studies presented hereinabove, specifically rats and rabbits are well known to have hepatic physiologies that closely resemble the hepatic physiology of humans.
  • the blood-pool selective oil-in-water emulsions of the present invention afforded no adverse response in the pig pulmonary hypertension model.

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Abstract

L'invention a trait à une émulsion huile-dans-eau du type lipoprotéine à modification de surface utile en tant que véhicule d'apport sélectif du pool sanguin pour agents d'imagerie lipophiles ou dérivés lipophiles d'agents d'imagerie hydrosolubles. Ce véhicule d'apport sélectif du pool sanguin, qui demeure dans le sang pendant plusieurs heures, ne fait montre que d'une séquestration hépatique précoce très réduite et est éliminé dans les 24 heures. Le diamètre moyen de la phase huileuse est inférieur à 150 nm, ce qui réduit au maximum une séquestration par le système réticulo-endothélial. La surface de la phase huileuse est modifiée par un phospholipide à modification polyéthyl glycol pour empêcher l'existence d'interactions normales avec les sites récepteurs des hépatocytes. En imagerie radiographique, il est possible d'introduire dans le noyau lipophile d'une particule d'émulsion du type lipoprotéine des triglycérides radioactives ou stables, synthétiques ou semi-synthétiques polyhalogénées, 2-oléoyglycérol-1,3bis[7-(3-amino-2,4,6-tri-iodophényl)heptanoate] par exemple, ou des dérivés lipidiques solubles d'agents de contraste hydrosolubles classiques, notamment des esters aliphatiques d'acide iopanoïque, diatrizoïque et acétrizoïque.
EP98918097A 1997-04-11 1998-04-10 Vehicule du pool sanguin pour agents d'imagerie lipophiles Withdrawn EP0973553A2 (fr)

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US4330597P 1997-04-11 1997-04-11
US43305P 1997-04-11
PCT/US1998/007239 WO1998046275A2 (fr) 1997-04-11 1998-04-10 Vehicule du pool sanguin pour agents d'imagerie lipophiles

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JP2001300287A (ja) * 2000-04-20 2001-10-30 Nof Corp パーフルオロカーボン乳化製剤
US7713517B2 (en) 2004-04-21 2010-05-11 Marval Biosciences, Inc. Compositions and methods for enhancing contrast in imaging
US8357351B2 (en) 2004-04-21 2013-01-22 Ananth Annapragada Nano-scale contrast agents and methods of use
US8734853B2 (en) 2008-11-17 2014-05-27 University Of North Texas Health Science Center At Fort Worth HDL particles for delivery of nucleic acids
US20160346219A1 (en) 2015-06-01 2016-12-01 Autotelic Llc Phospholipid-coated therapeutic agent nanoparticles and related methods

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US5356633A (en) * 1989-10-20 1994-10-18 Liposome Technology, Inc. Method of treatment of inflamed tissues
US5843473A (en) * 1989-10-20 1998-12-01 Sequus Pharmaceuticals, Inc. Method of treatment of infected tissues
US5776429A (en) * 1989-12-22 1998-07-07 Imarx Pharmaceutical Corp. Method of preparing gas-filled microspheres using a lyophilized lipids
US5478860A (en) * 1993-06-04 1995-12-26 Inex Pharmaceuticals Corp. Stable microemulsions for hydrophobic compound delivery
DE4408011C1 (de) * 1994-03-10 1995-11-02 Max Delbrueck Centrum Pharmazeutisches Mittel zur Tumortherapie
GB9509016D0 (en) * 1995-05-03 1995-06-21 Royal Free Hosp School Med Tissue entrapment
US5858397A (en) * 1995-10-11 1999-01-12 University Of British Columbia Liposomal formulations of mitoxantrone

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WO1998046275A2 (fr) 1998-10-22
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CA2286138A1 (fr) 1998-10-22
JP2001524957A (ja) 2001-12-04
WO1998046275A3 (fr) 1999-01-28

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