WO1999055230A1 - Hyaluronan-based imaging agents - Google Patents
Hyaluronan-based imaging agents Download PDFInfo
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- WO1999055230A1 WO1999055230A1 PCT/US1999/009177 US9909177W WO9955230A1 WO 1999055230 A1 WO1999055230 A1 WO 1999055230A1 US 9909177 W US9909177 W US 9909177W WO 9955230 A1 WO9955230 A1 WO 9955230A1
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- hyaluronan
- dtpa
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- WTPVYAUXMCZWJW-UHFFFAOYSA-N CC(C)(C1)C1(C(C(C1O)O)O)OC1[I]=N Chemical compound CC(C)(C1)C1(C(C(C1O)O)O)OC1[I]=N WTPVYAUXMCZWJW-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/006—Biological staining of tissues in vivo, e.g. methylene blue or toluidine blue O administered in the buccal area to detect epithelial cancer cells, dyes used for delineating tissues during surgery
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1806—Suspensions, emulsions, colloids, dispersions
- A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
Definitions
- the present invention relates generally to imaging agents and, more particularly, to Hyaluronan-based imaging agents useful for targeting specific cells.
- Hyaluronan also known as Hyaluronic Acid (HA)
- HA Hyaluronic Acid
- CD44 belongs to the link module class of HA binding proteins and RHAMM belongs to a separate protein family. The expression of these two receptors is elevated on some human tumors. Both CD44 and RHAMM are critically involved in regulating cell motility and proliferation, and both are overexpressed during experimental tumor progression and following response to injury.
- RHAMM on cell motility and proliferation absolutely require their HA binding capability.
- CD44 and RHAMM overexpression has also been linked to human tumors including myeloma, pancreatic, lung, and breast cancer.
- the role of overexpression of specific CD44 isoforms in breast cancer and their utility for prognostic indication are unclear, but RHAMM overexpression, particularly within small subsets of cells in the primary tumor and within metastasis is prognostic of poor outcome in two geographically distinct populations. Further, overexpression of RHAMM enhances the prognostic value of commonly used tumor parameters, including lymph node status and metastasis free survival.
- Magnetic resonance (MR) images are based on the signal from hydrogen nuclei contained in hydrogen-rich compounds in me body: water and lipids. Image contrast depends primarily on two inherent properties of different tissues, specifically the hydrogen content (spin density) and the proton relaxation times (Ti, T2, and T2*). MR contrast agents shorten proton relaxation times of tissues containing them and, therefore, alter the signal intensity on MR images.
- MR contrast agents are of two types, endogenous and exogenous, the first of which comprises the many forms of endogenous iron, such as ferritin. The second type is represented by small chelate complexes of (typically) gadolinium (Gd 3+ ) ions, and large particulates of coated iron oxides, generally introduced intravenously.
- Superparamagnetic materials exhibit extremely large magnetic moments, even in weak external magnetic fields.
- the large magnetic moments associated with superparamagnetic iron oxide particles in tissues cause local field inhomogeneities which are used to improve detection of lesions by increasing lesion conspicuity in magnetic resonance imaging .
- the more specific the accumulation of a contrast agent within the target tissue the better the resulting lesion-tissue contrast.
- a useful magnetic iron oxide contrast agent should preserve its integrity once injected, escape rapid metabolism, selectively recognize the target, and bind to it.
- most colloidal superparamagnetic iron oxides are taken up rapidly by cells of the macrophage monocyte phagocytic system, preventing useful amounts of label from being delivered to specific tissues.
- Typical carriers include proteins, antibodies, polysaccharides, cells, and liposomes.
- Amphiphilic molecules such as surfactants or lipids self-assemble in water forming structures such as micelles, bilayers, tubes or vesicles. Nature provides many examples of such molecules which play key roles in the stability and functions of all cells and organisms.
- chemists have prepared a large number of synthetic amphiphiles, in particular hydrophobically-modified polymers, consisting of a water-soluble polymer onto which a small number of hydrophobic groups are attached by a covalent bond.
- Liposomes are vesicles consisting of a lipid bilayer which encloses an aqueous pocket. These aqueous pools can be loaded with water-soluble agents such as drugs or imaging reagents.
- liposoluble molecules can be dissolved in the liposome bilayer.
- liposomes can be used to carry a broad range of molecules. This property has been exploited in the design of in-vivo drug delivery systems. A recent patent application of Hyal Pharmaceutical Corp.
- arabinogalactan a naturally occurring polysaccharide which seems to have an affinity for receptors on hepatocytes.
- Other polysaccharides such as fucoidan, mannan, and chitosan, have also been studied. Most of these compounds show predominant biodistribution to liver, spleen, kidneys, and lungs.
- hydrophobically-modified polysaccharides have been investigated and utilized in biotechnology and medicine. This research has focused on polymers such as pullulan, dextran, and mannan partially substituted by various hydrophobic groups, such as long alkyl chains and cholesterol. It has been demonstrated that these polysaccharides effectively coated liposomal surfaces, and rendered the liposomes more stable against external stimuli, such as pH, ionic strength, and in-vivo biodegradation by enzymes and serum proteins, compared with conventional liposomes. Similar effects have been found with liposomes protected with hydrophobically-modified poly-(N-alkylacrylamides).
- Paramagnetic metal complexes are exogenous MR contrast agents.
- Gadolinium is the paramagnetic metal that contains the largest number of unpaired electrons and is, thus, theoretically, the most efficient Ti relaxation metal that can be used in contrast media for MR imaging.
- the metal When administered as free Gd 3+ the metal is extremely toxic. However, when bound to a chelating agent, it loses its toxicity but still maintains its paramagnetic properties. As in the case of superparamagnetic iron oxides, stringent requirements are placed on e Gd 3+ -chelates for them to become acceptable contrast agents.
- the present invention provides HA-based contrast-enhancing imaging agents for targeting specific cells.
- HA-based contrast agents target specific cells, display sufficient relaxivity, and are otherwise characterized by a specific in vivo distribution, in vivo stability, excretability, and a lack of toxicity.
- HA-based contrast agents of this invention are of three types, HA-complexed gadolinium (HA-Gd or Gd-HA), HA-complexed superparamagnetic iron oxides (HA-Fe), and HA-Gd complexed with liposomes (HA-Gd-liposomes).
- Gadolinium can be prepared within an HA carrier, yielding an HA-Gd gel, and can also be bonded to complexing agents attached to HA. Liposomes can optionally be incorporated into the HA-Gd combination.
- Superparamagnetic iron oxides can be prepared within an HA carrier, yielding an HA- iron oxide nanocomposite which can be isolated as a viscoelastic gel, a ferrofluid, or a dried powder.
- HA-based magnetic resonance (MR) contrast agents are believed to enable HA-receptor-directed imaging of human breast cancer cells by targeting the HA receptors, CD44 and RHAMM.
- HA-Gd, HA-Fe and HA-Gd-liposomes with Magnetic Resonance (MR) imaging are disclosed.
- methods of preparing HA-based MR contrast agents are disclosed.
- the role of HA-based contrast agents in targeting CD44 and RHAMM in tumor cells is determined. Also, a method of determining whether a tumor cell is benign or malignant is performed by measuring the presence or absence of an HA receptor with an HA-based imaging agent and determining the presence or absence of an HA receptor.
- FIG. 1 is a table showing the characteristics of human breast cancer cell lines
- FIG. 2 is a scintigraphic image of a nude rat showing the association of radiolabeled HA with a tumor in the rat;
- FIG. 3 is a scintigraphic image of a nude rat showing the association of radiolabeled HA with the carotid artery of the rat;
- FIG. 4 is a graph showing the molecular weight distribution of hyaluronan after being injected intravenously
- FIG. 5 is a depiction of two reaction schemes whereby HA is linked to a gadolinium complexing agent
- FIG. 6 is a graph showing the effect of pH and concentration of phosphate buffer on the electrophoretic mobility of HA-iron oxide particles;
- FIG. 7 is a depiction of the hydrophobically-modified HA complexed with liposomes;
- FIG. 8 is a depiction of the pathway for modifying HA by attaching Dieffiylenetriamine pentaacetic acid (DTPA) and complexing to gadolinium;
- DTPA Dieffiylenetriamine pentaacetic acid
- FIG. 9 is a graph showing the MRI signal intensity of HA-Gd linked to sepharose beads and free Gd as a function of concentration
- FIG. 10 is a graph showing the signal intensity of HA-Gd and free Gd in the liver as a function of time
- FIG. 11 is a graph showing me signal intensity of HA-Gd and free Gd in skeletal muscle as a function of time
- FIG. 12 is a graph showing the expression of CD44 and RHAMM cell surface receptors in benign and malignant tumor cells using FACS analysis
- FIG. 13 is a graph showing the uptake of Texas red-labeled HA in benign and malignant tumor cells as a function of concentration
- FIG. 14 is a graph showing the uptake (internalization) of labeled HA by different cell lines that express differing levels of HA receptors as a function of time;
- FIG. 15 is a graph showing tfie MRI signal intensity of benign and malignant tumor cells as a function of percentage HA-Gd substitution levels
- FIG. 16 is a depiction of signal intensity at specific tissues within a rat injected with HA-Gd
- FIG. 17 is a graph showing t e effect of the anti-CD44 antibody, KM201 , on the signal intensity of labeled HA added to different cell lines that express HA receptors;
- FIG. 18 is a depiction of confocal fluorescent images showing the effect of the anti- CD44 antibody, KM201 , on the uptake of labeled HA in different cell lines.
- Hyaluronan (HA)-based magnetic resonance (MR) contrast agents are used to enable HA-receptor-directed imaging of cells.
- These cells can be tumor cells of different cancers, including breast, colorectal, and lung cancers.
- FIG. 1 shows certain characteristics of specific human breast cancer cell lines. As illustrated by FIGS. 2 and 3, it has been shown in rats that HA injected at a concentration of between 3-10 milligrams/milliliter (mg ml) '1 retains its high molecular weight for 48 hours, and, thus, is able to bind to d e hyaluronan receptors CD44 and RHAMM. Specifically, FIG. 2 shows the targeting of 125 I-HA to a rat colorectal tumor cell line that displays high levels of the HA receptor, CD44.
- FIG. 1 shows certain characteristics of specific human breast cancer cell lines. As illustrated by FIGS. 2 and 3, it has been shown in rats that HA injected at a concentration of between 3-10 milligrams/milliliter (mg ml) '1 retains its high molecular weight for 48 hours, and, thus, is able to bind to d e hyaluronan receptors CD44 and RHAMM.
- FIG. 2 shows
- FIG. 3 shows the targeting of 125 I-HA to balloon catheter- injured rat carotid arteries, when the site displays maximum levels of the HA receptors, CD44 and RHAMM. Both of the images of FIGS. 2 and 3 were detected using a phosphoimager.
- FIG. 4 shows die molecular weight distribution of 6 mg/kg hyaluronan to be greater than 750,000 daltons 24 hours after being injected intravenously.
- me graph shows the HA concentration (ng/ml) as a function of elution volume (ml) and molecular weight (kDa). The assay leading to this result was conducted on a B 16 Molecular Weight Calibration Sephacryl 500-HR 1.6x61.5 cm column. HA injected at this concentration retained its high molecular weight, and, thus, it will be able to interact widi HA receptors, CD44 and RHAMM.
- HA used in die present invention has an average molecular weight of between about 20,000 and about 200,000 Daltons. A more preferred value for the average molecular weight is about 50,000 to about 100,00 Daltons. It is believed iat sonication of HA to reduce die average molecular weight to between about 20,000 and about 50,000 would improve penetration of the HA-based imaging agent made from the sonicated HA.
- Hyaluronan-based contrast agents are of tiiree types, HA-complexed gadolinium (HA- Gd or Gd-HA), HA-complexed superparamagnetic iron oxides (HA-Fe), or HA-Gd complexes with liposomes.
- the contrast agents are prepared by me synthesis and characterization of polysaccharide-based iron oxides and of complexes of modified HA widi gadolinium, optionally wi liposomes. To test die use of these as target contrast agents, these contrast agents are administered intravenously.
- CD44 and RHAMM in targeting HA to breast carcinomas is assessed by attempting to interrupt me binding of HA-Gd (optionally with liposomes) or HA-Fe with either blocking antibodies specific to RHAMM or CD44, or antisense peptides prepared against e HA binding domains of these receptors (see the explanation of FIGS. 17 and 18 below).
- HA is an ideal drug carrier and targeting agent for a wide variety of indications and pharmacological agents.
- a process has been developed to administer a preloading dose of chondroitin sulfate that binds to the liver scavenger receptor responsible for me uptake of HA by the liver. Using this approach, the amount of HA targeted to die liver can be reduced by over 80% . This treatment does not alter die ability of HA to target to sites expressing CD44 and RHAMM, because diese receptors are molecular ly distinct from the scavenger receptors of me liver.
- HA complexed widi gadolinium can be used for imaging disease conditions in malignant tumors. Specifically, it can be used to preferentially detect malignant breast cancer cells.
- Malignant human breast cancer cells have been shown to express higher levels of HA receptors man benign human tumor cells and to take up labeled HA at much greater rates man benign tumor cells.
- HA-Gd This uptake has been shown to be specific to HA and to be mediated by HA receptors, such as CD44 and RHAMM. Further, human malignant tumor cells exposed to HA- Gd exhibit a stronger signal intensity in magnetic resonance imaging (MRI) man benign tumor cells, providing in vitro evidence of the effectiveness of HA-Gd in targeting tumor cells.
- MRI magnetic resonance imaging
- Gd was shown to be rapidly taken up in a dose-dependent manner by me liver, which expresses high levels of HA receptors.
- the signal intensity of HA-Gd was strongly increased from benign to malignant tumor cells relative to equivalent amounts of free gadolinium, which did not show a dose-dependent increase in signal intensity.
- the signal intensity was low and it was not increased wid increasing amounts of HA-Gd.
- HA-Gd was coupled to agarose beads, washed to remove unbound material, and imaged widi MRI compared to an equivalent amount of free gadolinium also coupled to beads and washed to remove unbound material. This test was performed wimout targeting to any cells.
- the signal intensity of the HA-Gd linked to sepharose beads is significantly higher d an that of free gadolinium at a concentration of greater than 1 mg/ml and, in fact, it increases with increasing concentration while free gadolinium remains virtually constant.
- HA-Gd administered in increasing concentrations to rats resulted in a dose-dependent signal enhancement in the liver, which contains large amounts of hyaluronan receptors.
- free gadolinium Gd-DPTA at 9.6 mg/kg and Gd-DPTA at 19.2 mg/kg
- the signal intensity was always lower dian that of HA-Gd even though die Gd-DPTA at 19.2 mg/kg corresponds to the amount of gadolinium complexed within 100 mg/ml of HA-Gd. This demonstrates that uptake of HA is receptor-mediated.
- the weight of the HA-Gd is based upon the weight of d e hyaluronan and does not reflect die amount of gadolinium that is complexed.
- the percentage of gadolinium relative to hyaluronan that is attached to the polysaccharide is between about 3.0 and about 12.0%, with a preferred range between about 5.6 and about 9.6% .
- FIG. 11 represents the same animal tests as shown in FIG. 10, except that, instead of liver cells, skeletal muscle was measured by MRI. In FIG. 11, signal intensity for HA-Gd uptake was much lower in the skeletal muscle tissue man in d e liver.
- HA-Gd signal did not change significantly with changes in dose and was more similar to that of free gadolinium.
- skeletal muscle unlike tumors and liver cells, does not express cell-surface hyaluronan receptors.
- MDA-MM-231 malignant melatin-2311
- MCF-7 benign tumor cells
- FIG. 12 shows that die malignant tumor cells (MDA-MM-231) express much higher levels of these receptors in vitro dian die benign tumor cells (MCF-7), thus demonstrating the importance of these receptors in imaging and treatment of malignant cells.
- Texas red-labeled hyaluronan was added to d e above two malignant and benign cell lines in vitro to assess whedier or not the labeled HA would be taken up differentially by the cell lines.
- bodi cell types showed an increase in uptake of die labeled hyaluronan with increasing concentrations and bodi cell types showed a plateau of this uptake.
- 10T1/2 fibroblasts (10T) were transfected with RHAMM/CD44 (LR21).
- Mutant active ras (c3) which highly express RHAMM and CD44 were compared to malignant (MDA) and benign (MCF) cells. Fluorescent dextran was used as a background control to - 10 -
- Malignant breast cancer cells (MDA-MB-231 cells) were grown as xenografts in nude rats and the animals were injected widi 100 mg/kg of Gd-HA (HA, 5.6% complexed, represented as "B”). The control was me nude rat before exposure to Gd-HA (represented as "A”). As shown in FIG. 16, tiiere is increased signal intensity witi in the tumor (its location indicated by the arrow in FIG. 16) and in liver cells. These results indicate a high probability of imaging malignant tumors in vivo with HA-Gd imaging agents.
- Texas red-labeled HA in die absence of antibody Similar results are shown in me confocal fluorescent images of FIG. 18. Specifically, when 50 ⁇ m/ml of the anti-CD44 antibody, KM201, is added to 10T1/2 parenteral and RHAMM transfected (LR21) cell lines, the uptake of Texas red-labeled HA by the cell lines is significantly decreased versus Texas red-labeled HA in die absence of antibody, i.e. , higher resolution in me unblocked images.
- RHAMM receptors and erk molecules occurs in the nucleus of MDA-MB-231 (malignant) cells, but is perinuclear in MCF-7 (benign) cells.
- overexpression of ras molecules correlates wid overexpression of erk molecules and RHAMM receptors in breast cancer cells.
- RHAMM expression correlates with ras proto-oncogene or mutant active ras expression in MCF-10A cells.
- RHAMM overexpression correlates with overexpression of active erkl, ras, and CD44. - 11 -
- HA-stabilized ferrofluids involves the preparation of superparamagnetic, nanosized iron oxides within the framework of HA-hydrogels and subsequent conversion of die gels into HA-stabilized ferrofluids. Reaction temperature, pH, and die namre of d e oxidant can all affect me Fe 2+ oxidation in die HA matrix and their effect is determined.
- the composites are characterized by elemental analysis (Fe content), powder X-Ray crystallography, transmission electron microscopy (TEM) and SQUID magnetometry.
- the ferrofluids obtained from die magnetic gels are characterized by capillary electrophoresis, dynamic light scattering, and TEM.
- HA-stabilized MR iron oxides Another way of making HA-stabilized MR iron oxides consists of preparing nanosized magnetite in the absence of any surfactant or polymer. The magnetite obtained by this route is stabilized subsequently in physiological media by controlled adsorption of HA. The colloidal stability of the ferrofluids is monitored as a function of polymer concentration using various techniques, including turbidity measurements.
- a solution of ferric chloride hexahydrate (1.0 g) and ferrous chloride tetrahydrate (0.5 g) in water (200 ml) is purged with nitrogen for 15 minutes.
- An aqueous solution of NH4OH (5% by weight) is added to me vigorously stirred solution to raise the pH to 8.0.
- the mixture is stirred for an additional 15 minutes at the end of the addition.
- the magnetite obtained can be separated by decantation in die presence of a magnetic field. The magnetite is washed widi water until the pH of die supernatant is between about 6 and about 7.
- the magnetite is then suspended in deionized water to form a stock solution (30 mg/ml).
- the size of the magnetite particles is 15 nm + 3 nm, as determined by transmission electron microscopy (TEM).
- An aliquot of the magnetite stock solution (1.0 ml) is added to a solution of HA-NH2 (its preparation is described below, 0.08 g) in water (5.0 ml).
- the resulting suspension is treated by sonication using a Microson Ultrasonic Cell Disruptor (2 minutes at power setting 15).
- the reselecting fluid is washed with a phosphate buffered saline (PBS) buffer (pH 7.33) and purified by elution tiirough a magnetic column.
- PBS phosphate buffered saline
- the resulting fluid is treated first with aqueous NaOH (0.1 M) to adjust the pH to 11, then with HC1 (0.1 N) to bring d e pH to 7.3.
- the fluid is then filtered dirough a 0.45 ⁇ m filter.
- a solution of NaN3 in water is added to the ferrofluid (final NaN3 concentration: 10 "3 M).
- FIG. 5 shows two possible routes for modifying HA by covalently linking to its backbone a compound which complexes with Gd 3+ .
- Route A an amine in which the primary amino group is linked to one of diree possible Gd 3+ complexing agents (1 , 2 or 3 in FIG. 5) is attached to HA which contains a carboxyl group.
- a primary amine may be introduced to the HA backbone and then reacted subsequently widi macrocycles bearing carboxylic groups.
- the modified polymers are then treated with gadolinium which will form complexes with the functional groups attached to HA.
- the resulting modified polymers are characterized by standard chemical means and the binding constant of Gd 3+ to the polymeric chelating agents is measured. These polymers are then evaluated as MR contrast agents.
- MR contrast agents There are three main factors to consider for making a complex of gadolinium which will be stable in vivo: a) me thermodynamic stability constant of the metal/ligand complex under physiological conditions, b) d e selectivity of the ligand for gadolinium, and c) the reaction kinetics.
- ferrofluids and gadolinium complexes are digested widi testicular hyaluronidase.
- the toxicity of the HA-based contrast agents is measured to determine die extent of complexation.
- HM-HA Hydrophobically-modified HA
- FIG. 7 in which die main chain of HA is partially substituted widi hydrophobic groups, is anchored on the outer surface of the phospholipid bilayer 4.
- These hydrophobic substituents can be inserted into die liposome bilayer, thus providing strong anchoring points 6 for the HA on die external membrane (liposome bilayer).
- Liposomes can be prepared by a variety of methods yielding eitiier unilamellar or multilamellar vesicles of narrow size distribution ranging in diameter from 50 nm to 1 ,000 nm. The liposomes can be obtained first in die absence of HM-HA 2 which can be anchored widiin die liposome membranes by incubation of "naked" liposome aqueous suspensions in me - 13 -
- HM-HA HM-HA 2
- Surface saturation and stability of the complexes can be determined by techniques including fluorescence spectroscopy, centrifugation, electron microscopy, and gel permeation chromatography.
- Drugs or imaging agents can be incorporated in me liposomes prior to stabilization widi HM-HA 2.
- the outer layer of HA serves as a targeting compound for me liposome.
- Encapsulated materials can be chosen among any known imaging agent, such as for example, the commercial MR imaging agent Magnevist ® , or the HA-iron oxide described above, the commercial MR imaging agent Magnevist ® along with HA-Gd or any HA-Gd complexing agent described above.
- the HM-HA 2 is prepared by covalent attachment of hydrophobic groups, such as n-alkyl chains of from about 10 to about 24 carbons or the cholesteryl group. It also may be prepared by activation of the carboxylic acid groups and subsequent conversion to amides and conversion of primary hydroxyls to ethers.
- a fluorescent group such as pyrene or naphthalene can be linked to HA to serve as a probe of the effective anchoring of die HM-HA 2 onto die liposome membrane.
- HA may be anchored onto the outer membrane of me liposomes dirough a crosslinking agent.
- the crosslinking agent may admixed in me liposomes before diey are coated widi HA, as discussed in U.S. Patent No. 5,603,872 to Margalit.
- HM-HA 2 The structure of the HM-HA 2 is as follows:
- R is (CH 2 )nCH3, 3-cholesteryl, or H and Rl is X(CH 2 )nCH3, -NHCeH ⁇ NH-
- die binding of the HM-HA 2 to liposomes can generally be obtained in two steps: (a) preparation of uncoated "naked" liposomes, and (b) incubation of me liposome suspension in me presence of HM-HA. Binding can be measured by fluorescence spectroscopy, gel permeation chromatography, and centrifugation assays. The physico-chemical stability of
- HM-HA coated liposomes against external stimuli such as pH, ionic strength, and in vitro degradation by serum protein, and surface samration can also be evaluated using me assays used to measure binding. It is believed diat MR contrast agents, such as gadolinium, are - 14 -
- modified liposomes eimer in die aqueous core of die liposomes or widiin die lipid bilayer.
- the resulting materials can men be tested following the protocols employed in die evaluation of me other HA-based contrast agents.
- HA has been modified with ethylene diamine, hydrazine monohydrate, and diethylenetriamine (as described below), where the complexing agent is linked to a small amount of the HA carboxylic groups. Alternatively, the complexing agent is linked to me C6 position of the HA disaccharide units.
- the final gadolinium-HA complexes are analyzed by all of die methods described above. Modification of HA with Ethylenediamine
- Ethylenediamine (1.2 g, 20 mmol) was added to a solution of sodium hyaluronan (200 mg, 0.50 mmol) in water (50 ml). The pH of the reaction mixture was adjusted to 4.75 using 0.1 N HC1. Then, l-ethyl-3-(3-dimethylaminopro ⁇ yl) carbodiimide (EDC, 382 mg, 2.0 mmol) was added in solid form. The pH of the reaction mixture was maintained at 4.75 by addition of 0.1 N HC1 and the mixture was stirred at room temperature for 12 hours. After this period, die pH of the reaction mixture was adjusted to 7.0 by addition of 1 N NaOH. The mixture was subjected to ultrafiltration using a YM30 membrane.
- the resulting viscous polymer solution was diluted with water. This aqueous polymer solution was added into a large amount of methanol. The solid precipitated polymer was separated by vacuum filtration, washed widi methanol, redissolved in die minimum amount of water, and lyophilized for 24 hours to obtain a yield of 350 mg of the ethylenediamine-modified HA.
- Diethylenetriamine (2.06 g, 20 mmol) was added to a solution of sodium hyaluronan (200 mg, 0.50 mmol) in water (50 ml). The pH of the reaction mixture was adjusted to 4.75 using 0.1 N HC1. Then, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 382 mg, 2.0 mmol) was added in solid form. The pH of the reaction mixture was maintained at 4.75 by addition of 0.1 N HC1 and me mixture was stirred at room temperature for 12 hours. After mis period, the pH of the reaction mixture was adjusted to 7.0 by addition of 1 N NaOH. The mixture was subjected to ultrafiltration using a YM30 membrane.
- the resulting viscous polymer solution was diluted with water and subjected to a second ultrafiltration.
- the resulting concentrated aqueous polymer solution was lyophilized for 24 hours to obtain a yield of 340 mg of the diethylenetriamine-modified HA. - 15 -
- the solution was transferred to a dialysis bag (molecular weight cutoff: 10,000) and dialyzed exhaustively against water.
- the resulting aqueous polymer solution was lyophilized for 24 hours to obtain a yield of 300 mg of the hydrazine monohydrate-modified HA.
- Sodium hyaluronate 200 mg, 0.50 mmol was dissolved in 20 ml of water. The pH of the solution was adjusted to between 3.5 and 4.5 with aqueous hydrochloric acid (0.1 N) and aqueous sodium hydroxide (0.1 N). To d is solution, EDC (93 mg, 0.50 mmol) was added, and die pH of the solution was adjusted to between 3.5 and 4.5.
- the resulting viscous polymer solution was diluted with water and subjected to a second ultrafiltration.
- the resulting concentrated aqueous polymer solution was lyophilized for 24 hours to obtain a yield of 240 mg of product.
- the three types of modified HA were obtained with yields between 300 and 350 mg, and after modification, diey were complexed to gadolinium.
- an aqueous solution of modified HA (5 ml, 10 g/L) was added dropwise to a stirred aqueous solution of gadolinium chloride (1 ml, 0.05 to 0.2 M). The mixture was kept at room temperamre for 2 hours.
- HA-Gd complex was isolated by lyophilization of me purified solution and a colorless material was obtained. It was dien dissolved in physiological buffer to prepare a MR contrast agent solution of concentration from 0.5 to 1.0 g/L. From - 16 -
- the DTPA pathway proceeds with NH2 groups being attached to the HA widi ediylenediamine (EDA) and forming several different preparations, some having different ratios of EDA to NH2 groups, as shown.
- EDA ediylenediamine
- the DTPA is then attached to die NH2 groups on the HA preparations. Finally, the gadolinium is complexed with these preparations to yield HA-Gd- DTPA complexes.
- Table 2 shows the water content, determined by gravimetric determination, DTPA molar percentage, obtained from 'H NMR spectroscopy, and gadolinium content from ICP analysis of die different preparations formed by the pathway described.
- Table 3 shows me reagent components used in the different preparations. Specifically, it depicts the amount of HA-COONa, DTPA, DCC (1,3-Dicyclohexylcarbodiimide), NHS (N-Hydroxysuccinimide), EEDQ (2-ed ⁇ oxy-l -ethoxy carbonyl-l,2-dihydroquinoline), and EDA.
- Table 4 shows die retention times of the different preparations on a column in which retention time is a function of die molecular weight of the preparation.
- HA-COONa 1.00g(2.4x 1.00g(2.4x 1.00g(2.4x 2.00g(4.8x
- the reagents used in die DTPA padiway were: Hyaluronic acid (sodium salt), TEAT (1,4,8,11 - Tetraazacyclotetradecane-l,4,8,ll-tetraacetic acid tetrahydrochloride tetrahydrate), DTPA (Diediylenetriamine pentaacetic acid), DCC (1,3-Dicyclohexylcarbodiimide), N- Hydroxysuccinimide, EEDQ (2-ethoxy-l-ethoxycarbonyl-l,2-dihydroquinoline), Gadolinium (III) Chloride hexahydrate, Arsenazo III ([2,2'-(l-8-dihydroxy-3,6-disulfonaphthylene-2,7-bisazo)- bisbenzene arsonic acid]), acetonitrile, methanol, ethylenediamine, triethylamine, HCl, and NaOH.
- HA-COONa 250 mg, 0.62 mmol COONa was dissolved in distilled, deionized H2O (20 ml). The pH (measured with pH paper) was adjusted to 3 with 1.0N HCl. EEDQ (0.309 g, 1.25 mmol) in MeOH (15 ml) was added dropwise to the HA mixture. Etirylenediamine (0.42 ml, 6.2 mol) was added and die mixture was stirred at room temperamre for 24 hours.
- the mixmre was dialyzed against 2L MeOH/H2 ⁇ (1:1, v:v) for 3 days, men against 2L H2O for 3 days (MWCO: 12-14,000 Da).
- the dialyzed mixtore was filtered by vacuum filtration using a sintered glass filter and lyophilized to yield a light, white solid (150 mg, 60% yield). Attachment of DTPA to HA-NH2 proceeds as follows:
- DTPA 0.5 g, 1.26 mmol
- acetonitrile 5 ml
- triethylamine 0.88 , 5X molar amount of DTPA
- DCC 0.72 g, 1.8 mmol
- N-Hydroxysuccinimide 0.208 g, 1.8 mmol
- HA-NH2 250mg, 0.62mmol NH2, assuming all COONa groups were aminated
- H2O 20ml
- d e pH was adjusted to 10 widi 1.0 N NaOH.
- a 1 % HA-DTPA solution in H2O was prepared.
- a 0.1 M GdCh solution was added dropwise, and this mixtore was subjected to ultrafiltration using a YM30 membrane.
- GdCh was added until the filtrate contained detectable amounts of Gd 3+ , as detected by a colorimetric test using Arsenazo III in acetate buffer, pH 3.89 at 0.1 ionic strength (NaCl).
- the solution was subjected to two additional ultrafiltrations, adding 20ml of H2O after each filtration cycle.
- the HA-DTPA-Gd was lyophilized, yielding a light, white solid.
- HA-COONa 1.0 g was dissolved in distilled deionized water (80 ml). The pH of the solution was adjusted to 3.0 using hydrochloric acid (HCl, 1.0 N). A solution of EEDQ (7.7 g) in methanol (150 ml) was added dropwise to die reaction mixtore. The mixtore was stirred at room temperature until it turned clear. Ediylenediamine (EDA) was then added to the - 20 -
- reaction mixtore which was stirred at room temperature for 24 hours.
- the solution was placed in dialysis bags (SpectraPore membrane with a molecular weight cut off (MWCO) of 6,000 to 8,000 Daltons).
- MWCO molecular weight cut off
- the solution was dialyzed against methanol/water (1/1 v/v (volume ratio), 2.0 L) for 3 days and against water (2 L) for 3 days.
- the resulting solution was concentrated by ultrafiltration.
- the polymer was isolated by freeze-drying (HA-NH2, 0.3 g)
- HA-COONa 1.0 g was dissolved in distilled deionized water (80 ml). The pH of the solution was adjusted to 3.0 using hydrochloric acid (HCl, 1.0 N). A solution of EEDO (7.7 g) in dimethylformamide (150 ml) was added dropwise to the reaction mixtore. The mixtore was stirred at room temperatore until it turned clear. Ediylenediamine (EDA, 8 ml) was then added to the reaction mixture, which was stirred at room temperatore for 24 hours.
- EDA Ediylenediamine
- the solution was placed in dialysis bags (SpectraPore membrane with a MWCO of 6,000 to 8,000 Daltons). The solution was dialyzed against isopropanol/water (1/1 v/v, 2.0 L) for one day and against water (2 L) for 3 days. The resulting solution was concentrated by ultrafiltration. The polymer was isolated by freeze-drying (HA-NH2, 0.46 g). 3(a) HA-COONa (2.0 g) was dissolved in distilled deionized water (2000 ml). The pH of the solution was adjusted to 3.0 using hydrochloric acid (HCl, 1.0 N). A solution of EEDQ (15.4 g) in dimethylformamide (150 ml) was added dropwise to the reaction mixtore.
- HCl hydrochloric acid
- the solution was dialyzed against isopropanol/water (1/1 v/v, 2.0 L) for one day and against water (2 L) for 3 days. The resulting solution was concentrated by ultrafiltration. The polymer was isolated by freeze-drying (HA-NH2, 0.96 g).
- the reaction mixtore was stirred at room temperatore for 24 hours.
- the mixtore was filtered and e filtrate was concentrated by ultrafiltration widi exhaustive washes with water.
- the polymer (HA-DTPA) was isolated by freeze-drying (0.24 g). - 21 -
- Superparamagnetic forms of iron oxides are readily prepared widiin ionic polysaccharides and they can be complexed with HA to obtain an imaging agent.
- the resulting nanocomposites can be degraded into stable aqueous ferrofluids.
- HA is used as a template for the preparation of magnetic nanocomposites, and subsequent ferrofluid formation.
- the crosslinking ion (Fe 2+ ) serves as the reaction center for the in-sito formation of nanocrystalline iron oxides.
- the oxidation of the ferrous ion leads predominantly to the formation of a superparamagnetic form of iron oxides, eitiier maghemite ( ⁇ -Fe 2 03) or magnetite (Fe3 ⁇ 4).
- the room temperatore satoration magnetization of dehydrated gels was 12 emu g "1 at 30 kOe for dry specimens containing 21 % (w/w) of Fe. These gels were converted into strongly magnetic, colloidally stable ferrofluids by sonication in water or treatment widi L-ascorbic acid.
- TEM Transmission electron microscopy
- TEM Transmission electron microscopy
- TEM Transmission electron microscopy
- the electron diffraction pattern of these particles showed diffraction rings characteristic of ⁇ -Fe2 ⁇ 3 and Fe3 ⁇ 4. It was difficult to distinguish between die two phases.
- Size distribution analysis of ferrofluids by quasi-elastic light scattering (QELS) revealed the presence of diree particle distributions.
- One of the particle distributions detected was in die size range of 20-50 nm. Because the QELS measurements yield die size distribution of die iron oxide particles stabilized by residual chains of HA, a slightly larger size, compared to die TEM results, is expected to account for the corona of polymeric chains surrounding die oxide particles.
- the histogram also reveals larger particles having a size range of 125-250 nm. This may represent agglomerates of the smaller particles.
- a further signal centered at 4 nm is attributed to free HA.
- the surface charge properties of the ferrofluids were determined by capillary electrophoresis.
- the HA-iron oxide particles (ferrofluids) elute as a broad band after caffeine used as a neutral marker, indicating diat this ferrofluid consists of negatively charged particles.
- the negative charge is attributed to the carboxylate groups present in die HA chains stabilizing die iron oxide particles. Electrophoretic mobilities and zeta potentials for particles were calculated from the electropherogram. To stody die influence of pH, ionic strengtii and applied - 23 -
- HA a 10 gram liter solution of HA was prepared in a 0.1 M solution of FeCh in methanol/water (50/50 v/v). Gelation occurred immediately, resulting in the formation of large, clear yellow, hydrogel fragments in me FeCh solution. This suspension was kept under a static nitrogen atmosphere at room temperatore for 3 hours. Subsequently, the HA gel fragments were thoroughly washed with methanol/water (50/50 v/v) to remove any uncomplexed Fe 2+ ions from the gel. A 0.5 M solution of NaOH in methanol/water (50/50 v/v) was added to the washed gel to hydrolyze the complexed Fe 2+ ions.
- the gel-like product was centrifuged and washed to remove methanol, and then redispersed in deionized water.
- An aqueous solution of L-ascorbic acid (1 mM) was added to diis suspension and die resulting mixtore was stirred at room temperatore for 24 hours, then sonicated for 30 minutes. It was purified by ultrafiltration through a 10,000 MWCO DIAFLO membrane at a nitrogen gas pressure of 60 psi. This resulted in a homogeneous suspension (ferrofluid) of iron oxide stabilized by HA.
- This ferrofluid had a solid content of 6.6 milligrams dry solid per milliliter of suspension and an iron (Fe) content of 40% w/w of dry solids as determined by elemental analysis.
- TEM samples were prepared by dispersing the ferrofluid in 2-propanol, and placing a drop of diis suspension on a carbon coated TEM grid and letting it dry.
- dilute suspensions of die ferrofluid were - 24 -
- CE Capillary Electrophoresis
- Samples for CE were prepared by adding 5% (v/v) of the HA based ferrofluid and 5% (v/v) of a stock solution of caffeine (1 mg/ml) to a sodium phosphate buffer.
- Caffeine acts as a neutral marker and can be used for the calculation of electroosmotic flow.
- Experiments were conducted at room temperatore in a pH range of 5.5 to 11 (ImM, 10 mM, and 25 mM phosphate buffer concentrations) and for applied voltages of 15 to 30 kV. As indicated previously, die electrophoretic mobility of the HA-iron oxides was approximately 3 x 10 "8 m 2 /V sec.
- magnetic gels may be prepared by degassing a solution of FeCh (0.2 M, 25 ml) in methanol/water (50/50 v/v) by bubbling nitrogen for 15 minutes.
- a solution of HA HA
- An aqueous solution of L-ascorbic acid (1.0 mM, 1 ml) was added and die resulting mixtore was sonicated for 30 minutes. It was purified by dialysis against water at room temperature. Bom suspensions are believed to be useful as MR Imaging agents.
- HA-imaging agents In order to determine the ability of HA-imaging agents to bind to human breast cancer cells, die fluorescent dye Cy3, Texas red, or any oti er suitable dye is coupled to HA-imaging agents complexed widi liposomes. Binding stodies include Cy3-HA alone to determine whether coupling widi MR imaging agents alters binding to die cell surface. It is believed diat HA- imaging agents complexed wid liposomes may have a longer duration in cells because of the encapsulation in d e liposomes.
- Cy3-HA is prepared widi a procedure identical to diat for preparing fluorescein isothiocyanate fluorochrome that emits at 590 nm witii HA (FITC-HA), which has been used extensively for studying HA/cell surface interactions. Labeling of HA with Cy3 should not affect die binding properties of die HA. Cy3 is superior to FITC, because it does not rapidly fade. - 26 -
- FIG. 1 The cell lines described in FIG. 1 are used for this stody because tiieir expression of HA receptors has been well described.
- Cells are prepared for flow cytometry (FACS) analysis, then incubated for 1 hour on ice with Cy3-HA imaging agents. Live cells are released from the substratum, incubated with FITC ILA ⁇ antibody, washed wi i phosphate buffered saline (PBS), and dien cell surface fluorescence analyzed widi the flow cytometer at a wavelengm of 590 nm.
- FACS flow cytometry
- the tumor is excised and analyzed for cell surface display of RHAMM and CD44 by obtaining single cells and the cell surface binding of RHAMM and CD44 antibodies is quantified widi FACS.
- protein lysates are prepared and die presence of RHAMM and CD44 is also detected in Western Blot assays.
- mRNA is obtained from tumor cells and RT-PCR analysis of both RHAMM and CD44 is conducted, using appropriate primers.
- the chosen cell lines (10 6 cells/rat) are men injected and tumors allowed to grow for varying periods of time in order to obtain tumors of increasing size.
- the HA-based imaging agents chosen from the above stodies are then labeled widi I25 I, so that die targeting ability can - 27 -
- mice are imaged wid MR, because diis mediod may be more sensitive than phosphoimaging.
- animals are initially injected with 10 mg kg "1 of chondroitin sulfate to down-regulate scavenger receptors that take up HA in die liver.
- the double-labeled HA imaging agents are men injected intravenously one hour later, phosphoimaged, and the animals taken for MR imaging. Timing should not be a problem because stodies indicate that HA targets tomors for 12-24 hours. It is believed diat ti ese experiments will show mat HA-imaging agents can target human breast tomors and that they will demonstrate die smallest size of tumor that can be imaged using MR imaging.
- Antibodies are injected into animals intravenously, one hour after the intravenous injection of chondroitin sulfate, and 0 to 15 minutes prior to injection of me HA. It is believed that the effect on targeting can be quantified at varying times after injection. Binding of HA to its receptors is a complex event and it is possible diat die antibodies that affect HA binding to myeloma cells do not affect HA binding to breast cancer cells. If these antibodies are not effective at blocking HA binding in vitro, anti-sense peptides to the HA binding domains of CD44 and RHAMM are used. This approach has been used successfully in vivo to block the AT-1 receptor.
- Bodi CD44 and RHAMM require die presence of a basic amino-acid motif to bind to HA, and anti-sense peptides to d is region are prepared. They are administered in vitro or in vivo, as described above for antibodies. The effect of peptides on die distribution of HA is assessed wid FACS. An effective concentration range is determined experimentally.
- a human combinatorial phage display library can be used to isolate antibodies diat recognize die HA binding motifs of CD44 and of RHAMM, which are conserved in mouse to human. The attainment of monoclonal and polyclonal antibodies to these sequences has been attempted and it failed to raise a polyclonal response.
- use of die phage library allows the attainment of human antibodies diat recognize tiiese domains. - 28 -
- tiiese antibodies may bind to the CD44 and RHAMM receptors and prevent HA or HA-based contrast agents from binding to them.
- the antibodies can clarify die role of CD44 and RHAMM in binding HA in cells. Role of HA-Gd Contrast Agents in Central Nervous System (CNS) Disorders
- HA-Gd contrast agents for CNS disorders include imaging of brain tomors, stroke-induced injury, and Alzheimer's type neural degeneration.
- Gadolinium is currently used to image CNS tomors, but is only effective at locations where there is breach of the blood-brain barrier making mis procedure less effective in visualizing die entire tumor encompassing intact vascular areas.
- current MR imaging can visualize only gross tissue atrophy which is only useful late in disease progression.
- compromised tissue can be visualized by MR. Nevertheless, this does not reveal die full extent of the vascular system that undergoes biomechanical changes that could contribute to further damage via secondary processes, such as tiiose mediated by vascular invasion of inflammatory cells.
- HA hyaluronan receptors
- these receptors include die HA receptors, RHAMM and CD44.
- Levels of intracellular adhesion molecule (ICAM-1) are also elevated in these conditions and ICAM appears to be identical to liver endothelial HA receptor which has been shown to bind to HA.
- HA/gadolinium conjugate will allow MR imaging of not only CNS tissue where gadolinium is currently useful, but also areas where the blood brain barrier is intact and exhibits increased HA receptors, which is indicative of alterations in vascular and tissue functions diat reflect potential local damage. This utility is based on evidence of an association between elevated HA receptors and tissue injury.
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
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JP2000545439A JP2002512942A (en) | 1998-04-28 | 1999-04-27 | Hyaluronan-based image forming agents |
AU38700/99A AU3870099A (en) | 1998-04-28 | 1999-04-27 | Hyaluronan-based imaging agents |
NZ507540A NZ507540A (en) | 1998-04-28 | 1999-04-27 | Hyaluronic acid based imaging agents containing gadolinium or iron oxide |
EP99921503A EP1075215A4 (en) | 1998-04-28 | 1999-04-27 | Hyaluronan-based imaging agents |
CA002328698A CA2328698A1 (en) | 1998-04-28 | 1999-04-27 | Hyaluronan-based imaging agents |
Applications Claiming Priority (2)
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US8331198P | 1998-04-28 | 1998-04-28 | |
US60/083,311 | 1998-04-28 |
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WO1999055230A1 true WO1999055230A1 (en) | 1999-11-04 |
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PCT/US1999/009177 WO1999055230A1 (en) | 1998-04-28 | 1999-04-27 | Hyaluronan-based imaging agents |
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EP (1) | EP1075215A4 (en) |
JP (1) | JP2002512942A (en) |
AU (1) | AU3870099A (en) |
CA (1) | CA2328698A1 (en) |
NZ (1) | NZ507540A (en) |
WO (1) | WO1999055230A1 (en) |
ZA (1) | ZA200006067B (en) |
Cited By (17)
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EP1233754A2 (en) * | 1999-12-03 | 2002-08-28 | The Regents of The University of California | Targeted drug delivery with a cd44 receptor ligand |
WO2003018640A2 (en) * | 2001-08-22 | 2003-03-06 | Aventis Pharma Deutschland Gmbh | Use of heparinoid derivatives for treating and diagnosing diseases that can be treated by heparinoids |
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US6875753B1 (en) | 1996-03-14 | 2005-04-05 | The Governors Of The University Of Alberta | Methods for cell mobilization using in vivo treatment with hyaluronan (HA) |
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5368840A (en) * | 1990-04-10 | 1994-11-29 | Imarx Pharmaceutical Corp. | Natural polymers as contrast media for magnetic resonance imaging |
US5449508A (en) * | 1991-02-01 | 1995-09-12 | Unger; Evan C. | Phosphorylated materials as contrast agents for use in magnetic resonance imaging of the gastrointestinal region |
US5593658A (en) * | 1992-09-04 | 1997-01-14 | The General Hospital Corporation | Medical compositions |
US5707604A (en) * | 1986-11-18 | 1998-01-13 | Access Pharmaceuticals, Inc. | Vivo agents comprising metal-ion chelates with acidic saccharides and glycosaminoglycans, giving improved site-selective localization, uptake mechanism, sensitivity and kinetic-spatial profiles |
US5756069A (en) * | 1993-07-23 | 1998-05-26 | Torchilin; Vladimir P. | Amphipathic polychelating compounds and method of use |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU3239895A (en) * | 1994-08-10 | 1996-03-07 | Roderick T. Coward | Method of using hyaluronic acid for the detection, location and diagnosis of tumors |
-
1999
- 1999-04-27 CA CA002328698A patent/CA2328698A1/en not_active Abandoned
- 1999-04-27 WO PCT/US1999/009177 patent/WO1999055230A1/en not_active Application Discontinuation
- 1999-04-27 JP JP2000545439A patent/JP2002512942A/en active Pending
- 1999-04-27 EP EP99921503A patent/EP1075215A4/en not_active Withdrawn
- 1999-04-27 AU AU38700/99A patent/AU3870099A/en not_active Abandoned
- 1999-04-27 NZ NZ507540A patent/NZ507540A/en unknown
-
2000
- 2000-10-27 ZA ZA200006067A patent/ZA200006067B/en unknown
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5707604A (en) * | 1986-11-18 | 1998-01-13 | Access Pharmaceuticals, Inc. | Vivo agents comprising metal-ion chelates with acidic saccharides and glycosaminoglycans, giving improved site-selective localization, uptake mechanism, sensitivity and kinetic-spatial profiles |
US5368840A (en) * | 1990-04-10 | 1994-11-29 | Imarx Pharmaceutical Corp. | Natural polymers as contrast media for magnetic resonance imaging |
US5449508A (en) * | 1991-02-01 | 1995-09-12 | Unger; Evan C. | Phosphorylated materials as contrast agents for use in magnetic resonance imaging of the gastrointestinal region |
US5593658A (en) * | 1992-09-04 | 1997-01-14 | The General Hospital Corporation | Medical compositions |
US5756069A (en) * | 1993-07-23 | 1998-05-26 | Torchilin; Vladimir P. | Amphipathic polychelating compounds and method of use |
Non-Patent Citations (1)
Title |
---|
See also references of EP1075215A4 * |
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Also Published As
Publication number | Publication date |
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EP1075215A4 (en) | 2002-04-17 |
EP1075215A1 (en) | 2001-02-14 |
JP2002512942A (en) | 2002-05-08 |
NZ507540A (en) | 2002-07-26 |
AU3870099A (en) | 1999-11-16 |
ZA200006067B (en) | 2001-08-23 |
CA2328698A1 (en) | 1999-11-04 |
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